US3645687A

US3645687A - Immunodiffusion plate apparatus

Info

Publication number: US3645687A
Application number: US642A
Authority: US
Inventors: Samuel T Nerenberg
Original assignee: SAMUEL T NERENBERG
Current assignee: SAMUEL T NERENBERG
Priority date: 1970-01-05
Filing date: 1970-01-05
Publication date: 1972-02-29
Anticipated expiration: 1989-02-28
Also published as: KE2643A

Abstract

A device for the quantitative determination of protein by radial diffusion thereof through a coating of antibody on a sheet formed of a paper-thin liquid-permeable chemically inert layer (e.g., cellulose acetate) on a resilient liquid-impermeable backing (e.g., flexible plastic). The device includes a wall and marginal framework therearound, having a slot for slidably receiving a perforate or solid plate into a fixed position above the wall. The device further includes means for slidably receiving said sheet between the wall and either of the plates in position. Blood serum may be applied via a capillary tube through the openings in the perforated plate onto a sheet in predetermined placement. In one embodiment, the device contains identical backto-back units as above described. In a second embodiment, one side of such back-to-back units contains parallel members mounted on one wall and cooperating therewith to form parallel slots adapted to receive a number of sheets.

Description

United States Patent 1 1 Feb. 29, 1972 Nerenberg [54] IMMUNODIFFUSION PLATE APPARATUS [72] Inventor: Samuel T. Nerenberg, 21 Haynard Court, Burlingame, Calif. 94010 [22] Filed: Jan. 5, 1970 [21] Appl. No; 642

[52] US. Cl ..23/230 R, 23/230 B, 23/253 R, 195/139 LE, 424/12 [51] 1nt.Cl ..C12b l/00,G0ln 33/16 [58] Field of Search ..23/230, 253, 230 B; 195/139 LE [56] References Cited UNITED STATES PATENTS 3,367,841 2/1968 Buissiere et al. ..23/253 X 3,389,966 6/1968 Saravis ..23/230 3,165,450 1/1965 Scheidt ..l95/139 LE Primary Examiner--Morris O. Wolk Assistant Examiner-R. M. Reese Attorney-Flehr, Hohbach, Test, Albritton & Herbert [S 7] ABSTRACT A device for the quantitative determination of protein by radial diffusion thereof through a coating of antibody on a sheet formed of a paper-thin liquid-permeable chemically inert layer (e.g., cellulose acetate) on a resilient liquid-impermeable backing (e.g., flexible plastic). The device includes a wall and marginal framework therearound, having a slot for slidably receiving a perforate or solid plate into a fixed position above the wall. The device further includes means for slidably receiving said sheet between the wall and either of the plates in position. Blood scrum may be applied via a capillary tube through the openings in the perforated plate onto a sheet in predetermined placement. In one embodiment, the device contains identical back-to-back units as above described. In a second embodiment, one side of such back-to-back units contains parallel members mounted on one wall and cooperating therewith to form parallel slots adapted to receive a number of sheets.

8 Claims, 9 Drawing Figures PATENTED FEB 2 9 I972 SHEET 1 [IF 2 INVENTOR. III." BY Samuel T." Nerenberg 74%, 9L4 w w orneys w [I m a x fi l D f IMMUNODIFFUSION PLATE APPARATUS In a method for using the above device for the quantitation of protein, the antibody layered sheets are placed into their holders in the device, the protective plate is slid into its groove of the device to seal the coating from the air during incubation period, the protective plate is removed and replaced with the perforate plate, blood serum is applied via a capillary tube through the perforations onto the coating, the serum and antibody reacts to form precipitin rings and the area of said rings (representing the quantity of antigen in the blood serum) is quantitated.

In another embodiment of the device, the above antibody layered sheet is placed at the bottom of an open-topped container, a lid is placed on the container for sealing during incubation, the lid is replaced with a perforate template, serum is applied through the perforations, and the resulting precipitin rings are quantitated.

