CA2104415A1 - Method for generating monoclonal antibodies from rabbits - Google Patents

Abstract

ABSTRACT OF THE INVENTION
This invention relates to a method for producing rabbit monoclonal antibodies employing a stable xenogeneic fusion partner. The stable xenogeneic fusion partner is a product of the fusion of a rodent myeloma cell and a non-transformed rabbit partner cell. A method for generating rabbit monoclonal antibodies is disclosed that comprises fusing a nontransformed rabbit partner cell with a rodent myeloma cell to produce a xenogeneic fusion partner, selecting a stable fusion partner producing undetectable levels of antibodies, fusing the stable fusion partner with a rabbit antibody producing cell and isolating an antibody producing cell line that produces antibodies directed to a predetermined antigen.

Description

2 ~ ~ e~

~E~OD FOR GENERATING ~ONOCLONAL ANTIRODXES FRO~ R~BBITS

The present invention relates generally to the field of monoclonal antibodies. More specifically, the invention relates to methods for producing rabbit monoclonal antibodies and to the cells used to produce them~
Monoclonal antibodies are typically produced by fusing an antibody producing cell wi~h an immortalizing cell to generate a ho~ogenous population of cells all producing antibodies directed to the same antigenic epitope. Mice and rats are traditional sources for the antibody producing cells. Cell hybrids produced from the fusion of the antibody producing cell and ~he immortal fusion partner, termed fusion products of hybrids, are sel~cted and tested for the quality and quantity of secreted antibodies reacting with the immunizing antigen. The hybrids are expanded in vitro or in vivo, as ascites for large scale antibody production. For a review of current procedures used to develop monoclonal antibodies, see Waldman, T., Science 252:1657-1662 (1991) and ~arlow, et al., Antibodies: A Laboratory Manual. Cold Spring Harbor, 1988.
New York.
Monoclonal antibodies are preferred over polyclonal antibodies for diagnostic assays. Monoclonal ankibodies represent a homogenous population of antibody with a defined specificity. The antibody can be used rep~atedly over ti~e with consistent results. Since the monoclonal population is homogenous it tends to have a more predictable reaction pattern in immunoassays over its polyclonal counterpart. ~oreover, in contrast to polyclonal antibodies, monoclonal antibodies of consistent quality can be generated over a prolonged period. The specificity of monoclonal antibodies makes them the preferred type of antibo~y for immunoassays such as enzyme linked immunosorbent assays (ELISA~, radioimmunoassays (RIA)/ western blot or im~unohistochemical assays.

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While rats and mice are the typical sources. for monoclonal antikody production, some antigens do not sufficien~ly stimulate th~ ~evelopment of high-affinity antibodies in mice and rats to make them useful in diagnostic assays. Examples of this type of antigen primarily include carbohydrate moieties. While amino acid epitopes tend to be more immunogenic than carbohydrates, there are amino acid epitopes ~hat do not stimulate high affinity antibodies in rodents. Therefore another convenient source o~ monoclonal antibodies would be of great benefit to the artO
Rabbits are an excellent source of polyclonal antibodies D Antigen stimula~ion, in rabbits, using a wide variety of immunogens, consistently generates high titer rabbit polyclonal anti~odies. Moreover, rabbits are a common and convenient laboratory animal. While rodents are less lik~ly to produc~ high-affinity antibody to carbohydrate moieties, ra~bi~s more consistently react positively to foreign carbohydrate determinants. The di~ference in affinity between rabbits and mice may be attributed to the fact that these organisms are evolutionarily disparate and may therefore respond differently to the same antigen. Thus rabbits may be better able to generat~ str~nger antibody responses to those human epitopes that are weakly reactive in mice.
Carbohydrate determinants are important markers for cancer diagnosis and infectious disease; thus there is an increa~ing need for high affinity anti~di~s to these antigens~
In addition ~o th~ different antigenic respons~s between rabbit and mice, antibodies produced in rabbits offers additional advantages to diagnostic assays. Some humans, for example, tend to have endo~enous levels of anti-murine antibodies lHAMA). In standard ELISA assays that employ two murin~ antibodies, this antibody cross links the two murine antibodies to generate false positive si~nals. Reduced false positives are an important goal in 2 ~

H-85~2 3 immunoassay development. Therefore mechanisms to reduce false positives in diagnos~ic assays are important to health care personnel. ~ndogenous human anti-rabbit antibodies are uncommon. Additionally, human anti-mouse antibodies may complicate therapeutic uses for murine antibodies as well. For example, the ability to detect an antigen associated with cancer in an individual who is i~aged or trea~ed with murine monoclonal antibodies to that same antigen may be compromised in a diagnostic assay that employs the murine monoclonal-monoclonal format. ~ rabbit-rab~it double monoclonal format or a rabbit monoclonal-murine monoclonal f ormat would obviate the need f or a mouse-mouse monoclonal format.
While monoclonal antibodies are generally pref~rred over polyclonal antibodies in immunoassays, there is no efficient method available to produce rabbit monoclonal antibodies in a consistent quantity or quality. There are no rabbit myelomas or suitable immortalizing fusion partners derived from rabbi~s. Other methods ~or producing rabbit monoclonal antibodies include B cell transformation in rabbits u~ing SV40 (simian virus 401 or EBV tEpstein Barr VirU5) and oncogene transfection. All o~ these methods have proven difficult and re~ult~ are inconsistent (~ee Collins, ~t al., Proc. ~latl. ~cad. S~i. 71-260-262, 1974 and Strosberg, e~ al., U.5. Patent No. 4,859,595, issued August 22, 1989). In the absence of a rabbit derived my~lom~ cell lin~, standard monoclonal anti~ody techniqu0s are not use~ul. Thus, at present there is no reproducible method for generating rabbit mono~lonal antibodies.
Some laboratories hav~ looked at the production of heterohybrids. Antibody seereting cells isolated ~rom one speci~s and fused with immortalizinq cells from another species yield interspecies, or heterohybridomas~ The term h~terohybrid fusion is used herein interchangeably with x~nog~neic fu~ions. Raybould, et al. disclose a meth~d for pr~du~ing rabbit-mouse hybridomas that secrete rabbit , :. :

2 ~ 3 monoclonal antibodies (Raybould, et al., cience 240: 1788-1790, 1988 and Raybould, et al., U.S. Patent No. 4,977,081, issued December 11, 1990). Rabbit monoclonal antibodies were produced by fusing mouse myeloma ce~lls with rab~it splenocytes. ~table antibody production was only obtained when the fusion product~ were grown i~ the pres~nc~ of rabbit serum. Raybould, et al. indicate in their conclusions that the use of rabbit serum is es~ential to rabbit monoclonal antibody production.
Rabbit monoclonal antibodies produced in the presence of rabbit serum is contaminated by endogenous rabbit antibodies present in the ~era. Rabbit sera contains between 10-30 mg/ml of immunoglohulin G (IgG) as compared with microgram quantities of IgG produced by a murine hybridoma or heterohybridoma. Nonspecific rabbit immunoglobulin present in the monoclonal antibody preparation increases background reactivity and reduces the sensitivity of assays designed to detect antibody production from the fu~ion product. To prepare monoclonal antibodies for diagnostic assays, ~he monoclonal anti~odies must be purified away from the nonspecific rabbit im~unoglobulin present in the sera. These techniques are difficult, labor intensive and increase production cost~.
Antibodies produced fro~ interspecies hybridomas such as those ~ethods disclosed by Raybould, et al. tend to be unstable. Becaus~ of the abnormal number of c~ro~oso~es, segregation does not always deliver identical sets of chromosomes to daughter cells and th~se chromosomes may be lost. Both the chromosomes containing the fu~ctional, rearranged immunoglobulin heavy-chain and light-chain genes and the chromosomes permitting drug resistance are needed to maintain cell replication and antibody production. As disclosed in t~e detailed d~scription of the invention, (see Tables 2 and 3) the interspecies fusion of Raybould ~as unstable over time and failed to produce antibody.
The cQ~ercial use of a ~onoclonal antibody depencls on the stability and quality o~ antibodies produced fro~ a 2 ~

particular clone. In addition to stability and quality, the concentration of antibodies produced from the clone should be high enough, prefera~ly greater than 25 ~g/ml, to obtain commercially useful quanti~ies o~ antibodies.
Usually the high~st levels o~ antibody production are obtained when the antibodies are produced as ascitesO
Ascites production is the most cost ~ffective and efficient way to grow large quantities of antibodies. Normally murine hybridomas are produced as a~cites in a closely related murine host. Heterohybridomas secrete antibodies derived from an animal o~her than a mou~e. An immunocompetent mouse will see the heterohybridomas as foreign or develop an immune response to the antibod:ies.
Thus a method for producing rabbi~ monoclonal antibodies as ascites would represen~ a significant advance in the art.
While there is some evidence that rabbit heavy chain-murine light chain chimeric antibodies can be produced as ascites in nude ~ice, the use of mice to generate antibodies comprised o~ rabbit light and rabbit heavy chain is heretofore undisclos d (Dreher, et al., J. Im~lQqy 130:442-447, 1983). Moreover, neither the chimeric clones nor those disclosed by Raybould were stabl0 over time.
There~ore useful quantities of antibodies for commercial diagnostic assays could not be produc~d. Ware, et al.
demonstrated tha~ rat X mouse hybridomas can be grown in severe combi~ed immunodeficient (SCID) mice. (J.
I~un~lQ~.~cal ~etho~s 85:353-361, 1985, her~by incorporated by re~erenc~). Antibody production for xenogeneic fusions was previou~ly limited to antibody production in tissue cultur~. The demonstration of high l~vel heterohybridoma rab~it antibody production in SCID mice is heretofore undisclosed.
The ability to consistently produce rabbit mcnoclonal anti~odies is undisclosed. Further, a method for reproducibly generating rabbit monoclonal antibodies in the absence o~ rabbit serum ~nd a method for grow:ing th~
antibody producing cells as an ascites tumor would b~ a .
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significant advancement in the art. ~abbit monoclonal antibodies could thus be used to overcome some of the difficulties associa~ed with generating antibodies to carbohydrate (Raybould, et al., supra).
The present invention discloses a stable xenogeneic fusion partner comprising the product of a cell fusion between a rodent myeloma cell and a non-transformed rabbit cell. In one preferred embodiment of ~he invention, the stable fusion partner produces undetectable le~els of antibody. The rodent myeloma cell is preferably mouse derived and in a particularly preferred embodiment of t:his invention, the mouse-derived rodent myeloma cell is the SP2/0 cell line. In another particularly pre~erred embodiment of ~his invention, the stable fusion partner is derived from a fusion between an SP2/0 cell and bovine somatotropin sensitize~ rabbit splenocytes. This fusion partner is hypoxanthine-aminopterin-thymine sensitive and hypoxanthine-guanine-phosphoribosyl transferase deficient.
Naturally occurring varian~s and mutants of this sta~le fusion partner are additionally contemplated within the scope of this invention. In yet another particularly pre~erred embodiment of ~his invention, the stable ~usion partner co~prise6 the cell line deposited with the American Type Culture Collection (Rockville, Maryland), under the ~5 Budapest Treaty, as cultur~ number 11086~
In another pr~erred embodi~ent of this invention, a stable cell line that secretes rabbit ~onoclonal antibodies is conte~plated. This stable cell line comprises the product of the fusion of a stable xenogeneic fusion partner which in turn co~prises the fusion of a rodent myelo~a cell and a non-transformed rabbit cell with a rabbit splenocyte that has been sensitized wi~h a predet~r~ined antigen and produces antibodies to that antigen. Preferably/ this stable cell line is the product of a fusion that utiliz0s a stable xenogeneic fusion partner that itself does not seGrst~ detecta~le quantitie~ of antibodies~ In addition, this stable cell line is preerably the product of a fu~ion ' '. :

