CA1201667A

CA1201667A - Diagnostic reagent and method

Info

Publication number: CA1201667A
Application number: CA000402420A
Authority: CA
Inventors: Chaya Moroz
Original assignee: Individual
Current assignee: Individual
Priority date: 1981-05-15
Filing date: 1982-05-06
Publication date: 1986-03-11
Also published as: JPH0751062B2; DE3218312C2; GB2103238A; US5871735A; DE3218312A1; US4882270A; FR2505871A1; JPH05260990A; IL62879A0; GB2103238B; US4954434A; JPH0614873B2; FR2505871B1; IL62879A; JPS57196154A

Abstract

ABSTRACT

There are provided monoclonal antibodies which react with human oncofetal ferritin and which do not react with human spleen ferritin or with liver ferritin; there are also pro-vided monoclonal antibodies which react both with human placenta oncofetal ferritin and with human adult spleen ferritin. There is provided a process for producing clones producing such antibodies and such clones, and an assay for the detection of human breast cancer based on the determination of oncofetal ferritin, which assay is based on such monoclonal antibodies.

Description

.The present inventlon relates to monoclonal antibodies which react with human embryonic ferritin derived from human placenta, which do not react with adult human spléen or liver ferritin: to monoclonal antibodies which react with human placenta embryonic ferritin and which cross-react with human adult spleen ferr;tin~
The invention further relates to an assay for the detection of human breast cancer and/or Hodgkins disease which comprises selectively determining in body tissues and/or lymphocytes derived from patients where human oncofetal ferritin is present, According to a specific embodiment of the invention the presence or absence of human oncofetal ferritin is determined by a cytotoxic assayO

According to a further specific embodiment of the invention the presence or absence of human oncofetal ferritin is determined by a radio-immunoassay, The assays according to the present invention are based on the use of the specific monoclonal antibodies, which can be used in any type of suitable assay for the determination of the presence or absence of oncofetal ferritin, the presence of ~hich is indicatiYe in human patients that a breast cancer of stage I or II is present, or of Hodgkins disease.

According to a preferred embodiment of the invention the presence of oncofetal type ferritin on thP surface of lymphocytes in the cir-culation of patients is determined9 the presence of such ferritin being indicative of breast cancer or of Hodgkins disease~

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STATE OF PRIOR ART :
Ferritin is the major iron storage protein in tissues, and small amounts (65-15~ ng/ml) can be detected in plasma. The analysis of normal tissue ferritin by isoelectric focusing has revealed cons-iderable heterogeneity. Marcus and Zimberg (Arch.
Biochem. Biophysic. 162, 493, 1974) showed that ferritin isolated from breast tumor tissues contained acidic isoferritin, not found in adult liver ferritin9 while Drysdale and Singer (Cancer Res.
44, 3352, 1974) demonstrated acidic isoferritin in Hela tumor cells and in placenta cells. They suggested the term "carcino-fetal" isoferritin for such isoferritin.
Marcus and Zimberg, (Clin. Res. 23, A 447, 1975) and Jacobs et al (Br. J. Cancer 34, 286, 1976) reported increased serum ferritin concentrations in patients with breast cancer, and suggested its assay as possible indicator for detecting breast cancer. However, slnce any heterologous anti-ferritin serum cross-reacts with anti-genic determinants associated with both adult and carcinofetal ferritin, it cannot distinguish between the two isoferritins.
Their results were therefore significant only in pat;ents in whom the ferritin level was above normal range (~>200 ng/ml). In a recent study9 Moroz e~ al., Cancer Immunol. and Immunotherapy ~101,(1977) have identified a subpopulation oF lymphocytes bearing ferritin on their surface in breast cancer patients. The ferritin is of the carcinofetal type and these ferritin positive lymphocytes appeared in early stages of breast cancer (stage I - II). They were not found in patients with benign breast disease7 or in healthy people (Giller et al9Surgery Gyn.Obst. 149,6555 1979~.

~,--~L2Q~6~7 The identification of ferr;tin bound to lymphocytes or ;ts presence in t1ss~e fluids is the basis of the assay for the early detection of breast cancer in human patients.
In recent years3 a method was developed by which one hybridizes mouse myeloma cells with hyperimmunized mouse spleen cells, (~ohler and Milstein, Nature, 256:495: 1975). Such a hybrid cell can produce a single antibody directed towards a single antigenic determinant. After cloning of such hybrid cell a clone of h~bri~
cells is thus obtained; producing a single monoclonal antibody.
The use of monoclonal antibody directed towards a single antigenic determinant available only on the carcinofetal ferritin, and not on adult ferritin, renders the identification of such an isoferritin in adult life (in plasma or on lymphocytes) a more specific and sensitive tool for the detection of a malignant disease. The chances of detecting a benign disease known to be associated with elevation of serum adult type ferritin are significantly decreased.

