WO1998005964A1

WO1998005964A1 - A method for preserving and enhancing visualization of immunodiffusion reactions

Info

Publication number: WO1998005964A1
Application number: PCT/US1996/012690
Authority: WO
Inventors: Curtis Sharon; Lisa Nakakihara
Original assignee: Qed Bioscience, Inc.
Priority date: 1996-08-02
Filing date: 1996-08-02
Publication date: 1998-02-12
Also published as: AU6842596A

Abstract

A method for preserving a visualization of an immunodiffusion reaction comprising (i) providing an immunodiffusion reaction in a hydrated semi-solid gel (6), (ii) placing a wetted water absorbent web, e.g. wetted blotting paper (17), on the surface of the reacted immunodiffusion gel (6), and (iii) drying the gel at an elevated temperature such that the gel dries evenly, is described.

Description

A METHOD FOR PRESERVING AND ENHANCING VISUALIZATION OF IMMUNODIFFUSION REACTIONS

FIELD OF THE INVENTION This invention relates to immunodiffusion technology which yields a permanent visualization of the immunodiffusion reaction.

BACKGROUND OF THE INVENTION Immunodiffusion refers to an immunological reaction in which one of the reactants diffuses into a supporting gel containing another reactant. The objective is to visualize and immobilize an jLn vitro reaction between, e.g., a precipitating antibody and a soluble antigen.

Immunodiffusion is carried out either as single radial immunodiffusion (SRI) or as double radial diffusion (DRI) . In SRI, only one reactant, usually the antibody, is included in the gel, i.e., the antigen, is contained in a well, cut into gel from which it diffuses radially. Alternatively, the antibody may be contained in the well from which it diffuses into the gel containing the antigen. A precipitate ring is formed around the well by contact of the diffusing reactant with the reactant contained in the gel. In DRI, each reactant diffuses from a different well into a neutral gel. The assay results of prior art SRI and DRI techniques are generally indicated by Figure l (prior art) .

The terms immunoelectrophoresis or electroimmunoprecipitation refer to methods in which immunoprecipitation is obtained after electrophoretic separation of proteins in an electric field. When electrophoresis of an antigen is performed in a gel containing the corresponding antibody, a precipitate is obtained with a rocketlike appearance, the height of which is proportional to the amount of antigen in the gel.

The antecedent of SRI is the observation of Petrie in 1932 that a ring of precipitate formed around a colony of an exotoxin-producing bacterium growing an agar which contained the specific antitoxin. In 1949, Ouchterlony and Elek independently adapted this observation to produce a double immunodiffusion technique. Wells were produced in an agar plate, using a template or cutter. Individual wells received either antigen or antiserum. As the two reactants diffused into the gel, a line of precipitate formed within the zone of overlap. This technique, although a useful qualitative technique for demonstrating and comparing antigens and antibodies, is inadequate for quantitative determinations.

Simultaneously, Oudin was transferring Petrie's observations to a tube, showing that rings of precipitate would form in a gel containing antiserum when it was overlaid with a solution of the corresponding antigen. This was single diffusion, as for the purpose of the method only the antigen diffusion was of consequence. It was a dynamic system, the precipitate rings migrating linearly down the gel with time; thus, single linear immunodiffusion. As the rate of the ring migration was determined by antigen concentration, it could be used for quantification of antigens against known standards.

In 1957, Feinberg combined Oudin's tube single diffusion with the Ouchterlony plate to produce a single radial immunodiffusion system. As in Oudin's technique, serum was incorporated in a molten gel, but as with Ouchterlony, the gel was poured into a plate and allowed to set. With a specially designed gel cutter, uniform wells were cut in the antiserum-gel and filled with antigen solution, using standard concentrations of the antigen for comparison with test antigens. Diffusion of antigen and formation of the precipitate rings were allowed to go to completion. Assay was based on a limiting dilution endpoint, i.e., the lowest concentration of antigen showing a ring of precipitate.

In 1963, To asi and Zigelbaum modified the Feinberg technique by eliminating the need to achieve an endpoint. The diameters of the precipitate rings were measured by placing a transparent ruler beneath the plate and plotting the diameters against antigen concentrations.

In 1965, Mancini and coworkers suggested that merely recording ring diameters could give erroneous results. They advocated projection of the rings onto strong paper, outlining the projected rings with pencil, cutting the paper discs outlined and weighing them. This so-called "Mancini technique" proved to be cumbersome, as subsequently acknowledged by Heremans. Because of this, in practice it has largely been abandoned, whereas the Tomasi and Zigelbaum modification of the Feinberg technique has generally been adopted as the method of choice and now forms the basis of commercially supplied immunodiffusion plates for the immunoassay of proteins in plasma and body fluids.