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wherein the stable xenogeneic fusion partner is in turn the product of a fusion tha~ utilized a mou~;e-d~rived rodent myeloma cell. More preferably, the c~ll line is the product of a fusion ~hat utilized an SP2/0 murina myeloma cell line and more preferably, ~he rabbit splenocytes were sensitized with a carbohydrate antigen and these splenocyte~ produced antibodies to that antigen. In a preferred embodiment, ~he stable xenogeneic fusion partner is the cell line deposited with the American Type Culture 10 Collection as cell culture number 11086. In a particular preferred embodiment, the rabbit splenocytes were sensitized with N-acetyl D-glucosamine and produced antibodies to the carbohydrate antigen.
~n yet another preferred em~odiment, a method is 15 provided Por producing rabbit monoclonal antibodies comprising the steps of fusing a nontransformed rabbit partner cell with a rodent myeloma cell to produce a xenogeneic fusion partner, selec~ing a stable ~usion partner producing undetectable levels of antibody, fusing 20 the stable fusion partner wi~h a ra~it antibody-producing cell and isolating an antibody producing cell line obtained from the fusing step tha~ pro~uces antibodies directed to a predetermined antigen. In one emb~diment, the selecting step additionally comprises generating a drug resi~tant 25 stable fusion partner and the isolati.ng step additionally comprises growinq the antibody producing cell in the drug.
Preferably, khe rodent myelo~a cell is mou~e derived and the rabbit antibody producing c~ll is obtained from a rabbit im~uniæed with the predeter~ined antigen. In a 30 preferred embodiment of this ~ethod, the stable fusion partner is the cell line deposited with the ~merican Type Cultur~ Collection as culture nu~ber 11086.
In another pre~erred me~hod for producing rabbit monoclonal antibodie~, a stabl~ fu~ion partner derived ~rom 35 a fusion between a SP2/0 cells and bovine somatotropin-se~itized rabbit splenocytes is hypoxanthine-aminopteri~-thymidine sen~itive and hypoxanthine guanine-phosphoribosyl ... .. .
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~ ~ ~L~ ~ 3 transfarase de~icient. The me~hod comprises fusiny the stable fusion partner wi~h rab~it antibody-producing cells, isolating the fusion products, growing the ~usion products and collec~ing antibodies ~rom the fusion products. In one embodiment the growing s~ep occ-lrs in vitro and in a preferred embodiment the growing step occurs in vivo.
Preferably, the in vivo growing step consists of generating ascites an~ more preferably, ~he growing step oonsists of introducing the fusion products into mice deficient in mature T and B lymphocytes.
Figure 1 is a photograph of a chromosome spread of metaphase chromosomes from the exemplary fusion partner cell line OMB-037. The arrows identify rabbit chro~osomes.
Acrocentric chromosomes are murine.
Figure 2 illustrates the results of a study to optimize the fusion efficiency of rabbit monoclonal antibody production. Tha culture conditions are provided on the horizontal axis~ P3.653, SP2/0 and O~B-037 fusion partners were tested together with different sera: rabbit sera (RS~, horse sera ~HS), fe~al calf (FCS) and splenocyte conditioned media ~SC~). The percentage fusion efficiency is expressed as the number o~ wells with viable hybrids divided by the total number o~ wells receiving hybrids in each group.
Figure 3 diagrams the results o~ ELISAs to dete~ine the stability o~ the he~erohybrid SA157-516.5 a~ compared with a co~mercially prepared rabbit anti~Group A
Streptococcus polyclonal antibodies at a concentration of 10 ~g/ml. NSB (non-specific binding) indicates background due to non-specific adherence of the antibodi~s in tissue culture supernata~t~.
Figure 4 is a photograph of a cellulose acetate gel containing aliguots of crude ascites. Lanes 1 and 2 contain aliquots of crude ascites ~rom heterohybridoma 5AlG7 516.5. Lane 3 is from fu~ion partner OMB-037.
Figur~ 5 illustra~es th~ results of an ELISA to t~st for the presence of rabbit i~unoglobulin in asc.ites fluid 2~

obtained from ~he heterohybridoma SAlG7-516O5 as compared with the fusion partner OMB-037. (N) and ~S) denote immunoglobulin produced in nude or SCID mice respectively.
Figure 6 illustrates the results of an ELISA to test for the presenc~ of mouse immunoglobulin in ascites fluid obtained from the heterohybridoma SAlG7-516.5 as compared with the fusion partner OMB-037. (N) and (S) denote immunoglobulin produced in nude or SCI~ mice respectively.
Figure 7 illustrates the results of an ELISA to t,est for the presence of rabbit antibodies recognizing Group A
Streptoco~cus as compared with fusion partner OMB-037. (N) and (S) denote i~munoglobulin produced in nu~e or SCID mice resp~ctively.
Figure 8 is a K~ plot for antibodies produced from heterohybridoma SAlG7-516.5.
Figure 9 compares the reflectance of di~ferent concentrations of bacterial lysate using four differen-t combinations of monoclonal or polyclonal rabbit anti~ody preparations.
Using methods disclosed herein, the generation of rabbit monoclonal antibodies is now possible through the employ of a stable, productive xenogeneic fusion partn~r.
The term xenogeneic fusion par~ner is used to describa the interspecies fusion of two or more cells to obtain an immortal cell line capable of ~using with spleen cells.
Stable, xenogeneic fusion partner cell lines derived in part from rabbit cells are required for the reproducible production of rabbit monoclonal antibodies.
The reproducible production of rab~it monoclonal antibodies is undisclosed in the art. The use of xen~geneic fusion partners involving more than the ~usion o~ two cells is disclosed for a few systsms, but not ~or rabbit~O In particular Oestberg, et al., (U.S. Patent No.
4,634,664, issued January 6, 1987 and Teng, et al. ~Proc.
Natl Aca~ Sci! ~USA) 80:7308-7312, 1983) disclose the use of xenogeneic ~usion partners to produce human monoclonal antibodie~. Xenogeneic fusion partners are also described `.. , ~ , ' .. . . . :
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~ ~ ~L~ 3 for the production of bovine and sheep monoclonal antibodies; however ascites production is neither disclosed nor described (Kennedy, et al., J. Gen. Virol. ~9:3023-3032, 1988, and Flynn, et al., J. Immunol. Metho~ds 121:237-246, 1989).
Success in human and large mammalian systems is not predictive of success in rabbits. The fusion of a myeloma cell with a host par~ner cell does not, of it~elf, guarantee success in generating a useful xenogeneic fusion partner. A useful xenogeneic fusion partner preferably exhibits cell stabili~y over time and preferably replicates with a ~oubling time o~ at leas~ 24 hours. Moreover, the cells are preferably sensitive to Hypoxanthine, Aminopterin and Thymidine (~AT). More pre~erably, the fusion partner contains at least one host species-specific chromo60me.
While it is recognized that not all fusion partners obtainsd by this method will be equally suited as fusion partners, methods are detailed for the testing and isolation oP stable fusion partners well suited for fusion.
The selection of a suitable fusion partner as disclosed herein facilitates ~he reproducible and consistent production of monoclonal antibodies. Therefore, methods are disclosed for selecting a suitable xenogeneic fusion partner useful for preparing rabbit ~onoclonal antibodie~, ~or testing the fusion partner and for using ths fusion partner in sub~equent fusions to produce hiqh quality and u~eful quantities of rabbit monoclonal antibodies. In addition, an exemplary xenogeneic fusion partner obtained using the methods outlined in this invention is deposit~d in the ~merican Typ~ Cul~ure Collection, Rockville, ~aryland tATCC) as CRL 11086.
The production and identification of a suitahle fusion partner is essential to the production of monoclonal antibodies obtained from xenogeneic fusio~s. Xenogeneic fusions o~ mouse or rat my~loma (or other rodent im~ortali2ing cells) wi~h splenocytes or other cells ~rom a dif~erent ~pecies produces cells that are often 2 ~

genetically unstable. Chromosomes are deleted ~rom tha cells with further cell passage in culture. Thus, the fusion product o~ a mouse myeloma with splenocytes derived from another immunized animal may initially secrete antibody. Over time, chromosome loss m,ay result in the loss of antibody expression. As disclose,d by Raybould, et al., media components such as species Ispecific sera or additional additives may be requirad to maintain these cell lines. Thus, for many xenogeneic fusions o~ a myeloma and a splenocyte derived from a different species, antibody production is not stable over time.
In a preferred embodiment of this invention, a stable xenogeneic fusion partner is produced by fusing a roclent myeloma cell with a non-trans~ormed rabbit partner cell.
It is contemplated within the scope of this invention that either rat or mouse myeloma cells may be used in practicing this invention. Myeloma cells are immortalized plasma cells and there are a varie~y o~ myeloma cells used in the art. Some, like the mouse myeloma cell line SP2/0, are a fusion product of a murine cell line P3 and murine splenocytes. Other myeloma cells secrete or produce light chain antibody protein. There are a number o~ mouse i~mortalizing cell lines known in the art that ar~ suitable for fusion with splenocytes. Possible mous~ myeloma cell lines that may be u6ed in this invention include, but are not li~ited to, IgG secreting mouse myeloma cells such as ~PC 21 or P3X63AG8 (ATCC #TlB9) and more pre~erably, non-antibody secreting cells such as SP2/0 (ATCC # CRL 1581), NS-1 ~ATCC #~I~18)~ P3.X63AG8.653 (ATCC # CRL 1580), F0, S194/5.XXO.BU-1 (ATCC # CRL 1580) and FOX-NY (ATCC # CRL
1732). These cell lines are available from A~CC
(Rockville, MD.). The following examples used SP2/0 as the im~ortalizing cell since the cell line is well characterized in the art and does not secrete antibody light or heavy chain protein. For a history of the SP2f0 cell lineage and its relationship to other myelomas, see Harlow, et al~ (~nti~dies: ~_k~or~tory~ Manual p.l45).