BACKGROUND OF THE INVENTION Immunological procedures for the quantitation of specific protein antigens have been widely used for both clinical and research analyses. Such procedures are based on the measurement of the degree of precipitation in the reaction between such protein antigens and their specifically reactive antibody. Perhaps the most widespread usage of this reaction is in quantitative single radial immunodiffusion. The first such technique, performed in agar gels, was described in Mancini et al., A Single Radial Diffusion Method for the Immunological Quantitation of Proteins," 11 Prot. Biol. Fluids 370 (1964). According to that technique, antibody was incorporated in uniform concentration in an agar gel with a thickness of about 1 mm. formed by means of plastic frames. After solidification of the gel into a plate, a series of spaced-apart wells were cut therein and filled with precise volumes of antigen solution determined by means of a calibrated micropipette. The immunodiffusion reaction between antigen and antibody was allowed to proceed in the gel in the fomi of a precipitin ring during incubation for about 14 days in an oil bath. The bath functioned to prevent drying. A photographic enlarger was used to project the thus-formed circular shaped precipitin line images onto photographic paper. These images were then cut out, weighed, and plotted on semilog paper for quantitation. The accuracy of quantitation was found to be on the order of i percent. The major drawback of the Mancini technique is that the 14-day diffusion period is impractical for clinical use since laboratory tests normally are required within a 12-24 hour period to gauge the need for immediate therapy.

In view of the pressing need for a more rapid assay procedure for the clinical determination of serum immunoglobulins, Hyland Laboratories, of Los Angeles, California, developed a product called an Immunoplate for quantitating the three common serum immunoglobulins: 'y A Globulin (IgA), y M Globulin (IgM), and 'y o Globulin (IgG). These plates embody the principles of Mancini but require only overnight incubation to yield complete ring precipitin reactions. The ring diameters are plotted on a linear portion of semilogarithmic paper with the values of the reference sera on the coordinate logarithmic axis to obtain a standard curve. Values for the unknown samples were taken from the standard curve. In contrast to the Mancini technique which employs precisely measured volumes of antigen solution in the sample wells, the Hyland technique is less accurate recommending measurement by the mere filling of wells to the gel surface. This technique is presently being used extensively.

In 1965, an analytical study of the Hyland Immunoplates was published by Fahey, .l. L., et al., in Quantitative Determination of Serum lmmunoglobulins in Antibody Agar Plates," 94 J. Immunol. 84 (1965). The probable error determined by Fahey et al. was about 1- percent compared to the 1-5 percent reported by Mancini in his more refined and prolonged procedure. ln particular Fahey, et al. calculated the standard deviations for the three immunoglobulins to be: IgA 1 70 mg./l00 ml., IgG 220 mg./l00 mL, and IgM 1 35 mg./ ml. Furthermore, experience in the clinical laboratory with Hyland Immunoplates, monitored by a quality-control procedure, indicate that the probable error is much larger than i 10 percent under actual hospital conditions, approximating 20-25 percent when a number of technologists are responsible for carrying out the procedure on a routine basis. Although fairly good straight-line relationships are obtained between precipitin ring diameters and logarithmic concentration of antigen, the slopes of the curves are very steep so that even small variations in reading precipitin ring diameters lead to significant differences in final results. A further source of error is attributable to divergence of the ring precipitin diameters from true circles. Although results could be improved somewhat by multiple readings of a single precipitin ring taken in different directions, this would prolong the procedure significantly when numerous samples are taken. In summary,

some of the major drawbacks to the use of the Hyland Immunoplates include its inaccuracy; refrigerated storage temperatures; relatively large volumes of undiluted serum sample (on the order of 0.25 ml.) is necessary to determine the level of all five immunoglobulins; and inaccuracies in the system.