In SRI, when antigen diffuses radially into a gel containing antiserum, immune complexes are formed and a precipitate begins to deposit as a ring at the interface between the two reactants. The higher the concentration of antigen in the well, the more rapidly precipitate takes place, i.e., rings appear sequentially in direct relationship to antigen concentration. Ongoing diffusion of antigen from the well builds up the precipitate at the outer edge of the ring, where the antigen encounters additional antibody. Therefore, the system is initially in a dynamic state in which the rings increase in size with time. A static stage is reached when all the antigen has diffused into the gel and precipitate is complete. At this stage, the rings are most distinct and remain constant in size, so readings can conveniently be taken at any time thereafter. At the static stage, the area circumscribed by the ring is proportional to the concentration of the antigen in the well. Since area is proportional to the square of the radius (and, hence, to the square of the diameter, D) , then the square of the diameter (D²) of the ring is proportional to the concentration of antigen. Such being the case, plotting D² against concentration on arithmetic graph paper will produce a straight line. Alternatively, to avoid the requirement of calculating D², se ilog graph paper may be used, plotting D itself on the linear sale against antigen concentration on the log scale.

Immunodiffusion reactions are generally carried out in an agarose gel. For example, in SRI techniques, the molten gel is cooled to -50 °C to avoid heat denaturation of the antiserum, which is warmed to a similar temperature and added to the molten gel in desired concentration, the two are mixed gently, but thoroughly, and poured into a dish, or onto a glass plate (e.g., a microscope slide) , supported on a perfectly horizontal surface to ensure uniform gel thickness. When the gel has set, wells are cut using a sharp cutting instrument designed for the purpose. Alteratively, a plastic mask with a pattern of holes can be floated on the molten gel, which is then allowed to set to form a fir seal between mask and gel. In this case, wells need not be cut, the antigen solution being applied to the exposed areas of gel .

The plates are incubated in a humid atmosphere, preferably until ring formation is complete.

Measurement of the ring diameters may be done with a transparent ruler or, more conveniently, with the wedge-shaped scale introduced by McSwiney. Mechanical aids for reading ring diameters are also available commercially. These include a modified jeweller's eye-piece with an in-built graduated scale and electronic plate-readers.

These prior art techniques require that the results of the assay be determined while the gel is still semi-solid and visualized by incident light. Efficiency is compromised by a frequent need to stain the precipitate rings. Because gels cannot be permanently stored in a hydrated or semi-solid state, the real data reflecting the immunodiffusion reactions is lost when the gel dehydrates or is destroyed. Accordingly, a need exists for technology which eliminates staining of the precipitate rings and allows the results of immunodiffusion reactions to be stored permanently. SUMMARY OF THE INVENTION

A focal aspect of this invention is the discovery of technology effective to provide a dried semi-solid gel including a visualized immunodiffusion reaction in which the precipitate rings are preserved. Preferably, the dried gel is adherent to a supporting substrate, such as a glass or synthetic resin sheet.

Another aspect of the invention is the discovery that staining of immunoassay precipitation rings may be eliminated by incorporation of a dye into the sample solution.

DESCRIPTION OF THE FIGURES Figure 1 is a general depiction of prior art single and double diffusion assays.

Figure 2 illustrates the production of a solidified gel layer on a support with holes provided therein.

Figure 3 illustrates the production of a diluted sample including a buffer and a dye.

Figure 4 represents the solidified gel having holes therein and further having the diluted sample solution pipetted into the holes followed by overnight incubation. Figure 5 illustrates the positioning of the web or blotting paper on the surface of the gel including the visualized reaction, the heating of the complex so formed, and the removal of the web or blotting paper leaving the preserved assay. DETAILED DESCRIPTION OF THE INVENTION

Pursuant to this invention, a support surface is provided with a layer of any gel having an aqueous continuous phase which allows free diffusion of molecules preferably in three dimensions. Agarose gel is preferred. Polyacrylamide gels are also useful in the practice of the invention.

Any solid support surface, including glass, to which the gel binds or adheres may be utilized. Sheets useful as support surfaces, preferably semi- rigid or flexible, may be of any desired thickness, preferably to mm. , and may be formed from any normally solid synthetic resin. Useful synthetic resins include polyolefins, e.g., polyethylene and polypropylene, polyvinyl resins. e.g., polymethylmethacrylate and polystyrene, and polycarbonates.

Synthetic resin support sheets coated with a thin layer of the gel used in the invention are particularly preferred. An example of such a support sheet is the Gel Bond^® product available from FMC Bioproducts, Inc., 5 Maple Street, Rockland, Maine. Gel Bond^® is a flexible synthetic resin sheet coated with a thin layer of dried agarose.

The gel may contain an antibody corresponding to an antigen present in a sample to be assayed.