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2 1 ~ 5 It is contemplated within the scope of thi~ invention that at least one rabbit chromosome or genes translocated from at least one rabbit chromosome onto an immortalized call chromosome is required for the production of a useful immortalizing fusion partner. Thus, candidates for an immortalizing ~usion partner could include immortalizing cell derivatives from any numb2r of species, such as human, mouse, goat or the like. This immortalizing fusion partner is then fused to a rabbit antibody producing cell.
However, others believe that chromosome translocations of essential portions of the rabbit chromosome that permit rabbit immunoglobulin expression are sufficient ~or rabbit antibody production. Thus, the identification of rabb.it chromosomes per se is not an accurate predictor for ~usion lS partner selection.
The non-transformed partner cell used in the fu~ion is a growth regulated cell, of the ~ cell lineage, derived fro~ a rabbit. In a preferred embodiment of thi~
invention, the cell is a rabbit splenocyte. Example 1 details the generation of a xenogeneic fu~ion partner that is the product of a fusion between rabbit splenocytes obtained from a rabbit im~uni zed with bovine ~omatotropin.
Bovine soma~otropin is an exemplary im~unogen. Therefore it is contemplated that another im~unogen could be used to obtain an immune response in the rabbit donor used to generat~ the fusion partner. It is further contemplated within the scope o~ this invention that the splenocytes used in a fusion with mou~e myeloma cells to obtain the fusion partner, need not be derived from an im~uni2ed rabbit since immunization is not a prerequisi~e for the identification of a suitable xenogeneic partner.
Cell fu~ion between the immortalizing cell and the non~transformed rabbit partner cell may be per~ormed using a number of method~ known in the art. Chemicals that promote fusion are commonly re~erred to a~ Eusogen60 These agents are extrem~ly hydrophilic and fac.ilitate ~embrane contact. As on~ method of cell fusion, the cells are lused 2`~ 3 using polyethylene glycol. In a preferred embodiment the m~thods for cell fusion follow those described in Example 5 and in the s0ction entitled "Fusion of Stable Fusion Partner with Rabbit Splenocytes". A specific example of the method used to generate an exemplary fusion partner, OMB-037, is provided in Example 1.
Other methods that could similarly be used to facilitate cell fusion inclu~e electrofusion. In this method, c~lls are placed in special buffers and are exposed to a predetarmined electrical ~ischarge that alt~rs the cell membrane potential and increases the likelihood of cell fusion. ~dditional methods for cell fusion contemplated for use in this invention are bridged-fusion methods or the like. As one example of a bridged-fusion method, the antigen is biotinylated and the myelomas are avi~inylated. When the cells are added together, an antigen reactive B cell-an~igen-biotin-avidin-myeloma bridge is formed. This permits the specific fusion o~ an antigen reactive cell wi~h an immortalizing cell. The ~ethod may addi~ionally employ ch2mical or electrical means to facilitate cell usion.
The fusion products are grown in a compatible media as outlined for the xenogeneic fusion of Example 1. As in ~xample 1, following fusion, the cells are selected for hypoxanthine a~inopterin and thymidine (HAT) sensitivity.
Pr~ferably, the ~usion products do not produce antibody.
If the non-transformed rabbit cells were derived from rabbit splenocytes, and in particular ~rom rabbit splenocytes obtained from an immunized rabbit, then it is necessa~y to check the cell supernatants for antibody production, including production of either light or heavy antibvdy chains. As one method for selecting fusion products that are HAT resistent and do not secreta antibody, the supernatant ~rom fusion products sensitive to EAT and 8~azaguanine resistant are tested for the presence of IqG using standard ELISAs well known in the art. The supernatant is addi~ionally electrophoresed using cellulose , '.

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aceta~ or polyacrylamide gel elec~rophoresis to visualize antibody protein. Finally, cell lysates from the ~usion products are tested for IgG specific mRNA. Cells testing negativ~ ~or IgG production are te~te~ for their efficacy as a fusion partner.
Useful candidates for xenogeneic fusion are those ~usion products that double preferably at least once in 24 hours, fail to produce antibody protein and are capable of becoming 8-azaguanine resistan~. A method for makincl a candidate fusion par~ner 8-azaguanine resistan~ is provided in Example 3. As an alternative to 8-azaguanine resistance, the cells can also be made 5BUdR (5-bromodeoxyuridine) resistant. Such methods are available in general procedural texts for mo~oclonal antibody production, including An~ibodies: A _La~oratory Manual (Harlow, et al., supra). Other selectable drugs suitable for selecting ~usion products are ~nown in the art. For an overview of other selection systems see Klein, J. (1982) in Im~Qlo~y: T~ Science of Self-N~nself Discriminatio~~ J.
Wiley & Sons, New York.
As an example of the development and sele~tion of a stabl~ fusion partner suitable for the production of rabbit monoclonal an~ibodies, the fusion partner OMB-037 was selected ~rom splenocytes obtained from a rabbit im~unized with bovine somatotropin usiny the immunization protocol provided in Example 1 and fused to mcuse myeloma cell line SP2~0.
The resulting fusion part.ner was tested for antibody secretion, growth characteristics and by karyotyping. The fusion partner was ~e~ted in subsequent rabbit splenocyte fusions and tested ~or rabbit monoclon~l antibody production. A photograph of a chromosome spread of the ~usion partner cell line O~B-037 is provided in Figure l.
Ra~bit chromosomes are identified by arrows. Mice have 44 chro~so~es ~hat are acrocentric while rabbit cells have 40 chr~mosoaes that are metac2ntric. Originally the cell line contained 6-8 rabbit chromosomes and this nu~ber o~

chromosomes was constant for 9 months of continuous culture. After approximately one year in culture the cell line contained one rabbit chromosomeO Despi~e this chromosome loss over time, the cells functioned efficiently as fusion partners and the monoclonal antibody producing cells generated by fusing the xenogene:ic fusion partner with an antibody producing cell have b~en stable in culture over one year. To ensure a constant fusion efficiency, the xenogeneic fusion partner was initially expanded and frozen down. A new frozen aliquot was used each 6 months. A
protocol for the long term use of a suitable xenogeneic fu~ion partner is provided in Example 2. Even though the karyotype for O~B-037 changes slightly overtime, new batches thawed each 6 months ensured consistent fu~ions.
To generate rabbit monoclonal antibodies, the skable fusion partner was fused to cells producing rabbit antibodies. Potentially any cell producing rabbit antibodies could be used in a ~usion with the xenogeneic fusion partner. Rabbi~ antibody producing cells were obtained by immunizing rabbi~s with antigen of choice using methods well known in the art. For a review of rabbit immunization strategies see Harlow, e~ al. (supr~, pp 92-114).
In general, i~munogens must ~ulEill two criteria to be i~munogenic. First, they must possess a site for antibody binding and second, they must po~sess a site for class II/T
cell interaction. Therefore, most ~oreign protein fulfill these criteria and are i~nunogenic. Immunization of rabbits with carbohydrate groups bound to protein or carbohydrate alone can be used to initiate an i~mune response. In general, bett2r immunogenic responses are obtained by immunizing rabbits with carbohydrate groups associated with protein. As an example of the ability of the fusion partner to facilitate rabbit monoclonal antibody production and specifically to produce monoclonal antibodie~ to carbohydrate moieties, the exemplary fusion .

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partner OMB-037 was fused with splenocyte~ from rahbits immunized with pepsin-digested Group A 5treptococcus (GAS).
In Example 4, outli.ned ~elow, pepsin~digested Group A
S eptococcus pyroqens ~cell wall remo~ed) was used to initiate an immune response. Purified preparations of Group A Streptococcus-specific epi.tope (N-acetyl glucosamine) were not used since nitrous acid extract enriched fractions of N-acetyl glucosaJnine (NAG~ would likely paralyze the immunized animal. It is well known in the art tha~ high affinity monoclonal antibodies directed to this epitope are dif~icult to develop in mice.
Strategie~ for im~unization with polyFeptide are well documented in the art. Gen~rally, animals are initially immunized by introducing the antigen in combination with an adjuvant such as Freunds (see Harlow, et al., supra.).
Other adjuvants are additionally described in the art.
Subsequent boosting injections of antigen are given in association with incomplete Freunds adjuvant, with another adjuvant known in the art or alone, without adjuvant.
U~ually, animals are given an initial injection. Two to four weeks later, when the immunoglobin response has developed and secre~ed antibodies have cleared, boosting injections are given to develop an IgG response.
Strategies for i~munizing against carbohydrate are different. One strategy suitable for carbohydrate immunization and particularly suited for Group A
Streptococcus is provided in Example 4 and adapted fro~
O~terland, et al. (J. E~p~ Med. 123:599-614, 1966 hereby incorporat~d by reference). The rabbits were tested three week~ into the immunization protocol. Adeguat~ titers were usually achieved within this time frame. If not, rabbits were intravenously given boosts of antigen every other week (IV), with the larges~ dose of antigen, until acceptable titers were measured. Modifications of the protocol provided in Example 4 and other i~muni2ation protocols could a~di~ionally be-used to generate useful antibody titers.

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It is additionally conte~plated within the scop~ of this invention, that rabbit cells may be immunized in vitro. Procedures are available for immunizing human lymphocytes in culture. For a review of in vitro immunization techniques, see James, et al. (J. Immunol.
Metho~$ 100:5-40, 1987, hereby incorporated by reference).

Rabbits are immuni2ed with antigen according to the method described above or other methods known in the art.
Rabbits con~aining suitable antibody titers, preferably within the range of 2-3 times greater than preimmunized sera from the same ra~bit, ~o the antigen of int~rest are selected ~or use. In Example 4, below; the selectad rabbit was boosted a final time with Group A Streptococcus (GAS) antigen three days before fusion.
Methods for obtaining and preparing single cell suspensions of rabbit splenocytes for cell ~usion are readily available in the literature. The methods used here were similar to ~hose used to generate mouse splenocytes.
Briefly, the rabbit was sacriiced and the spleen was physically teased and resuspended in a suitable media such as phosphate buffered saline or HB ~RO media (Irvine Scientific, Santa Ana, California). Cell concentrations were determined using me~hods well known in the art and the concentration was adjusted to ~.5x106 cells/ml. Leu-leu-o~e peptide (L-Leucyl-L-leucine-~ethyl ester, aoehrin~er Mannheim, Indianapolis, Indiana) was added to kill ~he lysosomal-enriched cells such as macrophages. The cells were spun down and mixed with the fusion partner.
There are many methods known in the art to facilitate cell fusion ~or hybridoma production. Polyethylene glycol (PEG) is khe standard fusion-mediating agent. Cell ratios of splenocytes to fusion partner cells will be optimized for the conditions of ~ach particular fusion. Cell to fusion partner ratios traditionally vary bet~een 2:1 to 10:1. Si~ilarly, the optimal concentratlons o~ P~G will vary dependîng on tha molecular mass of the PEG. In - - , ,, - . .
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.