SUMMARY OF THE PRESENT INVENTION :
The present invention deals with the production of two mouse mono-clonal antibodies, each specific for a different antigenic deter-minant on human placental ferritin.
l) CM-OF-H9- directed towards an antigenic determinant specific for human embryonic ferritin

2) CM-~F-3- directed towards a determinant mutual to hu~an embryonic ferritin and adult spleen ferritin.
The inYention further relates to a sensitive assay for the early detection of breast cancer. The assay also detects the presence of Hodgkins disease. The test is based on the identification of i67 "oncofetal" ferritin in the serum and in other body fluids , or bound to lymphocytes~ for the early detection of breast cancer and for the detection of Hodgkin's disease.
The unique specificity of the monoclonal antibody CM-OF-H9 to onco-fetal type ferritin, differentiates between elevation of serum ferritin caused by malignancy and the normal ferritin or that associated with benign diseases (i.e. Thalassaemia). CM-OF-3 can detect the elevation of ferritin in the two groups of diseases.
A test with both ant~boates differentiates between the malignant and non-malignant diseases.

MET~ODS :
Preparation o~ oncofetal ferritiD.
Ferritin was prepared from human placenta by a modif;cation of the method used by Beamish et al. (J. Clin. Path. 24~ 581, 1971).
Placenta tissue (500 gr) was sliced and water added to a total volume of 2000 ml. After homogenization the tissue suspension was heated to 75C for 20 mlnutes. The supernatant, after cooling and centrifugation at 10,000 rpm for 15 minutes, was treated wlth acetic acid to bring the pH to 4.6. The precipitated protein was remoYed by centrifugation at 10,000 rpm for 15 minutes and a clear super-natant was adjusted to neutral pH with dilute NaOH. When the clear brown supernatant was ultracentrifuged at lOO,OOOg for 240 minutes the suspended ferritin collected in a small button at the bottom of the tube. The precipitate was redissolved in 0.9% saline and further purified by passage through a Sephadex G200 column.

~, 12C~667 The ferritin fraction from this column was passed through a DEAE cellulose anion exchange resin using Tris-HCl buffer at pH 7.5 and a 0.02-0.5 M gradient. Three protein peaks were dbtained, the most acidic peak pI=4.8(No.III) was collected and used for analysis. Its purity was shown by ;soelectric focusing and immunoelectrophoresis against anti-ferritin serum and anti-human whole serum. This was used for the immunization of miceSsee below.
Preparation of Hybridomas Myeloma Cells :
Myeloma cells used for hybridisation - PB/NSl/~Ag4 -1 were grown in RPMI-1640 with ~0% ~Fetal Calf Serum (FCS).
Mice Balb/c Females 9 4-6 weeks old initially.
Immunization protocol - 3 weekly immunisations, of 50 ~g of acidic ferri~in in complete Freund's adjuvant, hydridising 3 days after the last injection of 10 ~g ferritin. Hyperimmune mice were rested at least one month before last boosting.

Cell Preparation Spleen Cells a. Spleens were removed from mice in RPMI-0;
b. Rinsed 2x in petri dish with RPMI-0;
c. Teased apart in RPMI-0 with 18 ga. needles, d. Cell suspension transferred to a tube and large chunks of tissue settled out;
e. Single cell suspension removed to a new tube spun at 800 RPM
(160 xg) 5 min;
Red blood cells lysed with 0.83% NH4Cl, pH 7.~;

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f. Cells washed 3x with RPMI-0, resuspended in same;
g. Cells counted with Trypan Blue.