Antigen containing sample, preferably containing a dye, e.g., Coomassie blue, which does not interfere with the immunoprecipitation reaction, and diluted with aqueous buffer to provide a pH of from about 6.8 to 7.4 is placed in a hole in the gel. A preferred buffer is IM Glycine 20 mM Tris 0.1% sodium azide. Other buffers which may be used will be known to the art. The antigen diffuses from the hole or holes into the antibody containing gel where it undergoes an immunoprecipitation reaction to provide Coomassie blue dyed precipitate rings around the hole in the gel from which it diffuses.

A sheet of absorbent material, e.g., blotting paper, is placed in contact with the gel surface, and wetted, preferably with distilled water. This procedure provides a complex comprising the gel and the sheet of absorbent material which facilitates capillary action and which facilitates even drying of the gel and tends to avoid deforming the precipitate rings. The complex is placed in or on a warming device to dry the gel. As the temperature of the complex is elevated, water evaporates from the surface of the absorbent material which concurrently absorbs water from the gel . The dried gel, preferably on a support surface, is a permanent record of the precipitate rings formed in the immunodiffusion reaction.

The invention is now more fully explained by reference to Figures 1 to 5.

Referring to Figure 2, a Gel Bond^® Sheet 1 comprising a flexible synthetic resin substrate having a surface area 2 coated with a thin layer of dried agarose which provides a hydrophilic surface to which liquid or molten agarose binds is selected to support an agarose gel layer. The Gel Bond^® Sheet 1 is warmed by lamp 3 to a temperature of 40 to 55°C. Liquid agarose 4 containing an antibody is poured from container 5 onto the adhesive area 2 of the Gel Bond^® Sheet 1 in an amount sufficient to provide an antibody containing agarose gel layer 6 approximately 3 mm in thickness. Reagent holes 7 are provided in the gel layer 6.

Figure 3 illustrates a container 8, such as a test tube, containing a sample 9 which may include an antigen to be assayed. A second container 10 containing an aqueous solution 11 buffered to a pH of about 7.4 of a dye, e.g., Coomassie blue, is separately provided. Appropriate buffers include Tris, Hepes, PBS, etc. The sample 9 and buffered dye solution 11 are combined in container 12 to provide a diluted and buffered sample 13 containing the antigen and the dye.

Figure 4 depicts the solidified antibody containing agarose gel layer 6 supported by the Gel Bond^® Sheet 1 and provided with reagent holes 7. Diluted and buffered antigen sample 13 containing the dye is transferred by pipette 14 from container 15 to some, but not all, of the reagent holes 7 in the solidified antibody containing gel layer 6. The gel is incubated at a temperature of about 20 to 24 °C for about 24 to 48 hours to permit diffusion of the diluted antigen containing sample into the antibody containing gel layer 6. The assay is visualized by precipitate rings 16 which form where the antigen of the diluted sample reacts with the antibody in the solidified gel. The rings are visualized by the dye.

Figure 5 shows the application of blotting paper 17 to a moist surface of the gel 6 which includes the visualized precipitate rings 16. The gel is heated and dried on a warmer 18. The dried gel including the visualized precipitate rings is removed from the warmer and may be retained as a permanent record.

Prior art immunodiffusion assays do not yield convenient and permanent records of the original data. Pursuant to this invention, the visualized assay per se is stored and constitutes the truest raw data available. This is particularly advantageous in those fields that require comparison of raw data from a plurality of assays.

Claims

1. A method for preserving a visualization of an immunodiffusion reaction which comprises:

(i) providing a hydrated semi-solid gel in which an immunodiffusion reaction is visualized,

(ii) placing a wetted water absorbent web on the surface of said semi-solid gel in which said immunodiffusion reaction is visualized to provide a complex comprising said semi-solid gel and said absorbent web; and

(iii) subjecting said complex to an elevated temperature to dry said semi-solid gel in which said immunodiffusion reaction is visualized, wherein, as the temperature of said complex is elevated, said gel dries evenly to provide a dried gel sheet in which said visualization of said immunodiffusion reaction is preserved.

2. The method of claim 1 in which said gel is an agarose gel and said absorbent web is blotting paper.

3. The method of claim 1 or claim 2 in which said visualized immunodiffusion reaction occurs between an antigen contained in a sample and an antibody contained in said gel.

4. The method of claim 1 or claim 2 in which said visualized immunodiffusion reaction occurs between an antibody contained in a sample and an antigen contained in said gel.

5. A method of claim 1 or claim 2 in which said immunodiffusion reaction occurs between an antigen containing sample solution including a dye and an antibody contained in said semi-solid gel, wherein said visualization of said immunodiffusion reaction is colored by said dye.