., , 2 ~ Ol~ 3 Example 5, below, 35% PEG 1500 was selected, however o~her concentra~ions of other molecular mass PEG, such as 50% PEG
4000, could alternatively be used to facilitate cell fusion.
It is important ~o maximize fusil3n efficiencies, particularly for epitopes that do not readily generate an immune response, such as carbohydrate. The fusion partner and the culture media are two of the most important variables in obtaining good fusion e~f:iciencies in the instant fusions. To reduce ~he risk that rabbit antibody producing cells go undetec~ed, fusion efficiencies should approach 100%; in o-~her wor~s, grow~h in all of the ti~;sue culture wells containing cells. U.S. Patent No. 4,977,081 to Raybould, et al. teach the production of rabbit monoclonal antibodies through the fusion of a mouse immortalizing cell with rabbit splenocytes in the presence of rabbit serum. Moreover, Raybould, et al. indicate that normal rabbit serum is required for rabbit monoclonal antibody production.
The rabbit monoclonal antibody procedure of Raybould was co~parad to other fusion combinations and conditivns.
In a study to determine the optimal combination o~ fusicn partner and media conditionsl a fusion partner, 0~ 37, obtained using the method~ o~ thîs invenkion, was compared with murin~ myeloma fusion partners SP2/0 and P3.653.
Rabbit serum supplem~nted media (RS), horse serum (HS), ~etal calf serum (FCS~ and spleen-conditioned medium (SCM) were compared in combina~ion with the fusion partners.
Spleen conditioned media was harvest~d as supernatant from normal splenocytes seeded at 1 x 106 cells/ml. in spinner cultures and cultured for 48h in standard medium (HB-GR0).
Cultures wer~ us~d at a final concentration of 20~.
The ~usions were performed as de~cribed in Example 5 except that rabbit splenocytes were obtained from a non-immunized animal. The culture condition modifications tested are denoted on the horizontal axi~ of Eigure 2.
Raybould's combination is identified by an arrow. As exemplified by Figure 2, the best ~usion efficiencies were obtained using ~usion par~ner OMB-037. In contrast to Raybould, optimal fusinn efficiencies were not dependent on the presence of rabbi~ serum. Results are expressed as the number of wells containing viable hybrids relative to the number of wells plate~ ~or each sample. A minimum of 288 wells were used ~or each sample group. Fusion products were identified in the wells by their size and the presence of cell growth in the wells. These methods provide an exemplary strategy for testing fusion efficiency in cell fusions selected for the production of rabbit ~onoclonal antibodies.
Subsequent fusions employing OMB-37 used fetal calf serum (FCS) as a growth supplement, as outlined in Example 5. Rabbit serum was found to complicate the hybridoma identification process since antibodies present in the rabbit serum reduced the specificity of the assay.
once fusions are complete, the wells containing antibody producing cells are grown for approximately 2-3 weeks and positive clones are selected and subcloned for further analysis. Methods for selecting a particular clone of intere~t are well known in the art of hybridoma t~chnology. For a review of strategies and selection techniques see generally Nakamura, et al., Immunoc~emical ~ssays and ~io~ens~ Tec~nol~qy for the l990s. American 50ciety for Microbiology, 1992. Washington D.C. A~
described in Exa~ple 5, the hybrido~a supernatants were screened for specific antibody production after day 18.
The wells ccnta.ining hybrid~ went throu~h three levels of testing designed ~or this particular antigen. It is conte~plated that those with skill in the art of assay development will similarly be able to develop suitable assays for a variety of antigens. Select clones that were positive in each of the three stages of testing, were carried in culture continuously and subcloned at least once to ensur~ their monoclonality.

.
, . .
.

2 ~ 3 Clones expressing GAS specific antibodies were detected using a series of three successive tests. These ELISA formats were developed to detect the presence of Group A Streptococcus specific antibodies in polyclonal sera, hybridoma superna~an~s, and ascites fluid. All three of the assays are detailed in Example 6. T~e first test detected ~he presence of antibodies to th~ pepsin-digested Group A Streptococcus. The results of the fir~t screen are provided in Table 1 below. In the second screen, antibodies produced ~rom clones passing ~he first test were retested using two forms of antigenO Pepsin-digested bacterial extracts were used, as in the first screen, and in addition ~he antibodies were tested using a nitrous acid extract (NAE) of Group A Streptococcus. Methods for preparing nitrous acid extracts of Group A Streptococcus are provided in Example 6. The nitrous acid extract is an enrichment o~ cell-derived carbohydrate that includes the epitope conferring group- specificity and i~ presumptive evidence that positive clones are producing antibodies specific for Group A Streptoc3ccus. Positive wells were expanded and were confirmed to be group specific by inhibition with N-acetyl-D-glucosamine which is the epitope recognized by antibodies specific to Group A Str~ptococcus.
Screening is preferably per~ormed over a p~riod of weeks and this is an initial indicator of clone ~tability~ In the third test, antibodies obtained from the positive clones identified in the s~ond test were further te~ted in an inhibition a~say with N-acetyl-glucosa~ine to ensure that ankibody speci~icity was directed to the GAS
carbohydrate epitope. The results from one fusion identified as SAlG are provided in Table 1 below.
As an alternative to these ELISAs r the samples could additionally be tested using a sandwich ELISA coating the ra~bit monoclonal antibodies of inter~st, followed by the antigen and subsequently follow d by antigen-specific monoclonal antibodies containing a suitabl~ label to facilitate quantitation.

T~BLE 1 FUSIO~ SUM~ARY
Initial Screen Retest Final Antigen PepsinPepsin ~ NAE Inhib.ition (by N-acetyl-D
gllucosamine) .
Purpose Detection Confirmation Specificity . _ Result 122/2844 (4%) 30/122 (25%) 9/30 (30%) (SAlG) - __ In order for a rabbit monoclonal antibody producing cell line to be use~ul, it must be stable. Often the products of xenogeneic fusions are unstable~ For fusion SAlG, performed using OMB and FCS, as outlined in Example 5, one clone of the nine, identified from the screening tests outlined in Example 6 and in Table 1, column 3 was identified as 5AlG7~516. This clone was chosen for subcloning. Subcloning is important ~or the development of commercializable antibodies because successful subcloning ensures that the clone is stable and homogenous.
Therefore/ it is contemplated that most antigen-specific rabbit-antibody producing clones identified using the m~thods of this invention will be subcloned.
As an example of a su~cloning strategy, Clone SAlG7-516 was sub~loned by limi~ing dilution (1/3 cell~well) 4 mo~ths after fusion~ Cells w~re distributed into 6, 96-well plates and 55 subclones were screened for antib~dy specificity. From these 55 subclones, 15 were positive (27%). It is anticipated that other clones will generate di~ferent percentages of subclone positivity~ It is expected that x~nogeneic fusions will likely ~ave a somewhat reduced rate of subclone positivity as compared with allogeneic or syngeneic fusions involving murine x murine or rat x rat.
These clones were followed for long term stabili-tyO
Stability is preferred for consistent com~ercial assay . ,. , ' . . ' - . ~ . ,' ' :
. ~
: . ,. ' , ' ' . , . :
.
.,, .
-: . . : ,. , . ~
.' '- .
. , , , . ,: , development. One subclone, SAlG7-516.5 was followed for long term stability with periodic testing for reactivity on pepsin~digested Group A S~reptococcus ~See Example 6 for methods). This clone as well as others generated by this method was stable for over 12 months in culture and continues to produce GAS specific antibodies.
Figure 3 illustrates the stability of the clone SAlG7~
516.5 as compare~ with a positive control. The positive control is commercially available rabbit polyclonal anti-Group A Strepto~occus serum diluted to a constant concentration for each testing date. The positive cont:rol serves as a measure of assay consistency. The clone SAlG7-516.5 was kept in culture for nine months and periodically tested for rabbit IgG secretion in an antigen specific assay such as one of those described in Example 6. Pepsin-digested Group A Streptococcus was coated onto a plate.
Culture supernatant was detected using goat anti-rabbit IgG
horseradish peroxidase conjugake using ortho-phenylamine diamine (OPD, Sigma, St. Louis, Missouri) as a substrate.
Re~ults provided in Figure 3 were read from an ELISA plate reader at OD490. Increasing optical density corresponds to increasing quantity of antibodies. Figure 3 indicates that as co~pared with the positive control, the clone SAlG is stable over ti~e.
As an additional measure of the usefulness of the ~ethod for generating rabbit monoclonal antibodies as compared with o~her methods available in the art, a comparison was ~ade of the stability and antibody output of an exemplary clone prepared and deposited by Raybould, et al. as a fetal calf serum adapted cell, ATCC ~B 9696. The experimental details are provided in Exa~ple 7. HB 9696 was grown in Dulbecco's Mini~um Essential Medium (DMEM) with 10% fetal calf as directed by Raybould. Table 2 provides a comparison of clones derived from the methods of this invention a~ compared with ATCC clone H~ 9696. The re~ults of Table 2 were generated u~ing an antigen specific ELISA of either NAE or pepsin-digested Group A

, - .
: .

L~ ~ L 3-Streptococcus. Rabbit Group A Streptococcus p~lyclonal antibodies and rabbit monoclonal antibody derived fro~
clones SAlF 7-205, 7-479, 7-490 and 7-572 were compared to antibody from HB g696 as well. Like SAlG clones, the SAlF
clones were derived ~rom the fusion of rabbit splenocytes with OMB-037 using methods identical to t'hose disclosecl for SAlG (see Example 5).
Table 3 provides the results of assays to quantitate the amount of rabbit immunoglobulin produce~ by the cultures. Group A Streptococcus polyclonal antibodies ranging from 1.6 - 1000 ng/ml was used to determine the IgG
concentration by ELISA. ATCC ~ 9696 failed to produce detectable antibodies using the growth con~itions provided by ATCC as compared to the SAlG clones that had similarly been frozen and thawed at least once.

:.. : - . .