In order to overcome certain of the aforementioned disadvantages, C. Vergani et al. developed a technique which was published in a paper entitled Quantitative Determination of Serum Immunoglobulins by Single Radial lmmunodiffusion on Cellulose Acetate, 4 lmmunochemistry 233 (1967). According to this technique, strips of unbacked cellulose acetate membranes are utilized as the carrier medium for specific antiserum instead of the agar gel in Immunoplates. In the Vergani method, the cellulose acetate strips are stretched between clamps to form a bridge in a moist boxlike chamber wherein diluted antiserum is sprayed onto the strips. Since the antiserum soaks though the strips, they must be suspended to avoid contamination of surfaces therebelow. Following spraying, the strips are left at room temperature in the moist chamber sealed via a lid. The test sample is then applied to the strip by means of a micropipette. In order to avoid drying, the strips are then placed into a mineral oil bath where they remain throughout the period of diffusion. After diffusion and the accompanying precipitin ring formation the strips are removed from the oil, immersed in a detergent solution to wash off traces of oil, and washed under tap water. The standard deviation for the results of this procedure was stated by Vergani et al. to be comparable to that of the aforementioned Fahey et al. study (:10 percent). The obvious advantages of this procedure as compared to agar immunodiffusion include: uniform thickness of the cellulose acetate supporting medium which yields a sharp outline of precipitin rings; significantly greater serum economy (e.g., a 4X28 cm. strip, accommodating 50 determinations, consumes only 1.2 ml. of diluted antiserum); and a 2-day procedure compared to 14 days in the original Mancini technique.

In spite of the aforementioned advantages, the Vergani technique has not achieved widespread use for a number of reasons. Perhaps the most important is that unbacked cellulose acetate membranes are quite brittle and delicate which renders them extremely difficult to handle without breaking. Furthermore, the strips lack rigidity and so must be stretched to apply the sample accurately In addition, the strips must be suspended on a bridge in a rather cumbersome apparatus to avoid contamination of surfaces therebelow since fluid will travel through the strips. Also, there is a certain loss of precision resulting from incomplete equilibration of the suspended strips and from the requirement for direct measurement by calipers rather than use of photographic enlargement prior to performing a reading. Finally, Vergani et al. instructs that to use the strips, the antigen (e.g., blood serum containing immunoglobulins) must be diluted prior to application, a time-consuming step which multiplies in a procedure designed to screen the blood serum of a large number of patients entering a hospital.

SUMMARY OF THE INVENTION AND OBJECTS This invention relates to certain holding devices for use in a single radial immunodiffusion technique on a paper-thin, liquid permeable, inert layer (e.g., cellulose acetate) on a resilient liquid-impermeable backing (e.g., flexible plastic).

In one embodiment of the invention, a holder is formed of a wall with a marginal framework on each side thereof extending outward therefrom. The framework is provided with plate slots on the lateral sides of said wall adopted to receive either a perforate template or a solid protective plate. Sheet slots are also provided internally of the plate slots for receiving an immunodiffusionsheet formed of a cellulose acetate layer on a flexible plastic backing. Multiple elongated slots in parallel,'

spaced-apart relationship may be mounted on either surface of the wall for slidably receiving a plurality of the aforementioned immunodiffusion sheets. Thus, a variety of sheet sizes may be utilized in the holder in accordance with the number of samples selected to be run on a single sheet (described hereinafter) resulting in a conservation of antiserum.

According to one procedural embodiment of the invention, antiserum is spread evenly onto the above immunodiffusion sheet which is then inserted, acetate layer up, via the sheet slots into the aforementioned holder. A protective plate is then slid into the plate slots above the sheet to form a compartment which protects the sheet from drying. The antiserum is then allowed to diffuse evenly in an equilibration stage, after which the protective plate is removed from the holder and replaced with the aforementioned template During a serum application stage, measured amounts of antigen sample (e.g., blood serum) are applied by means of micropipettes through the openings in the template. In a diffusion or incubation stage, the sheet is removed from Y the holder and placed under mineral oil for about 18 hours. The precipitin ring-containing sheet is then washed and stained and the ring 1 images enlarged onto photographic paper for viewing. During thisprocedure, the backed film is extremely easy to handle, much like photographic film.

In another embodiment of the technique and apparatus of the invention, quantitation can be performed on the above immunodiffusion sheet in a base dishlike device such as a conventional flat-bottomed Petri dish. Thus, the sheet is cut to fit snugly into the dish. The cut sheet is coated with antiserum and placed at the bottom of the base dish, a protective cover lid is placed thereover to protect the sheet from drying, and the antiserum is allowed to equilibrate. Then the lid is removed, replaced with a template lid having a series of radially placed holes, and serum is applied. Thereafter, mineral oil is poured in the dish for coverage during incubation during which the immunodifl'usion reaction is completed without drying out of the antiserum. The remainder of the steps performed on the sheet are as described above.