Myeloma cells a. Cells were removed from culture flasks with gentle pipet-t;ng into 50 ml Falcon/Corning tube3 b. Spun down at 900 RPM (200 xg) 5 minutes, c. Washed lx with RPMI-0, resuspended in same and counted with Trypan Blue.
Spleen Cell-Myeloma combination a. Spleen and myeloma cells were combined in a 10:1 ratio in a single 50 ml conical FalcontCorning disposable centrifuge tube;
b. Cells were pelleted at 900 RPM (200 xg) for 5 minutes;
c. Medium was aspirated as completely as possible;
d. All solutions and media used from now on were at room temperature; tube with cell pellet was immersed in a bath at 37C, and the following was added accompanied by gentle stirring;
0.2 ml 33% PEG 150Q for 1 minute, centrifuged at 200 9 for 5 minutes. Cells were resuspended and stirred gently for 1 minute followed by the addition of 5 ml RPMI-0 gentle stirring and addition of 5 ml RPMI-0 20~ Fetal Calf Serum.
Hybrid mixture looked like a poorly, resuspended cell suspension at this point with many small clumps;
e. The mixture was pelleted at 200 xg 5 minutes;
f. Cells were resuspended in RPMI-HY-HATD (at 37C) at a con-centration of 3x106/cc by squirting medium onto the cell pellet;

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g. Hybrids were plated out in flat bottom 96 well plates by adding 2 drops of cell suspension from a 5 ml pipet or with multi-p;pettor using cut off tips (about 65 microliters), containing 100-120 RPMI-HY-HATD (Approx. 2x105 cells);
h. Control wells containing NS-l cells + RPMI HY-HATD at lxlO6/m1 were set up;
i, Plates were cultured for 7 days;
j. On Day 8 and twice a week therefrom half of the culture nedium was removed by careful aspiration and fed with 80-100 microliters of RPMI-HY-HT medium;
k. Positive wells were screened for at 3 and 4 weeks after hybridisation.

Media & Solutions 1. RPMI-O (No FCS) 2. RPMI 1640-HY

500 ml sterile distilled water 55 ml 10 x RPMI-1640 6 ml I.ON Sodium Hydroxide 14 ml 7.5% Sodium Bicarbonate 6 ml Pen/strep 10 ml Glutamine ~ DMEM
86.5 ml FCS

3. RPMI-HY - HATD - day O -~ay 7 For 100 ml of medium 95 ml RPMI - 1640 ~ 20% FCS
1.0 ml Pyruvate (lOOx) 2.0 ml 50 x HAT
2:0 ml 50 x deoxycytidine r~
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4. RPMI - HY - HT - day 8 ~ day 14 For 100 ml of medium 97 ml RPMI-1640 + 20% FCS
2.0 ml 50 x HT
1.0 ml Pyruvate (lOOx) For Hybrids from dayls/onwards use RPMI-1640 ~ 20% FCS
and Pyruvate, or maintain in RPMI-HY-HT.

5. PEG 33 and 25% w/v Must be odorless and white. For 100 ml autoclave relevant wt in grams in a glass bottle at 15 lbs for 10-15 minutes.
When bottle is cool enough to hand hold(about 50C) add RPMI 1640-0 to make up to 100 ml, swirl to mix, store at RT.

6. HATD - Final concentrations of reagents H = Hypoxanthine 10 4M
A = Aminopterin 10 6M
T = Thymidine 2xlO 5M
D = Deoxycytidine 2xlO 6M
HT Stock lOOx - 100 cc Thymidine M.Wt,242.33 - 0.04846 g Hypoxanthine M.Wt 136.1 - 0.1361 9.
dd H20 up to 100 ml and warm to 60-70C to dissolve.
Readjust final volume with dd H20. Dilute to 50x and filter ~0.2 y) sterilise. Make 2 ml aliquots, store at -20C.

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6~7 A Stock 1000 x - 100 cc Aminopterin F.Wt 440.4 - 0.44 9 Bring to 50 ml with dd H20, add 0.1 N NaOH dropwise until am;nopterin ~issplves. Bring f;nal volume to 100 ml w;th dd H20.
Adjust volume to 100 ml f;lter (0.2 ~) ster;l;se. Store at -20C.

D Stock 100 x - 100 cc Deoxycyt;dine M.Wt 227.2 0.00454 g Dissol~e in dd H20, adjust to 100 Cc9 dilute to 50x stock, sterile (0.2 ~) Filter, store at -20C.

HAT - 50x - 200 ml Comb;ne 100 ml 100 x HT with 10 ml 1000 x A + 90 ml dd H20 = 50 x HAT, F;lter (0.2 ~) ster;l;se make 2 ml aliquots and freeze at -20C.