. . ~ : -' ~ . ' , ~ , ' ' ~

. . . .
.' , '' 2 ~
H~8522 24 TABL~ 2 EYAL~TION OF R~-B52 Ag specific assay NAE (OD @ 490 nm) Pep ~OD @ 490 nm) NSB 0.0 -_ _ Positive (10 ~g/~l) 3.9 + 0.05 2.9 ~ 0 1 Negative 0.0 ~
Our clones:
SAlF 7-2052.9 0O7 SAlF 7-4792.6 1.2 SAlF 7-49G2.8 0.7 SAlF 7-5722.6 1~2 .
ATCC clone:
.
RMH-B520.0 ~ 0.0 - ~ ~
. _ ~B~ 3 Rab~it I~unoglobulin ~ssay standar~ OD ~ ~9O n~
Po~itiV~1000 ng/ml 3~1 ~ 0.23 200 ng/ml 2.9 + 0.25 40 ng/ml 1.~ -~ 0.04 8 ng/ml 0.4 + 0.0 1.6 ng~l 0.1 + 0.0 ~eg~iv~.O ng/~l 0.O + O.O
~ [- ~j~Culture sup O.O + O.O

Antibodies can be produced in quantity in vitro or in vivo. ~owever, in vivo production of antibodies as ascites qenerates higher concentrations of antibodies more quickly than in vitro expansion~ Most xenogeneic ~usions do not grow well in ascites in part because the ~ous~ hosting ascites yrowth recognizes the fusion product as ~oreign.

.

- . - , - : , .

. ... - . , ~ - : . -: . : . . ., .
: . . .
. - . : . .

.. : - ~ .-.: ,, : -Determinan~s expressed on the surface of the clones are similarly seen as foreign, hence the efficiency of ascites production is compromised by humoral and cellular immune responses.
To circumvent the immune responses associated with xenogeneic antibody production, the clones were grown as ascites in either nude or severe combined immunodeficient mice (SCID). Techniques for handling immunodeficient mice are known in the art, ~herefor~ these precautions will not be reiterated h~re.
Stable hybridoma clones from xenogeneic fusion partners are preferably injected into nude or severe combined immunodeficient mice (see Example 8). Methods for ascites production are also well known in the art (see Harlow, et al. supra.). Briefly, the clones were prepared in growth media (HB-GR0 with 10% FCS) and injected into the nude or SCID mice that were primed with incomplete freunds adjuvant five days before receiving the clones.
Samples of ascite~ were tested for the presence oP
rabbit-derived and mouse-deriY~d antibodies and for antibody specificity to the particular antigen o~ interest.
Two to three weeks after injection of the clones into the mice, the peritoneal ascite~ fluid was collected and a sample was loaded onto a cellulose acetate gel to check for the presence of antibody protein. Cellulose acetake gel electrophore~is separates molecules based on charge.
Figure 4 is a photograph o~ a cellulose acetate gel containing aliquots of crude ascites using clone SAlG7-516.5. The photograph indicates that the SAlG cells produce antibodies as ascites in both nude and SCID mice.
The axrow indicates th~ presence of immunoglobulin in the rabbit samples from both nud~ and SCID mice. ~oving to the right, the next band is transferrin and the heavy band on the Par right of the photograph is albumin The ascites derived ~rom thx clone~ was additionally tested by ELISA
using the methods of E~ample 6 to confirm the speci~icity o~ the rabbit antibodie~ against Group A Streptococcus.

.
- : . ' . , ~ ' : ' . -.. . ......... . . . . .
.

The antibody content and the presence of rabbit monoclonal antibodies in the ascites was confirmed using the three assays descri~ed in Example ~. In the first assay, pepsin-digested GAS (a~ a concentration of 1:400 of 5 1 OD = A66~j was coated onto a plate. The ascites was added and probed with goat anti-rabbit antibodies conjugated to horseradish peroxidase (~IRP) (Fischer Biotech Pittsburgh, Pennsylvania using 0.4 mgJml s~ock diluted 1:1000). The titration of ascites in both nude and SCID mice was compared with ascites production using OMB-037 as a control. The results indicated that the SAlG preparations contained large amounts of rabbit antibodies. Results of this assay are provided in Figure 5.
The presence of contaminating murine immunoglobulin and the use~ulness of SCID mice as ascites hosts as compared with nude mice was determined using an assay for mouse immunoglobulin. This ELISA used a goat anti-murine IgG (Fischer Biotech, Pittsburgh, Pennsylvania, at 1 mg/ml, 250 ng/well) as a coating antibody. Ascites samples were added and mouse-derived antibodies were detected using a second goat anti-murine an~ibody labelled with HRP (Fischer Biotech, 0.4 mg/ml, 20 ng/well). ~esults are provided in Figure 6. Samples OMB-037 and SAlG, both from nude mice, contained large a~ounts of murine immunoglobulin.
Conversely~ the SAlG ascites obtained from SCID mice contain~d very little murine immunoglobulin.
Ascites fro~ hybrids of a murine x ~urine fusion contain antibodies from both the hybrid cell and from the hosk producing the ascites. This is true when xenogeneic hybrids are injected as well and the~e mouse antibodies could pose a problem in purification since murine antibodies would be purified together with rabbit antibodies. The use of SCID mice, having a blockage of both T and B cell maturation, is shown ~see Figure 7) to reduce th~ background of murine im~unoglobulinu Thus, it is contemplated wi~hin the ~cope of this invention that rabbit monoclonal antibodies generated by the methcds of .
. : ~. . : .

, . . ~-:.

H-8~22 27 this invention, are advantageously grown as ascites, preferably in SCID mice, to obtain commercial quantity and quality of antibodies.
The specificity of the rabbit antibodies produced by ascites was determined by a capture assay for antigen-specific immunoglobulin. Pepsin-digested group A
Streptococcus or NAE ex~rac~ed car~ohydrate was used to coat the ELISA plates. Aliquots of ascites were added and the reactive antibodies were detected with goat anti-rabbit IgG labelled with HRP. Results of these comparisons are provided in Figure 7. While ascites using the OMB--037 clone failed to react to ~he speci~ic antigen, SAlG clones grown in either the nude or SCID mice produced antigen specific antibodies.
Purified monoclonal antibodies are required for consistent reactivity in diagnostic assays. Contaminating protein, including immunoglobulin, decreases the level of sensitivity in immunoassays by increasing non-specific reactivity. For many applications, column purification is not always necessary, however ~or the repeatable and consistent perfor~ance o~ monoclonal antibodies, purification is generally r~quired. There are a variety of methods known to those with skill in the art for antibody purification and therefore, the proposed methods ~or antibody purification outlined below should not be construed as limiting upon the scope o~ the invention.
The ascites f luid contairling antibodies wa6 clarified with saturated ammonium sulfate and lipoprotein was removed by dextran sulfate precipitation. Rabbit antibodies were puri~ied by separation on Q Sepharose columns (Pharmacia, Piscataway, New Jersey) or on N-acetyl-glucosa~ine agarose im~unoa~finity columns. ~rocedures for separating and purifying antibodies using column chromatography are known in the art. Basic methods in column chromatography that can be adapted to acco~oda e the rabbit monoclonal antibody purification include tho~e of Cle2ardin, et al.
and Tasaka, et al. (~L_ÇhrQ=~Q3~ 319-67-77 1985 and ~
. .

.

His~ E~_Cytochem 17:2B3-286 t 1984 respectively, which are hereby incorporated by re~erence). The various fractions were eluted from the column and tested by ELISA.
The single peak containing rabbit immunoglobulin was tested using methods described in association with Figure 5 and the Group A anti-streptococcus activity was confirmed using methods described in association with Figure 7. An exemplary methodology for rabbit monoclonal antibody purification is provided in Example 3.
The R~ of the antibodies was determined using techniques well known in the art (see Lindmo, et al., J.
Immunol. Method~ 72:77-89, 1984) ~riefly the Ka ~as determined using a solid phase assay by immobilizing the antigen, here the NAE extract, onto polystyrene beads precoated with rabbit polyclonal anti-GAS antiboclies (Immucell, Portland, Maine). Procedures for conjugating antibodies to polystyrene beads are available from manufacturers and are well known in the art. The polyclonal antibodies were saturated with NAE obtained from the Group A Streptococcus. Il25-labelled and unlabelled SAlG
monoclonal antibodies were titrated against a constant amount of antigen conjugated to the bead. The K~ was calculated using Scatchard analysis. In this example, antibodies produced from clone SAlG7-516.5 was determined to be 2.74 ~- 0.6~ x 109. The Kn plot i5 provid~d in Figure 7.
Rabbi~ monoclonal antibodies produced using the methods of this invention can be used as substitute antibodies for murine polyclonal or monoclonal antibodies or for rabbit polyclonal an~ibodies or employed for any number of uses known in the art of im~unologyO Therefora, the rabbit monoclonal antibodies produced by the methods of this invention can be used in fluorescent assays, as capture molecules for the purification of antigen, and in 3~ diagnostic as~ay$ such as West~rn blot, radioimmunoassays~
enzyme link~d i~munosorbent assays and in i~munochromatographic assays. For an example of the use of .

2 1 ~

the rabbit monoclonal antibodies of this invention in applications suited ~or diagnostic assay development, see the assays disclosed in Example Ç.
Other devices or one-step immunoassays that can incorporated the rabbit monoclonal ant.ibodies of this invention include the Concise~ Device (Hybritech, LaJolla), the TestPack~ device of Abbott La~oratories (North Chicago, IL), described in European Paten~ ~pplication No. 217,403, published ~pril 8, 1987 or similar test devices. Still other devices containing porous membranes that can be adapted to employ the rabbit monoclonal an~ibodies of the present invention include the ~evices of Bauer, et al., U.SO Pat. No. 3,811,840, issued May 21, 1974; Brown, III, et al., U.S. Pat. No. 4,916,056, issued April 10, 1990;
Cole, et al., U.S. Pat. No. 4,407,943, issued Oct. 4, 1983;
Cole, eg alO, U.S. Pat. No. ~,2~6,339, issued Jan. 20, 1981; Intengan, U.S. Pat. No. ~,440,301, issued April 3, 1984; Jolley, U.S. Pat. ~o. 4,704,255, issued Nov. 3, 1987;
Ratz, et al., U.S. Pat. ~o. 4,~96,6~4, issued Jan. 29, 1985 and Tom, et al., U.S. Pa~. No. 4,366,2~1, issued Dec.
28, 1982, all of which are incorporated herein by reference.
The rabbit monoclonal antibodie~ of the present invention can be used in chromato~raphic methods such as, for example, those described in Weng, et al., U.S. Pat. No.