It is an object of the invention to provide an improved device and technique for quantitating proteins by radial diffusion through specifically reactive antibodies which overcomes the disadvantages of the prior art.

It is a further object of the present invention to provide a device of the above type which eliminates the need for the cumbersome equipment of the prior art and which conserves serum and antiserum.

It is a further objection of the invention to provide a device of the above type which decreases the time of quantitation of protein in blood serum whereby a large number of patients may be screened simultaneously.

Additional objects and features of the invention will appear from the following description in which the preferred embodiments are set forth in detail in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a top view of one embodiment of a holder according to the invention.

FIG. 2 is a cross-sectional view of the device in FIG. 2 taken along the line 22.

FIG. 3 is a cross-sectional view of the device in FIG. 2 taken along line 33.

FIG. 4 is an enlarged portion of the device in FIG. 3 taken in the area 4-4. FIG. 5 is a top sectional view of FIG. 4 taken along the line 55.

FIG. 6 is a top view of another embodiment of a holder according to the invention.

FIG. 7 is a cross-sectional view of the device in FIG. 6 taken along the line 7--7.

FIG. 8 is a top view of a dishlike container embodiment ac- I cording to the invention.

FIG. 9 is a cross-sectional view of the device in FIG. 8 taken along the line 99.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Referring to the drawings, rectangular immunodifi'usion sheet 11 includes a paper-thin diffusion layer 12 integral with a resilient backing 13. Layer 12 is formed of any coatable material'that is chemically inert and readily permeable to liquid, such as cellulose acetate. Backing 13 is formed of any suitable material that is both flexible and impermeable to liquid such as plastic. I have found that a particular effective material for use as sheet 11 is a cellulose acetate layer on a backing of polyethylene terephthalate plastic (trademark Mylar). This integral sheet is sold under the name Titan IH-X-IOO by Helena Laboratories, Allen Park, Michigan.

Referring to FIGS. l-4, holder 14 includes facing upper and which cooperates with wall 16 to form an upper container 20 and a lower portion 21 cooperating with wall 17 to form a lower container 22. Since containers 20 and 21 have identical construction and function, the description herein of container 20 applies equally to container 21 with like part numbers assigned to each container. Holding means for sheet 11 are provided at opposing edges of wall 17 and comprising in this instance bent portions at

lateral edges

23 and 24 forming

lateral grooves

26 and 27 respectively. The forward edge of wall 17 is also bent over forming a similar groove 29.

Grooves

26 and 27 serve to slidably receive sheet 11 at the rear edge 30 of wall 16 while groove 29 functions to stop the sliding sheet in a reproducible fixed position relative to holder 14. A recess 31 is provided at edge 30 to facilitate removal of sheet 11 from holder 14.

Framework 18 includes an integral frame member 32 with inwardly extending ridges 33 on all four sides which separates wall 16 from wall 17. Member 32 includes lateral sections 34 and 36, front section 37 and rear section 38. The inner surfaces of sections 34 and 36 contain

slots

39 and 40 respectively and lips 35 which overlap and, thus, retain wall 16 in a fixed position. A flat coverplate 41 is attached to section 38, suitably by bolting to upwardly projecting ridges on the lateral sides of section 28 to form an opening 45 therebetween communicating with

slots

39 and 40. Hinged member 42 and the outer edge of section are suitably interconnected by means of a flexible fabriclike tape 43 with adhesive backing forming the pivotal line of the hinge. A slot 44 is provided in member 42 as a stop for a plate sliding through

slots

39 and 40 and opening 45. Such a plate in a stopped position would form an essentially sealed container.

Holder 14 may be formed of any rigid material such as wood, plastic or metal. It will be apparent that the above holder construction may be varied without altering the function thereof. For example, the entire holder could be formed of one integral structure as of a plastic material.

Template 46 is of a suitable rectangular shape and size for sliding through opening 45 and along

slots

39 and 40 and into slot 44. Template 46 is provided with spaced-apart fluid application holes 47 through which serum may be fed in a predetermined placement onto layer 12. By way of example, 42 holes, 1 mm. in diameter, may be drilled through the template. A handle 48 may be attached to template 46 to facilitate gripping on sliding into and out of holder 14. Template may be formed of any rigid or semirigid material, such as plastic. For visual monitoring of applied sample, as described hereinafter, the material is preferably transparent. It is understood that although template 46 is illustrated in position in upper container 20, that interchangeably it could be placed into lower container 22 for applying serum to a sheet 11 placed therein.