6. A method of claim 1 or claim 2 in which said immunodiffusion reaction occurs between an antibody containing sample solution including a dye and an antigen contained in said semi-solid gel, wherein said visualization of said immunodiffusion reaction is colored by said dye.

7. A method for providing a preservable record of an immunodiffusion reaction, said method comprising: (i) providing an immunodiffusion reaction visualized in a hydrated semi-solid gel sheet on a synthetic resin substrate;

(ii) placing a wetted water absorbent web on the surface of said hydrated semi-solid gel sheet in which said immunodiffusion reaction is visualized to provide a complex comprising said semi-solid gel sheet and said water absorbent web ; and

(iii) subjecting said complex to an elevated temperature to dry said semi-solid sheet in which said immunodiffusion reaction is visualized, wherein, as the temperature of said complex is elevated, said dries evenly to provide on said substrate a dried gel sheet in which said visualization record of said immunodiffusion reaction is preserved.

8. The claim 7 method in which said immunodiffusion reaction is a single radial immunodiffusion reaction, a double immunodiffusion reaction, or an electrodiffusion reaction.

9. The claim 7 or claim 8 method in which said semi-solid gel sheet is an agarose gel sheet and said absorbent web is blotting paper.

10. The claim 7 or claim 8 method in which said synthetic resin substrate is a flexible or semi-rigid sheet having a thickness of 1 to 5 mm formed from a polyolefin, a vinyl polymer, or a polycarbonate.

11. The claim 7 or claim 8 method in which said synthetic resin substrate is polyethylene, polypropylene, polyvinylchloride, poly ethylmethacrylate, or polycarbonate.

12. A method for providing a preservable record of a visualization of an immunodiffusion reaction which comprises

(i) providing a support surface; (ii) providing a layer of a hydrated semi-solid gel containing an antibody on said support surface;

(iii) providing at least one hole in said layer of a semi-solid gel on said support surface ; (iv) preparing a buffered liquid sample containing a dye, said sample comprising an antigen which reacts with said antibody in said semi-solid gel to form a precipitate colored by said dye; (v) placing an aliquot of said buffered liquid sample in said at least one hole in said semi-solid gel;

(vi) permitting said antigen and said dye in said sample to diffuse from said at least one hole into said semi-solid gel containing said antibody to produce a precipitate ring colored by said dye which is formed around said at least one hole in said semi-solid gel, said ring indicating reaction of said antigen with said antibody; and (vii) placing a wetted absorbent web on a surface of said semi-solid gel from which said visualized immunodiffusion reaction is apparent to provide complex comprising said semi-solid gel and said absorbent web.

13. A method for providing a preservable record of a visualization of an immunodiffusion reaction which comprises

(iii) providing at least one hole in said layer of a semi-solid gel on said support surface;

(iv) preparing a buffered liquid sample containing a dye, said sample comprising an antibody which reacts with said antigen in said semi-solid gel to form a precipitate colored by said dye;

(v) placing an aliquot of said buffered liquid sample in said at least one hole in said semi-solid gel;

(vi) permitting said antibody and said dye in said sample to diffuse from said at least one hole into said semi-solid gel containing said antigen to produce a precipitate ring colored by said dye which is formed around said at least one hole in said semi-solid gel, said ring indicating reaction of said antibody with said antigen; and

(vii) placing a wetted absorbent web on a surface of said semi-solid gel from which said visualized immunodiffusion reaction is apparent to provide complex comprising said semi-solid gel and said absorbent web.

14. The method of claim 12 in which said semi- solid gel is an agar gel and said absorbent web is blotting paper.

15. The method of claim 12 or claim 14 in which said absorbent web is blotting paper.

16. The method of claim 12 or claim 14 in which said support surface provided in step (ii) is formed from a synthetic resin.

17. The method of claim 12 or claim 14 in which said support surface is a flexible synthetic resin sheet.

18. A preservable record of an immunodiffusion reaction, said preservable record comprising:

(i) a dry agar sheet comprising a substantially undistorted visualization of an immunodiffusion reaction; and

(ii) a polymeric substrate for said dry agar sheet.

19. The preservable record of an immunodiffusion reaction of claim 18, wherein said immunodiffusion reaction is a single radial immunodiffusion reaction, a double immunodiffusion reaction, or an electrodiffusion reaction.

20. The claim 18 or claim 19 preservable record of preservable immunodiffusion reaction in which said polymeric substrate comprises a flexible synthetic resin substrate having a hydrophilic surface coat to which said dry agar sheet adheres.

21. A dry agar sheet bearing a visualized immunodiffusion reaction.

22. A visualization of immunodiffusion reaction in a sheet formed by drying an agarose gel including a visualization of an immunodiffusion reaction.

23. A substantially undistorted visualization of an immunodiffusion reaction in a dry agar sheet.