Screen;ng and determ;nation of the spec;f;city of the monoclonal ant;bod;es was performed by a haemagglutination test.
Enbryon;c placenta and adult spleen ferr;t;n were coupled to Ox red blo!od cells Ox RBC by CrC12.
50 ~1 of ;ncreas;ng dilut;ons (startln9 at 1:10 of hybr;doma culture med;um supernatant were mixed with 1~ ~1 of adult or embryonic ferr;t;n Ox RBC and haemaggluti~ation determined.
Supernatants of clones gi~ing a haemaglutination titer of at least 1:1000 were selected.
A clone designated CM-OF-3 was selected.
The clone CM-OF-3 is specific for embryonic ferritin and it cross-reacts with both adult and embryonic ferritin.

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~20~ii67 Th~ monoclonal ant~body obta~ned, C~l-OF-3 was used to block th~
cross-react~ve det~rIll~nants of fetal and adult ferr~tin, ~n order to produce a dl Fferent monoclorIal aIlt~body~ cM-oF-lls~ Wh~CIl ls dlrected to a spec~flc fetal d~term~nant.
The follow~ng ~mmun~zatlon procedure was used:
A! Imnunlzat~on and Fuslon Protocol.
Embryon~c fcrr~t~n Isolat~d from human Placenta/a prote~n oF pl 4.B(see p.4~5) was reacted w~th monoclonal antibodles CM-OF-3 In the follow~ng ratio:
embryon~c ferrltln (90 119 ln PBs)was m~xed with Asc~tes flu~d from r~ALBIc mouse conta~n~ng CM-OF-3 ant~ferrltln monoclonal ant~bod1es 10 mgrlml ).
The m~xture was ~ncubated for 30 m~n at 37C fo~lowed by overn~ght ~ncubation at 4C. The m~xture was centr~fuged at 10000 G, the pre c~p~tate ~orn~d, was dlscarded and the supernatant was used for ~n~nuniza~on. Each BALB/c mouse was ~nununlzed wlth the above super-natant'm~xed w~th complete Fre~nd's adjuvant, ln~ected lntradermally onca a week for 3 s~eeks. A booster ~mTun~zatlon of f~fth o~ the abDve dose was ~n~ected lntraper~toneally.

After 3 days fronl boost~ mouse spleen was asept~cally removed and fus~on was perfornled by ~ncubat~ng 108 spl~en oells w~th 107IP3-~SI/l-Ag4 nlyeloma cells as set out above ~n the hybr~lzat~on proc@dure and the same subsequent procedures for pos~t~v~ clone ~dent~f~cat~on was followed. Thus a clone des~gnated as CM-OF-H9 was obta~ned, B. Character~zation o~ the Ilonoclonal Antibody Obta~ne(I.

Character~stlcs of the CM-OF-H9 monoclonal ant~bodles:
The CM-ûF-I19 monoclonal antibody belongs to the IgG~class~ ~t does not fvrm precip~tates w~th ferr~.in, ~t binds rabb~t comi-lement~ In the ascltlc fluid obtained th~ ant~body cont~nt was a~out 7 mg pe~r ml. On~ ml of ascitlc flu~d blnds about 2 mg o~ ~mbryclllc ferrlt~n and non~ o~ a(lult spl~en or 1 i ver f~rri ti n.

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Reactivity of Monoclonal Antibody Source of human ferritin CM-OF-H9 CM-OF-3 1. Adult spleen from Thalassaemia - +
2. Normal serum - ~
3. Breast cancer (PBL)* ~ +
4. Breast cancer (serum) +
5. Hodgkin's Disease (spleen) +
6. Benign breast disease (PBL)*

7. Benign breast disease-serum - +

~PBL: Peripheral blood lymphocytes The two antibodies make possible a rapid and convenient detection of malignancies of the breast and of Hodgkin's disease, and a differentiation of these from Thalassaemia, which results in an increase of ferritin.

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P~inciples'of'Serolo~ical Test Procedures 'Determlnation'of'Lymphocyte Bound Ferritin (LBF) :

The presence of lymphocyte bound ferritin is indicative of the presence of breast cancer in human patients.
The determination o~ such ferrit;n is effected as follows:

a. ' Lymphocytes are isolated from peripheral blood;
b~ Lymphocyte bound Ferritin is determined by a conventional type of assay based on the use of the novel specific monoclona1 antibodies, specific towards ferritin derived from human placenta, Accord~ng to a preferred embodiment the test is carried out as follows:

a~ Lymphocytes are isolated from peripheral blood by Ficoll-Hypaque gradient centrifugation;

b, The presence or absence of LBF is determined by any con-vent;ona1 type of assay, such as cytotoxic test~ radio-immunoassay, Such assay can also be carried out with serum or other body fluidso Test for the Early Detection of Breast Cancer and Hodgkins Disease.
Collection and Preparation of Cells.
1, Blood collection tubes~ 15 ml containing heparin.
2, Conical centrifuge tubes, 25 ml.
3. Pasteur pipettes and bulbs.
4. PBS~ of pH 7.2 5. Ficoll-Hypaque ~ens;ty solution 1.077 gm/ml.