Protective plate 49 is of approximately the same size as template 46 and may be provided with a similar handle 50 The plate material may be rigid or semirigid. Plate 49 slides into the same slots as template 46 to cooperate with either

container

20 or 22 to form chambers shielded from substantial contact with surrounding air. Plate 48 is illustrated in FIGS. 1-4 to be in a reserve nonfunctional position. Plate 49 would be functional if a second sheet 1 1 were placed in container 22. Alternatively, if template 46 is slid out and replaced with plate 49, container 20 would be sealed thereby.

Referring to FIGS. 6 and 7, an alternative holder 14a is illustrated. Only the components in holder 14a that vary from those in holder 14 will be described in detail or assigned different part numbers. Lower container 22a has the same structure and function as that of container 22. On the other hand, upper container 20a functions to slidably receive three different size sheets 11a of the same material as sheet 11 but transverse to sheet 11. To accomplish this, three

elongated members

51, 52 and 53 having overhanging portions on both sides thereof are mounted in place of wall 16. The overhanging portions form slots 54 which, along with

grooves

26 and 27, form channels for three sheets 11a. In contrast to stationary section 36, section 36a is hinged in the same manner as member 42 to provide ready access to sheets 11a. Since these sheets 11a are received from the side, there is no reason to hinge member 42a and so it is stationary (in contrast to hinged member 42). The only functional distinction between

holders

14 and 14a is the ability to utilize variable sheet sizes in the latter, thus conserving antiserum for runs involving a small number of samples.

The foregoing description relates to a two-chambered holder with a convenient slot for reserve storage of the template or protective plate when not in use. It is to be understood that a single-chambered device (e.g., formed of the upper or lower half of the

holder

14 or 14a) when used for quantitating proteins on a sheet similar to sheet 11 is considered to be within the scope of my invention.

In one embodiment of the technique of the present invention performed either on holder 14 or 140, large sheets of cellulose acetate backed with Mylar plastic (commercially available as Titan III-X-lOO from Helena Laboratories, Allen Park, Michigan) were cut into rectangular segments to fit slots 20 of holder 11. The upper left-hand corner may be notched or cut off for orientation purposes.

In a saturation step, monospecific antibody is spread across the cellulose acetate by suitable means such as a plastic disposable razor with the blade removable. The antibody is reactive with a specific protein to produce a precipitin. For purposes of this description, commercial antisera monospecifically reactive with the three immunoglobulins, lgG, IgA, and IgM of blood serum are used as the antibody for the quantitative determination of the same immunoglobulin. The antisera are preferably diluted with saline solution prior to application to the cellulose acetate. The optimum dilution (i.e., the one producing the heaviest precipitin rings) must be determined for each commercial antiserum batch by trial and error since there are certain variations from batch to batch. By way of example, it has been found that using antisera obtained from l-Iyland Laboratories, Los Angeles, California, a dilution with a 0.9 percent saline solution is effective in the following ratios of antiserum to diluent; lgA 1:10, lgG 1:20, and IgM 1:10. The diluted antiserum is then spread uniformly onto an immunodiffusion sheet as with a plastic razor with its blade removed. As a quantitative example, 1.5 ml. would be suffcient quantity for a 4X5 inch sheet.

In an equilibration step, the coated sheet is slid into

grooves

26 and 27 of holder 14 by pivoting member 42 out of the way. Protective plate is then slid into its guiding slots thereover into the slot of member 42 in a seated position. Thus, antiserum is prevented from drying during equilibration or uniform saturation throughout the cellulose acetate layer. Equilibration takes approximately 10-15 minutes at room temperature or about 5-10 minutes at 37 C.

The reason that equilibration may be performed in efficient holder 14 is that backing 13 of sheet 11 is impermeable to liquid and so there is no soaking therethrough onto wall 16. This is a marked contrast to the unbacked cellulose acetate membrane of Vergani which necessitates the use of a cumbersome apparatus and technique for quantitation.