4,740,468, issued April 26, 1988, incorporated herein by reference, and published European Application No. 186,100 to Yue, et al., published July 2, 1986.
Those with skill in the art of assay development will be readily able to make the appropriate modifications to these assays to use rabbit monoclonal antibodies directed to other antig~n or antibodies. The rabbit monoclonal antibodies can be used in therapeutic applications as targeting molecules for therapeutic modalities and ~or imaging reagents.
In a particularly use~ul application of th~ rabbit monoclonal antibodies of this invention, it is contemp:Lated that the antibodies can be used in solid-phase immunoassay devices. Such devices include the non-chromatographic ICON~ and like devices described in Valkirs, et al., U.S.
Pat. Nos. ~,632,901 and 4,727,019, issued December 20, 1986 and February 23, 138R, respectively, herein incorporated by reference. ICON~ is a trademark of Hybrit:ech Incorporated (San Diego, CA) for the devices described in the Valkirs, et al. In ~hese assays, a firs~ an~ibody or antigen is bound or fixed to a porous member such as a porous membrane, filter or the like. A porous membrane may be comprised of a flexible or rigid matrix made from any of a variety of filtration or chromatographic materials including glass fibers, micro-fibers, and natural or synthetic materials. Fluids should be able to flow into and pass easily through the porous membrane. The membrane should also preferably have pore sizes of at least 0.1~ and preferably no more than 20~. The porous membrane can be used alone or as part of a more elaborate deviceO
The test sample fluid, applied to the porous member, flows through the member and contacts the antibodies or antigen thereon. A test analy~e present in the test sample fluid is bound by the first antibody on the porous member.

Following the application of sample fluid, a second solution is added that preferably contains a solution o~
antibodies. This seoond antibody pre~erably binds the test analyte at an epitope that does not interfere with the binding of the first an~ibo~y or antigen. In another preferrad embodiment, the test sample is first processed to facilitate analyte de~ection and mixed with the second antibody befor~ the test sample iB applied to the porous member. The antibodies present in the detecting antibody solution is preferably labelled with a detection tag such as an enzyme for colormetric analysis, radionucleotide, fluoreseent label, colored latex particles or the like, .
Additional steps are added, if necessary, to visualize the labelled tag bound to the test analyte on the porous ;r member. The presence of the analyte in the sample fluid is detected as a positive signal on the surface of the porous membar.
Applications of such assays in ~he art are well known and are detailed in publications by Anderson, et al.and by Valkirs (Clin._ Chem. 32(9):1692-1695, 1986; Laboratory Medicine 19:564-567, 1988 respectively, both publications are hereby incorporated by reference). In Example 10, the rabbit monoclonal antibodies of this invention recognize Group A Streptococcus-specific antigenO It is anticipated that these assays, employing rabbit monoclonal antibodies, will be developed for any number of antigen or antibody detecting schemes including assays to detect human choriogonadotropin (HCG), lutropin (LH), or antibodies directed to HIV-1 or the like.
Solid-phase immunoassays are available in a number of different configurations. In a preferred configuration, the a~say format follows the format of ~nder~on et al. and like Anderson et al., includes an internal reference. This assay format employs the rabbit monoclonal antibodies of thi~ invention in the ICON~ assay methodology for solid-phase immunoassays as either a first antibody (the capture antibody) or as a detecting antibody. In another preferred embodiment, both antibodies employed for capturing and detecting antigen in a solid-phase immunoassay are rabbit monoclonal antibodiesO
Example 10 details the use of the ICON~ met~odology for the identification of ~roup A Streptococcus infection.

Table 4 illustrates the results of an ICON~ format assay for Group A Streptococcus using rabbit monoclonal antibodies for both the capture and detecting antibodies.
~acteria and yeast samples were processed as disclo~ed in Example 10, using method similar to those employed if the bacteria were isolated fro~ a throat swabO The bacteria were applied as test sample fluid to the porous member.
The +/~ d~signation in Table 4 indicates the pre~ence or ' :
.

2 ~

absence o~ rabbit monoclonal antibody reactivity to the organisms listed in the le~t column. The results indicated that the rabbit monoclonal antibodies are Group A
Streptococcus specific. It is recognized in the art that some preparations o~ antibodies directed to Group A
Streptococcus anti~en N-acetyl glucosamine may also bind to antigenic determinants present on Staphy~Lococcus aureus.
Since S. aureus may be present in sample ~Eluid, such as a resuspended throat swab, assays to cletect ~roup A
Streptococcus will have some level of false positives if the antibodies employed in ~he assay addition recognize S.
aureus. Results obtains from assays using antibodies that additionally recognizes S. aureus extracts should be confirmed by another method. Advantageously, the rabbit monoclonal an~ibodies of this invention, did not bind to S.
ure~ which, as note~ above, is traditionally a concern in Group A Streptococcus immunoassays. Thus, the use o~
rabbit monoclonal antibodies of this invention, generated to Group A Streptococcus obvia~es the need for confirming positive results by a second test method.
Table 4 STREPTOCOCCUS A SOLID PHASE IMMUNOA5SAY (MONO~MONO) SPECIFICITY TESTING
l. Streptococcus Group D, CDC SS754 2. Streptococcus Group A, Non-~eta (clin isolate) 3. Staphylococcus saprophytilus, ~TCC 15305 4. Streptococcu~ Group ~ II, SS 619 ~. Escherichia coli, ATCC 25923 6. Streptococcus Group D, ATCC 19434 7. Streptococcus Group ~" SS868 8. Streptococcus Group C, SSl88 9. Streptococcus Group B Ib, SS618 10. Streptococcus Group B, SS700 11. Streptococcus Group A, ATCC 19615 +
12. Streptococcus Group B III, SS462 13. Staphylococcus Group ~, Cowan I CDC
14. Klebsiella pneumoniae~ ATCC 23357 15. Streptococcus Group C, SS189 16. Candida albicans, HMCC 063 17. Streptococcus ~roup A, SS721 18. Streptococcus Group A, SS410 19. Streptococcus Group B Ia/ SS615 . ' :

2 10 ~

~-8522 33 Current assays for Group A Streptococcus use rabbit polyclonal antibodies for both the detecting antibody and the capture antibody. Figure 9 illustrates the results of a study to compare di~ferent rabbit antibody format assays.
Group A Streptococcus was diluted from ~xlOA colony forming units (CFU)/ml to 1.56x~06 CFU/ml as provid~ed below in Table 5. Twenty microliters of this suspension was processed in the ICO~ format assay (See Example 10 or commercially available from Hybritech, Incorporated, San Diego, California) using various combinations of rabbit monoclonal and rabbit polyclonal antibodies as detecting or capture antibodies. These particular ~our ICON~ format assays were prepare using rabbit polyclonal antibodies as detector antibodies with rabbit polyclonal antibodies as capture antibodies (poly~poly), rabbit monoclonal antibodies as detectvr antibodies with rabbit polyclonal antibodies as capture antibodies (mono/poly), rabbit monoclonal antibodies as detector antibodies with rabbit monoclonal antibodies as capture antibodies (mono/mono) and rabbit polyclonal antibodies as detector antibodies with rabbit monoclonal antibodies as capture antibodies (poly/mono).
Increasing numerical value on the Calibrator scale in Figure 9 corresponds to increasing bacterial concentrations. The reflectance (~) of the signal on the immunoassay was determined using a Model 1500 Plus reflectometer (Macbeth, Newburgh, New York) and data was expressed as 100~%R. The results indicated that all combinations o~ capture and detecting antibodies are well suited for solid-phase immun~assays. Therefore, rabbit monoclonal antibodies, produced by the methods of this invention, can replace rabbit polyclonal antibodies in a Group A StreptocQccus solid-phase immunoassay. It is contemplated that those with skill in the art of immunoassay developm~nt will similarly be able to make comparisons between rabbit polyclonal and rabbit monoclonal antibodies and between rabbit monoclonal and mouse monoclonal antibodies and select the desired antibody ' 2 1~ 3 population based on its effectiveness and convenience in a particular assay.

ICON~ Format Assay Comparing Rabbit Polyclonal and Rabbit Monoclonal Antibodies Calibrator Bacteria concentration Bacterial Concentration Number CFU/ml CFU/Assay 1 1.56x106 3.. 12x104 2 3.13x106 6O26X104 3 6.25x106 1.25x105 2.50x10~ 5.00x105 8 2.00x108 4.00x106 Particular embodiments of the invention are discussed in detail and reference has been made to possible variations within the scope of this invention. rhere are a variety of alternative techniques and procedures available to those of skill in the art which would similarly permit one to successfully perform the intended invention.

Examp.Le 1 Gene~ation of Fusion Partner ~MB-037 To generate the fusion partner, a rabbit was immunized with bovine so~atotr~pin (BST~ using the following immunization protocol. Three days prior to fusion, the rabbit was given a 50 ~g I.V. boost of BST. One day prior to the fusion, mouse murine-derived macrophAge ~eeder cells were collected and plated at 10,000 cells/well in 10% fetal calf serum (GI~CO, heat inactivated 5~0C, 30, mn) plus lX HAT~ The spleen yielded 300X106 c~lls. The cells were divided in half and 2 separate fusions were performedO A 5:1 ratio of spl~en cells to SP2/0 cells was fused in 43% P~G. The cells wer~ plated at 10$ cells per w~ll in 15% Rabbit sera, : .- . :

: . : : , - :

lX HAT on top of the feeder cells. Two weeks post fusion, the media was removed and r~placed with 10%FCS, lX HT.
Fusion #1 had approx. 10% wells with growth and fusion ~2 had approx. ~0% wells wi~h growth. The plates were screened in an ELISA using wells coated with 250 ng/well BST in carbonate buffer. All were negative for anti-BST
Ab. The cells were grown in 20 ~g/ml 8-azag~anine to find a HAT sensitive mutant (method provided in Example 3). One HAT sensitive line was tested for the presence of rabbit IgG by capture assay as described in Example 6. No Ab was detected down to 80 ng IgG/ml supernatant. At this point the cells were frozen using the methods described in Example 2.

Exam~le 2.
Long~Term Sto~age and Use of Fusion Partner Cells grown in log phase were centrifuged (300xg, 10 minutes) and resuspended in freezing medium (MEM + 30 horse serum ~ 10% DMSO). Cells w~re frozen at a cell concentration of sxlo6 cells~ 1 ml. vial ~NUNC, Vangard International, New Jersey). Cells were frozen in a controlled rate ~reezing chamber (~ degree/min) ~Cryomed, Inc., New Baltimore, MI) and were stored in liquid nitrogen.
Vials were thawed from liquid nitrogen storage in a 37 C water bath. Cells were diluted with 1 ml of HB-GRO
medium (Irvine Scientific, Santa Ana, California) and 10%
horse serum and transferred to a 15 ml conical tube containing 10 ml of HB-G~O + 10~ HS. Cells were centrifuged and plated at 2x105 cells/ml for culturing~

Example 3 Establishing 8-azaguani~e ~esistance and HAT Sensitivity 1 . 2 X 107 cells grown in log phase were centrifuged (300xg, 10 minutes) and resuspended in 12 ml of HB~RO +
10% horse serum supplemented with 20 ~g~ml of 8-Azaguanine ~L~ 4~

(Calbiochem, La Jolla, California). The cells were plated in 24 well plates at 2 ml/well. The cells were fed with HB-GR0 me~ia supplemented with 8-azaguanine every 3 days until colonies appeared. The colonies were grown to confluence and split into two portions. This was repeated or 5 cycles. The media was replaced in l/2 of the wells with medium containing HAT (Sigma, For lOOx. Hypoxanthine 10 mM, Aminopterine 0.04 mM, Thymidine 1.6 m~). Cell death indicated HAT sensitivity an~ the corresponding wells not exposed to HAT were expanded for fusion.