Procedures for Collection of Cells 1, Collect 15 ml blood into a heparin-containing blood collection tube, dilute 1: 2 i n PBS pH 7,2 2. Underlay the cell susp~nsion with 10 ml Ficoll-Hypaque density solution.
3. Centrifuge for 30 minutes at 300 x 9. room temperature.
4. Collect mononuclear cells from the medium: Ficoll-Hypaque interface with a Pasteur pipette and transfer to a new 15 ml tube.
5, Wash cells 3 times by suspension in 15 ml wash medium and centri-fugation at 300 x 9 for 10 minutes, at 4C.
Ç, Resuspend in wash medium and determine cell number, Radloimmuneassay ( RIA ) Requ;red Supplementary Material:
1. Minisorp test tubes 100 x 15 mm.
2, RPMI 16~0 3, Bovine serum albumin (BSA) 5% in PBS of pH 7.2; containing 0.025 %
sodium azide 4. Normal rabbit serum (NRS) 5. CMH-9 monoclonal antibodies 6, Blanc Ascites fluid 7, 125 1 anti-mouse IgG, * Sodium phosphate buFfered saline of 7.2 pH
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Radlo-Imllluilo Assay - 1 Peripheral blood monDnuclear cells are isolated by Ficoll-Hypaque gradlent centrifugation.
Test is performed in triplicate, A B~ank B Test.
1, Dispense 2 x 106 to 3 x 106 cells into each of si~x test tubes, pellet cells ~y centrifugatlon at 300 g for 10 min, 2~ Add NRS 20 yl diluted 1:10 in PBS, incubate 60 min, at 4C, 3, Add 30 yl of asc~tes fluids (diluti.on 10 5 ~n 5% BSA ) to each of 3 tubes.
A, Control ascites fluid contain;ng an IgGl non-specific mono-clonal antibody, non-reactive with oncofetal ferritin B, CMH-9 monoclonal antibod~es mix well and incubate at room temperature during 2 hours, 4~ Wash cells twice wlth.10 ml RPMI-16~0 by centrlfugation at 300g for 10 min. at 4C, 5, Add 0,1 ~Ci of I125 rabbit anti-mouse IgG (125-I Rabblt IgG I~Ci/~g) ~ncubate 60 min, at 4C wash twlce with cold RPMI 1640 as in 4, count radloactlvlty, Posltive test Cpm A- CpmB q~ 500, Rad~o Immuno Assay - 2 Af~er Stage 1, RIA - 1, the test procedure-is contlnued as follows:
CMH-9 F(ab)2 is obtained b~ peptic digestion of CMH-9 IgG according to Utsuml and Karush (Biochem, 1965 4? 1766) and ~rom the non speclflc lg(~l (see control of RlA-l)o The F~ab)2 fragments thus obtained are used as Follows:
Tube A: ContrGl F(ab)2 in S % BSA in PBS of pH 7.2;and 0,~25% sodium azide, Tube B: C~H-9 F(ab)2 in 5 ~ BSA in PBS o~ pH 7,2~ and 00025~ sodium azide, `~

i7 Incubate during 60 minutes at room temperature, wash once with 2 ml of 1 % BSA in pH 7~2 PBS;
add 125I-labelled ligand to test tubes A and B (giv;ng about 105 cpm);
eîther 125I-labelled oncofetal ferritin or a complex of 125I~polyclonal anti-oncofetal ferritin with oncofetal ferritin ,The complex is preformed a~ antigen/antibody molar ratios of 1:1 or up to 1:2,pr~-incubated with each other at room temperature for l hour, Incubate the labelled ligand together with cells for 1 hour at room temperature9 wash twice with 1 %
BSA in pH 7,2 PBS to remove unbound labelled ligand and count, If B exceeds A the test is positive, Cytotoxic Assay Test is performed in duplicates.
A. Control B, Test.
1. Suspend PBL at a density of 5 x 106 cells/ml in RPMI-1640 2, Place 150 ~1 of PBL into each of 4 12x75 mm test tubes Add Ascites fluid (30 ~1 dilution 10~4) A. Cohtrol Ascites fluid (2 tubes). B. CMH - 9 (2 tubes). Incubate 45 min. at ~C.
3. Add rabbit complement (100 ~1 diluted 1:5 in PBS) and incubate 60 min, at 37C with slow agitation.