Following equilibration, the protective plate is removed from the holder and the surfaces of layer 12 may be inspected by an oblique light. If an irregular layer of superficial fluid is seen on the surface, that is allowed to evaporate by leaving the sheet exposed to air for approximately 3 to 5 minutes. If the layer of fluid is unusually heavy, it may be spread more uniformly as with the plastic razor and allowed to evaporate. Drying of focal areas should be avoided to prevent distortion of the ultimate precipitin rings. Excess drying is recognizable by the appearance of occasional opaque, white spots on a dull grey background. If drying occurs, the oblique spots may be moistened with a drop of antiserum and uniformly spread with the plastic razor.

In preparation for a serum application step, a transparent template 46 is inserted into holder 14 via

grooves

26 and 27. The template prevents rapid drying during application of the blood serum sample. Concentrated reference samples of each immunoglobulin are diluted for application to form three precipitins for construction of standard curves in the anticipated range of sample values as explained hereinafter.

Referring to FIG. 5, in decreasing order of concentration points 56 a-c represent applied reference sera and the corresponding ultimately stained precipitin rings are represented at 57a-c. In like manner, points 58a-c represent applied unknown sera and precipitin rings 59a-c represent corresponding stained precipitin rings. Sera, both reference and unknown samples, may be labeled with bromphenol blue dye by mixing a few grains of dye powder with each sample for easier viewing of the precipitin.

Sample is applied through the template holes suitably by means of a tube which accurately measures small quantities of fluid. A preferred applicator is a microcapillary tube holder fitted with disposable 0.5 p.l self-filling capillary tube such as the Drummond type sold by Helena Laboratories, Allen Park, Michigan. For the most accurate control of samples, it has been found that mouth pressure exerted through narrow gauge tubing is more effective in forcing the sample from the capillary tubes than is the use of a compressible rubber bulb as supplied with the Drummond capillary tube holder. Sample in the microcapillary tubes is applied through template holes directly onto the cellulose acetate surfaces. During application of the dye-colored samples, total emptying of each capillary tube was determined by monitoring or viewing through the transparent plastic template. Each sample forms a concentric blue-colored spot on the surface of the cellulose acetate sheet.

For incubation, the template is slid out of the holder, member 42 is pivoted outwardly, and sheet 11 is removed from the holder and placed under the mineral oil. The sheet is then incubated overnight (e.g., about 18 hours) suitably at the following temperatures: about 25 C. for lgA, about 410 C. for lgG, and about 37 C. for IgM. The purpose of the mineral oil is to shield the sheet from drying out during incubation. It is noted that if holder 14 could be completely air sealed with the protective plate, incubation could be performed directly in the holder.

Following incubation, the sheet is removed from the mineral oil and suspended as from a clamp for a few minutes to allow residual oil to drip free of the sheet. In order to remove any remaining oil, the sheet may then be washed in a detergent-saline solution for approximately minutes. In a second wash the sheet may be retained in a 0.9 percent saline solution for approximately 30 minutes to remove unreacted proteins. The saline solution can be removed from these sheets by rinsing in tap water. To render the precipitin rings clearly visible, they may be stained as with Thiazine Red in a 1 percent acetic acid solution for approximately 30 minutes followed by rinsing as with 1 percent acetic acid solution. After allowing the sheets to dry, the stained precipitin rings represented at 57 and 59 are readily visible.

To quantitate the precipitin, it is preferred to project the dried unstained cellulose acetate sheets through a standard photographic enlarger for final viewing on photographic paper. In one system, the images projected onto sensitized paper such as Ectamatic SC paper produced by Eastman Kodak Co. and then passed through an Ectamatic Processor which provides enlarged images on a fixed photographic paper in a semidried state in less thana minute. The photographic paper may then be removed from the dark room for analysis. Diameters of each precipitin ring may be compared in several directions with a compass to confirm the formation of true circles. If so, the magnitude of the ring diameter may be measured. 0n those infrequent occasions when the ring precipitin do not form true circles, the area enclosed by the rings may be cut out along with the reference areas for weighing. A standard curve may be prepared by plotting the ring diameters and/or the weights of the paper areas circumscribed by the precipitin rings 57 of the reference specimens on the linear axis of two-cycle semilog graph paper against their known protein values plotted on the logarithmic axis. Unknown samples may then be read directly from the standard curve.