Exampl~ 4 Exemplary Immunization Protocol ~or Rabbit Monoclonal An~ibodies Directed to Carbohydrate The following immunization protocol was used to immunize New Zealand white rabbits (Siminek, Vista, California) with pepsin-digested Group A Streptococcus variant A486.
Pepsin-digested Stre~tococcus pyo~e~ (ATCC 19615) was prepared by growing the cells in Todd-Hewitt Broth at 370C.
The cells were heat killed at 700C for 1.5 hours. The bacteria were resuspended in 1 mg/ml pepsin at p~l 2.8 in O.85% NaC1 and incubated ~or 2 hours at 370C. The cell digest was wash~d 3 times with PBS (Phosphate Buffered Saline). The digest was diluted to 24~=0.38-0.40.
The im~unization protocol is provided below:
~ay: 1 3 5 0.5ml/day IV
8 9 11 l.OOmljday IV
12 15 16 l.OOml/day IV
l~ 19 29 l.OOml/day IV
Rabbits were bled on day ~2 and spleens were removed on day 32. The rabbi~ serum was tested by ELISA using pepsin- digested Group A Streptococcus Pvoqe~s (GAS~ as described in Example 6.
~a5~e 5 Fuslon_o~ ~ab~ ~_Ant,i~y~y~e5~y~¦n~ Cells with Exe~ y r~5 L~o~ =gll .
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A single cell suspension of rabbit splenocytes was obtained ~rom a rabbi~ immunized with ~roup A Streptococclls using the immunization protocol provided in Example ~. The splenocyte cell concentration was adjusted to 2. 5xlO6/ml in PBS. Leu-leu-ome peptide was added at 25t) ~M/l to kill the lysosomal-enriched cells such as macrophages. The mixture was incubated for 15 minutes at room temp. The c211s were spun down and the pellet was mixed with the fusion partner OMB-037 at a ratio of 4 spleen cells per OMB-037. The fusion was accomplished by slowly adding lml of 35% PEG
1500 (Aldrich, ~ilwaukee, Wisconsin) to tha cell pellet/OMB-037 mixture for one and half minutes and gradually diluted with serum ~ree media followed by serum containing medium. The ~usion mixture was brought up to a cell density of 8x105 cells/ml in 150 mls. in HB GRO
containing 15% fetal calf serum and HAT, as disclose in Example 3, and distributed into g6 well plates at 2.0 X 105 cells/well. The cells were incubated at 37 C in an atmosphere containing 5-10% COz.
After fusion, the cultures were examined for hybridoma ~rowth. The presence of larg~r cells and evidence o~ cell replication were the primary criteria used to assess hybrido~a growth. The cells were fed with H~T containing mediu~ on days 5, 8, and 13. The hybridoma supernatants were screened for specific anti~ody production after day 18. The product of one fusion experiment, used in subse~uent analysis i~ designated SAlG.
E~ample 6 Testinq FusiQns ~or ~tibody P~oduct~Qn Three tests were used to asses~ rabbit monoclonal antibody production directed to Group A Streptococcus (GAS~
1) ELISA for Group A Streptococcus (GAS~ antibodies:
Two forms of antigen were used to ~est for the presence of specific antibodies. The first preparation ~mployed pepsin-digested Strep~ococcuq,eyglçna~ cells. A method for preparing pepsin-diges~ed cells is provided in Example 2, .

.

2 ~ ~ ~ 9¢1 r-Nitrous acid extract preparations of Group A Streptococcus carbohydrates were obtained by incubating Stre~tococcus pyoqenes in lM HCL and 6M NaNO2. lM potassium Phosphate was used to stop the reaction. The lysate was centrifuged and the supernatant collected and filtered through a 0.2 ~m filter, concentrated and dialy2ed against 'PBS. Th~ nitrous acid extract (NAE) was diluted to a concentration of 1.6 mg/ml.
Either the pepsin digest preparation or the nitrous acid extract was used to coat the wells of Falcon 96 well assay plates. To coat the plates, 50 ~l of antige~ o~E a 1:400 dilution of stock pepsin-digested GAS or stock NAE in carbonate buffer pH 9.5 was added to each well. The plates were incubate~ at 37C overnight. Free absorption sites were saturated with 2% bovine serum albumin (BSA) in phosphate buffered saline containing 0.1% Tween (PBS/Tween). Fifty ~1 of each test sample was added to the plate and incubated at 37C for an hour. Unbound materials was removed by washing three times with PBS/Tween. Goat anti~rabbit IgG antibodies conjugated to horseradish p~roxidase in 50 ~l was added and incubated for an additional hour. After washing, 100 ~l of the substrate o-phenylenediamine dihydrochloride (OPD) was added and incubated at room temperature for 15 minutes. The presence of antibodies to Streptococcus A was demonstrated by an increase in absorbance at 490 nm.
2) Sandwich ELISA test for IgG: Falcon Probind assay plates were coated with 50 ~1 per well o~ goat anti-rabbit IgG in sodium phosphate buffer pH 7. 0 . Test samples were added and the procedure followed the assay described above.
A similar sandwich assay for detecting mouse IgG was also used to confirm the species of antibody production both for cell culture supernatants and ascites. No cross reactivity was found in either of the rabbit and ~ouse IgG assays.
3 ) Inhibition ELISA for Group A Streptococcus Antigen: The procedure was the same as Group A
Streptococcus assay (#1, above) with the exception that :.
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~ 3 equal volumes of 20~ N-acetyl--D-gluco!3amine and test samples were co-incubated at 37C for one hour. The resulting decrease in absorbance at 490 nm indicated the presence of antibodies to ~he Group A Streptococcus specific carbohydrate epitope.
After three stages of testing, the desired hybridomas were carried in culture continuously and subcloned at least once to ensure the monoclonalityO All subcloning was performed by limiting dilution to assure monoclonality using 1/3 cell per well.
Exam~le 7 Co~ar son of Rabbit Mo~oclonal An~ibo~y_P~~oducin~.C~lo~
HB 9696 with Rabbit MonQclonal An~ibody Producin~ Clone SAlG7.
Nitrous acid extracts of Group A Streptococcus (GAS) (the antigen used to produce clone HB 9696) or pepsin-digested Group A Streptococcus were coated onto ELISA
plates. The concentration of NAE product per well was 125 ng. The supernatant from cells grown ~o confluence in D~EM
~edia supplemented with 10% fetal calf seru~ was added to the wells. Following a lh incubation at 37C, the plates were washed and bound antibodies were detected with a goat anti-rabbit HRP conjugate. As positive controls~ rab~it Group A Strep~ococcus purified polyclonal antibodies and several clones from the fusion protocol generated with OMB-037 were added. The results are provided in Ta~le 2. HB
9696 did not produce antibodies specific for Group A
Streptococcus, nor were the cells stable in culture, even in the presence of rabbit sera.
To determine if HB 9696 was making antibodies, an initial scre~n quantitative ELISA was prepared in which a standard curve (~AS polyclonal) ranging from 106 - lOOOng was used to determine the immunoglobulin concentration.
The results are provided in Table 3. HB 9696 did not produce detectable antibodiesO The clone was either inherently unstable or particularly susceptible to ~reeze/thaw procedures. ~oth qualities weigh strongly a~ainst the use of these fusion partners and antibody producing cells for the production of commercial quantities of antihod.ies.