4. Count viable cells with Trypan blue, Positive Test:
= No, viable cells in A - No, viable cells in B x 100-? 4 %
No. of viable cells in A.

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CITOTOXICITY TEST KIT:

1. Monoclonal antibodies CMH-9 2~ Monoclonal antibodies, non-speciFic 3. Rabbit complement 4, Conventional adjuvants in standardized solution.

RIA TEST KIT:

1, F(ab)2 of CMH-9 29 F(ab~2 f non-specific monoclonal 125I-labelled ligand RIA-l TEST KIT:

1, Monoclonal antibodles CMH-9 20 Monoclonal antibodies~ non-specific 30 125I-antimouse IgGl 4. Adjuvants and standardized solutions

Claims

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A process for producing a clone to produce anti-human oncofetal ferritin monoclonal antibodies which react with human oncofetal ferritin, and which do not react with human spleen ferritin or with liver ferritin, comprising:
(a) immunizing mice with embryonic ferritin derived from human placenta or from breast cancer or Hodgkins disease spleen after having reacted the said ferritin with monoclonal antibody CM-OF-3 as herein defined;
(b) hybridizing hyperimmune mouse spleen lymphocytes of such mice with suitable myeloma cells; and (e) selecting a clone producing antibodies reacting only with human embryonal ferritin.

2. A process for producing a clone to produce anti-human oncofetal ferritin monoclonal antibodies which react with both hu-man placenta oncofetal ferritin and which cross-react with human adult spleen ferritin, comprising:
(a) immunizing mice with embryonic ferritin derived from human placenta, from human breast cancer or from human Hodgkins disease spleen;
(b) hybridizing hyperimmune mouse spleen lymphocytes of such mice with suitable myeloma cells; and (e) selecting a clone producing antibodies reacting with human embryonal ferritin, and cross-reacting with adult spleen ferritin.

3. A clone producing monoclonal antibodies when produced by the process of claim 1 designated herein as CM-OF-H9.

4. A clone producing monoclonal antibodies when produced by the process of claim 2 designated herein as CM-OF-3.

5. A process for the production of anti-human oncofetal ferritin monoclonal antibodies which react with human oncofetal ferritin and which do not react with human spleen ferritin or with liver ferritin comprising:
(a) immunizing mice with embryonic ferritin derived from human placenta, human breast cancer or from human Hodgkins disease spleen, after reacting the above ferritin with monoclonal antibody CM-OF-3 herein defined;
(b) hybridizing hyperimmune mouse spleen lympho-cytes of such mice with suitable myeloma cells;
(c) selecting a clone producing antibodies react-ing only with human embryonal ferritin;
(d) cultivating such clone so as to obtain anti-human oncofetal ferritin monoclonal antibodies; and (e) injecting the clone to mice and removing asci-tic fluid containing the monoclonal antibody.

6. A process for the production of anti-human oncofetal ferritin monoclonal antibodies which react with both human placenta oncofetal ferritin and which cross-react with human adult spleen ferritin, comprising:
(a) immunizing mice with embryonic ferritin derived from human placenta or from human breast cancer, or from human Hodgkins disease spleen;
(b) hybridizing hyperimmune mouse spleen lympho-cytes of such mice with suitable myeloma cells;
(c) selecting a clone producing antibodies reacting with human embryonal ferritin, and cross-reacting with adult spleen ferritin;
(d) cultivating such clone so as to obtain anti-human oncofetal ferritin monoclonal antibodies; and (e) injecting the clone to mice and removing asci-tic fluid containing the monoclonal antibody.

7. A process according to any of claims 1, 2 or 6, where-in the mice used are Balb/c females.

8. A process according to any of claims 1, 2 or 6, where-in the immunization of the mice is by a sequence of immunizations with oncofetal ferritin in complete Freund's adjuvant.

9. A process according to any of claims 1, 2 or 6, where-in the spleen and myeloma cells are combined in a ratio of from 20:1 to 1:1.

10. A process according to any of claims 1, 2 or 6, where-in the spleen and myeloma cells are combined in a ratio of about 10:1.

11. Anti-human oncofetal ferritin monoclonal antibodies, when produced by the process of claim 5.

12. Anti-human oncofetal ferritin monoclonal antibodies, when produced by the process of claim 6.