A statistical analysis of commercial standard reference sera for the immunoglobulins lgA, IgG, and IgM, was performed by applying blood samples to antisera-coated, backed cellulose acetate sheets according to the aforementioned technique and using reference sera as above-described for points of the standard curve. The results of this analysis are summarized in Table I.

Referring to Table I, it is apparent from the above standard deviations that a significant improvement in precision of quantitation is achieved by the present device and technique in comparison to that of the aforementioned agar gel technique as studied by Fahey et al. with standard deviations of IgA i 70 mg./l00 ml., lgG-220 mg./l00 ml., and IgM35 mg./l00 ml. The present invention has the same superiority of precision over the Vergani unbacked cellulose acetate technique since the latter is comparable in accuracy to that of Fahey.

An important factor in the improved accuracy of the technique of the present invention is the ability of the plasticbacked cellulose acetate sheet to be handled as a regular photographicfilm. Thus, projection and enlargement of the stained precipitin rings onto the photographic paper enables the enlarged photographically developed precipitin images to be more accurately measured than precipitin rings viewed through a microscope. It is noted that even a slight error in direct measurement of a ring diameter would introduce a relatively large error in the final result due to its logarithmic value.

Furthermore, recording of precipitin rings on photographic paper may be performed almost instantaneously with great precision compared to the tedious and relatively inaccurate technique of tracing the images in a darkroom recommended by Mancini et al. In addition, the time utilized for processing the photographic paper (less than a minute) is exceedingly more rapid than the tracing of individual images of many samples. Finally, increased accuracy and speed of an analysis are provided by the use of the compass to detect distorted".

precipitin rings. It is only those infrequently occuring distorted rings which require the time consuming process of weighing the closed areas for quantitation.

Referring to FIGS. 8 and 9, another embodiment is illustrated of a device which may utilize the aforementioned backed cellulose acetate sheet in a technique analogous to that described above. This dishlike device includes a container dish 60, an overlaying replaceable perforate template lid 61, and a solid protective lid (not shown) interchangeable with template 61. Dish 60 is suitably a flat-bottomed, open-topped rigid or semirigid dish such as disposable plastic, commercially available Petri dish. The size of dish 60 may vary in accordance with the number of samples to be applied in a single run. To conserve antiserum, an mm. diameter dish 60 may be employed along with template lid 61, with 16 holes to accommodate a like number of samples as described hereinafter. Template 61 (and the protective lid) may be of a flat-bottomed dishlike shape similar to dish 60 but with a slightly larger diameter so that an essentially snug fit is created with lid 61 overlying dish 60.

A circular disc 63 of the plastic backed, cellulose acetate material described above may be formed to conform to the inner diameter of dish 60. A spot may be applied at one point on the edge of the dish to orient the application of samples. Discs 63 may be stored in individual dishes 60 ready for use.

Application of antiserum may be performed as described in the aforementioned technique. The equilibration may be performed by placing disc 63 into dish 60 and covering the dish with protective lid. The precautions to prevent drying are described above.

Referring to FIG. 8, template lid 61 may be drilled with a series of holes 64 in a spaced-apart relationship. This spacing is planned so that when sample is applied through holes 64 onto disc 63, there is sufficient clearance for the spreading of each precipitin ring without contact with adjacent rings. Although the dishlike device is described with reference to a circular walled dish, it is to be understood that other shapes, such as rectangular, may be employed to the same effect.

In the incubation stage, template lid 61 may be removed and mineral oil directly spread over disc 63 in dish 60. The timing of incubation is as described above. The remainder of the quantitation procedure including washing, staining, and projecting the results onto photographic paper, may also be performed as described above.

Quantitating by means of

holders

14 or 14a has certain advantages over quantitation with dish 60. For example, specimen spacing, to prevent overlapping of precipitin rings, is easier to plan on a rectangular template and the corrolation of precipitin rings with the proper specimens is also simpler with a rectangular shape. Furthermore, the disc shaped sheet is somewhat more difficult to handle during a procedure, e.g., during removal from dish 60.