Example ~
Growth of Xe~o~eneic Ant.ibody Produci~_Clon.e as Ascites The stable hybridoma clones were prepared in growth medium, injacted into nu~e or SCID mice (pri~ed with incomplete Freunds adjuvant, 0.5 ml/mouse, fiva days prior to inoculation), at a concentration of 2Xl06 cells per mouseO After two to three weaks, peritoneal ascites ~luid was collected and quick-checked on cellulose acetate gel for the presence of antibodies. The anti~ody-containing ascites was Purther tested using the ELISAs described in Example ~ to detect rabbi~ antibodies against Group A
Streptococcus. . .
Ascites characterization: ~scit~s from both nude and SCID mice were run in an antigen-speci~ic assay (GAS
antigen coated down) and both sources appeared to have good anti-Group A Streptococcus activity (Fi~ure 7). To determine the origin of the antibodies (host versus fusion partner), assays were run ~o detect rabbit immunoglobulin (Figure 5) and mouse iT~munoglobulin (Figure 6). In the mouse and rabbi~ species specific assays, the capture antibodies (either goat anti-rabbit or goat anti-~ou~e) ~ere coated onto ~h~ plate to capture the appropriate species of an~i~ody in the ascit~s. The captured antibodies were detec~ed using goat anti-species antibodies conjugated to ~P. The results indicated that the ascites from both nude and SCI~ mice contained high amounts of rabbit antibodies (Figure 5). Conversely, only ascites from nude mice contained high amounts of host antibodies (Figure 6). ~hese results also confirm ~hat xenogeneic fusion partner OMB-037 did not produce antibody.
In our hands~ the heterohybrid, SAlG7-516.5, producad at least l~g/ml of purified antibodias from ascitesO The ''., - '' ' ' ' ' : . ~.
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total volume of ascites produced per mouse was variable and ranged from 1-6 mls /mouse.
Example 9 Rabbit Monoclonal Antibody P _ fication Ascites fluid from the rabbit x mouse x rabbit heterohybrids grown in nude or SCID mice were clarified of proteins using a single salt cut with saturated ammonium sulfate. Lipoproteins were removed using dextran sulfate precipitation and rabbit immunoglobulin was purified by either of two different mathods. In the first method, rabbit immunoglobulin was purified by ion exchange chromatography The buffer of the clarified ascites was exchanged with load/wash buffer ~20mM Tris, pH 8.0) in an ~micon Cen~riprep-30 column (Amicon, Beverly, Massachusetts) and loaded onto a Q Sepharose Fast ~'low column (FFQ; Pharmacia) and eluted with a gradient buffer of 20~M Tris, 0.5M NaCl, pH 8~0. Appropriate fractions were pooled for k~sting. In the second method, the clarified ascites was purified by immunoaffinity chromatography using an N-acetyl glucosamine (NAG) agarose column (Si~ma, St.Louis, Missouri). The loaded column was washed with phosphate buffered aline (PBS) and the rabbit anti-Group A Streptococcus antibodies were eluted with 10% N-acetyl gluco~amine (Sigma~ and dialyzed with PBS. Appropriate fractions were pooled for t~sting.
All samples were tested for species identity (rabbit or mouse) as described in association with Figures 4 and 5.
Antigen specificity was confirmed by ELISA (see Example 6 and the discussion associated with Table 1). Rabbit antibody fractions were pooled and any remaining murine immunoglobulin was removed using sheep anti-mous~
i~munoglobulin-coated polystyrene beads.
Example lQ
Use of Rab~ onoclonal Antibodies and Rabbit Polyclonal Antibodies in Solid-phase Immunoass?ay~ ?
: In this Example, Rabbit monoclonal antibody SAlG7-516O5 was used as the detecting antibody fcr Group A Streptococcus in a solicl phase immunoassay. 10 mg of the monoclonal anti~ody preparation was labeled with 10 mg o~ alXaline phosphatase. The conjugation of the alkalin2 phosphatase to the monoclonal antibodies followed procedures well known in the art of immunology. Both the antibodies and the alkaline phosphatase (AP) were dialyzed in P~S and filtered through 0.~5 ~m filters. The concen~rations of the antibodies and AP were determinad on a spectrophotometer at A280~ The antibody solution was dilute~ by weight to 5 mg/ml in PBS.
It is contemplated that this procedure could be used for either monoclonal or polyclonal antibodies and that the methods c~uld similarly be used with antibodies recognizing any number of antigen.
The A~ was conjugated to ~he heterobifunctional reagent Succinimidyl 4~(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC, Pierce, Rockford, Illinois) using ~ 10:1 initial molar ratio of SMCC to ~P. SMCC was suspended in dry acetonitrile to a concentration of 10 mg/~l. The SMCC
and AP were combined under Argon and incubated for 30 mn at room temperature. The mixture was applied to a Sephadex G-25 column, saturated with Argon gas, to separate free from bound enzy~e. The concentration of conjugated AP (SMCC-AP) was determined by spectrophotometer at A2~0-Rabbit monoclonal antibodies were conjugated to the heterobifunctional reagent N~uccini~idyl 3~(2-pyridyldithio~propionate (SPDP, Pierce). SPDP was also resuspended in dry Acetonitrile. The concentration of SPDP
was determined at ~26D. SPDP was mixed with the monoclonal antibodies at a molar ratio of 20:1 and incubated for 30 mn at room temperature under Argon. A 0.10 ml Dithiothreitol (DTT) solution was added to the antibody-SPDP mixture to give a final concentration of lmM DTT. Following a 30 mn incubation at room temperature, the conjugated antibodies were separated from unlabelled antibodies and free SPDP on a Sephadex G 25 colu~n. The conGentration of conjugated antibodies was determined at A2~0.

':,, . , . ~ " ' ~ ' . ' ' -The SMCC-AP and the SPDP-antibodies were separately diluted by weight to 1.5 my/ml with PBS. Equal volumes of SMCC-AP and SPDP-antibodies were mixed and deoxygenated under Argon. The antibody and enzyme solutions were mixed and stirred for 90 mn at room tempera~ure. Unreacted SPDP
groups were blockad using a 125 mg/ml solution of N-ethlmaleimide (NEM, Pierce) to yield a final concentration in the reaction of 0.01 NEM/ml reaction solution.
Unreacted SMCC groups were blocked using an equal volume o~
O.~OM 2-mercaptoethanol. The ~P-rabbit monoclonal antibody conjugate was concentrated on a Centricor 30 column (Amicon) at 20C-80C to no greater than ~5 mg/ml. The conjugated an~ibodies were ~ialyzed in PBS and purified by column chroma~ography. Peak ~ractions were pooled and the conjugated antibodies were diluted to 20mA where lmA is equivalent to AaBo=~ l .
Cap~ure ~ntibody Preparation: Rabbit monoclonal antibodies or rabbit polyclonal antibodies to Group A Streptococcus were coated onto la~ex beads as capture antibody. To prepare 0.36ml. of coating solution, 0.012 ml o~ a 7.65 mg/ml stock antibody solution was combined with 0.309 ml coating buffer (50mM ethanolamine in 0.9% NaCl pH 10.0) and O.036 ml latex and incubated overnight at 450C. 0.309 ml of backcoating buffer (0.3% bovine serum albumin in PBS) was added to the mixture and incubated for ~ hr. at room te~perature. The labeled latex was spun down and resu~pended in 005ml PBS and stabilization buffer (10%
sucrose, 2% nonfat dry milk in P~S) was added to obtain a final volume o~ lOml.
Solid~has~ Im~unoassay: For an immunoassay to Group A
Streptococcus, 3~1 of the Group A Streptococcus antibody-latex solution as a 0.30% latex solid solution was spotted onto porous members such as those disclosed in U.S~ Patent No. 4,727,019. In this example POREX~ (Porex Technologies, Atlanta, Georgia) filters were spotted with the Group A
Streptococcus solution, as well as the positive and negativ~ controls. An equal volume of Streptococcus-: ' :' . '- . , .
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specific antibody conjugated to latex and preincubated with nitrous acid extract o~` Streptococcus was spotted in a second location to as a. posi~ive control. Purified Streptococcus-negative rabbit polyclonal antibady was spotted onto a third position on the porous membrane as a negative control.
Group A Str~ptococcus nitrous acid extracts or extracts from other organisms were prepared using methods disclosed in Example 6 and neutralized with potassium phosphate. 20~1 of a solu~ion, equivalent to 4X106 CFU/ml, was added to 1 drop of antibody conjugated to alkaline phosphatase. The sample was filtered through a 0.2~m filter and the total sample was applied to the porous member. Once the liquid passed through the porous member, the surface was washed with wash solution (1% Triton X-100, 0.002% Nitroblue Tetrazolium (wt/vol), and sodium azide in PBS). Detection of bound sample was determined by the addition of the AP substrate, indoxyl phosphate, in PBS
(6.66 gm Tris, 0.~56 ml of a 90% solution of 2-amino-2,methyl-l,propanol, 1 gm NaCl, 0.1 ~m. sodium ~zide, 0.290 gm indoxyl phosphate and 0.411 ml conc. HCl). ~ purple dot at the positive control position indicated that the correct technique was used and that the reagents were functional.
A purple dot in the negative control position indicated that the sample did not contain human anti-rabbit antibody.
A purple dot in the latex bound anti-~roup A Streptococcus position indicated the presence of Group A Streptococcus in the test sampl2.
While particular e~bodiments of the invention have been described in detail, it will be apparent to those skilled in the art that the~e embodiments are exemplary rakher than limiting, and the true scope of the invention is that defined in the following claims.

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Claims (21)

1. A stable xenogeneic fusion partner that produces substantially undetectable levels of antibodies comprising the product of a cell fusion between a rodent myeloma cell and a non-transformed rabbit cell.

2. The stable xenogenic fusion partner of claim 1 comprising a fusion partner wherein said rodent myeloma cell is the SP2/0 line.

3. The stable xenogenic fusion partner of claim 2, comprising a fusion partner that is:
a. derived from a fusion between a SP2/0 cells and bovine somatotropin-sensitized rabbit splenocytes;
b. hypoxanthine-aminopterin-thymidine sensitive;
c. hypoxanthine-guanine-phosphoribosyl transferase deficient;
and naturally occurring variants and mutants thereof.

4. The stable fusion partner of claim 3, comprising the cell line deposited with the ATCC as culture number 11086, and the naturally occurring variants and mutants thereof.

5. A stable cell line that secretes rabbit monoclonal antibodies, comprising the product of the fusion of:
a. a stable xenogeneic fusion partner, which in turn comprises the fusion product of a rodent myeloma cell and a non-transformed rabbit cell that itself does not secrete substantially detectable quantities of antibodies;
and b. a rabbit splenocyte that has been sensitized with a predetermined antigen and produces antibodies to the antigen.

6. A cell line of claim 5, which is the product of a fusion wherein the stable xenogeneic fusion partner is in turn the product of a fusion that utilizes a mouse derived rodent myeloma cell.

7. A cell line of claim 6, which is the product of a fusion wherein the stable xenoganeic fusion partner is in turn further the product of a fusion that utilized an SP2/0 murine myeloma cell line.

8. A cell line of claim 7, wherein the rabbit splenocytes were sensitized with a carbohydrate antigen and produced antibodies to such antigen.

9. A cell line of claim 8, wherein the stable xenogeneic fusion partner is the cell line deposited with the ATCC and designated as cell culture number 11086.

10. A cell line of claim 9, further comprising a cell line wherein the rabbit splenocytes were sensitized with N-acetyl D-glucosamine and produced antibodies to the carbohydrate antigen.

11. A method for producing rabbit monoclonal antibodies comprising the steps of:
a. fusing a nontransformed rabbit partner cell with a rodent myeloma cell to produce a xenogeneic fusion partner;
b. selecting a stable xenogeneic fusion partner producing substantially undetectable levels of antibodies wherein the fusion partner is the product of a fusion between a rabbit cell and a rodent myeloma cell;
c. fusing said stable fusion partner with a rabbit antibody-producing cell; and d. isolating an antibody producing cell line obtained from step (c) that produces antibodies directed to a predetermined antigen.

12. The method of Claim 11, wherein said selecting step additionally comprises generating a drug resistant stable fusion partner and said isolating step additionally comprises growing said antibody producing cell in said drug.

13. The method of Claim 12, wherein said rodent myeloma cell is mouse derived.

14. The method of Claim 13, wherein said rabbit antibody producing cell is obtained from a rabbit immunized with said predetermined antigen.

15. The method of Claim 14, wherein said stable fusion partner is the cell line deposited with the ATCC as culture number 11086.

16. The method of Claim 15, additionally comprising the step of growing the antibody producing cells in vivo.

17. The method of Claim 16, wherein said growing step consists of generating ascites.

18. The method of Claim 17, wherein said growing step consists of introducing said fusion products into mice deficient in mature T and B lymphocytes.

19. A rabbit monoclonal antibody produced according to the method of claim 11.

20. A immunoassay comprising the rabbit monoclonal antibody of Claim 19.

21. A method for producing a cell line which produces rabbit monoclonal antibodies comprising the steps of:
a. fusing a nontransformed rabbit partner cell with a rodent myeloma cell to produce a xenogeneic fusion partner;
b. selecting a stable xenogeneic fusion partner producing substantially undetectable levels of antibodies wherein the fusion partner is the product of a fusion between a rabbit cell and a rodent myeloma cell; and c. fusing said stable fusion partner with a rabbit antibody producing cell.