It will beapparent from the foregoing that I have provided a number of embodiments of a holding device with which accurate and simple immunodiffusion quantitations may be performed on a flexible plastic sheet covered with a cellulose acetate film. Quantitation according to this invention is exceedingly accurate, easy and rapid to perform, and economical of serum and antiserum. Although the invention is described with respect to specifically constructed holders, it is understood that equivalent holders are within the present scope.

We claim:

1. In a method for the quantitative determination of protein, the steps of layering antibody specific to said protein onto a paper-thin cellulose acetate coating on a liquid impermeable resilient backing, placing said backed layered coating into a frame, sliding a protective plate into a groove in said frame, sealing said coating from the air, incubating said sealed coating for a sufficient time to cause the antiserum to be uniformly absorbed into the coating, removing said protective plate from said groove, sliding into said groove a perforate plate having a plurality of openings in spaced-apart relation, applying an unknown protein antigen through said template to said coating, reacting said antibody and antigen on said coating to form precipitin rings, and quantitating the area of said rings.

2. A device for the quantitative determination of protein by radial diffusion comprising open-topped containermeans, a sheet disposed in said container means, said sheet being formed of a cellulose acetate layer on a resilient impermeable backing, said layer being saturated with antibody specifically reactive with said protein, a perforate template adapted to fit over said container means, said template having holes in spaced-apart relation for applying protein sample to said layer in a predetermined pattern.

3. In a device for the quantitative determination of protein by radial diffusion thereof through a coating of antibody specifically reactive with the protein, a sheet formed of a paper-thin liquid-permeable chemically inert layer on a resilient liquid-impermeable backing, wall means, means forming a marginal framework on each edge of said wall means extending outwardly therefrom, said framework means having slot means adapted to slidably receive a plate, holding means disposed at lateral edges of said wall means at a level between said wall means and said slot means, said sheet being inserted in said holding means.

4. A device as in claim 3 wherein said coating is formed of a cellulose acetate and said backing is comprised of a pliable plastic.

5. A device as in claim 3 together with a perforate plate having a plurality of openings in spaced apart relationship and slidably received in said slot means whereby liquid sample may be applied via a tube through said perforations onto said sheet in predetermined placement.

6. A device as in claim 5 wherein said sheet is disposed between said wall means and said perforate plate.

7. A device as in claim 3 including a solid protective plate slideably received in said slot means, said marginal framework means, wall means and protective plate cooperating to form an air-sealed compartment for said sheet.

8. A device as in claim 7 wherein said sheet is disposed between said wall means and said protective plate within said air-sealed compartment.

Claims

2. A device for the quantitative determination of protein by radial diffusion comprising open-topped container means, a sheet disposed in said container means, said sheet being formed of a cellulose acetate layer on a resilient impermeable backing, said layer being saturated with antibody specifically reactive with said protein, a perforate template adapted to fit over said container means, said template having holes in spaced-apart relation for applying protein sample to said layer in a predetermined pattern.
3. In a device for the quantitative determination of protein by radial diffusion thereof through a coating of antibody specifically reactive with the protein, a sheet formed of a paper-thin liquid-permeable chemically inert layer on a resilient liquid-impermeable backing, wall means, means forming a marginal framework on each edge of said wall means extending outwardly therefrom, said framework means having slot means adapted to slidably receive a plate, holding means disposed at lateral edges of said wall means at a level between said wall means and said slot means, said sheet being inserted in said holding means.
4. A device as in claim 3 wherein said coating is formed of a cellulose acetate and said backing is comprised of a pliable plastic.
5. A device as in claim 3 together with a perforate plate having a plurality of openings in spaced apart relationship and slidably received in said slot means whereby liquid sample may be applied via a tube through said perforations onto said sheet in predetermined placement.
6. A device as in claim 5 wherein said sheet is disposed between said wall means and said perforate plate.
7. A device as in claim 3 including a solid protective plate slideably received in said slot means, said marginal framework means, wall means and protective plate cooperating to form an air-sealed compartment for said sheet.
8. A device as in claim 7 wherein said sheet is disposed between said wall means and said protective plate within said air-sealed compartment.