GB2193315A

GB2193315A - High sensitivity detection of antibodies indicative of AIDS exposure

Info

Publication number: GB2193315A
Application number: GB08716051A
Authority: GB
Inventors: Ronald Charles Fitzgerald; Iii George B Lamotte
Original assignee: Bio Rad Laboratories Inc
Current assignee: Bio Rad Laboratories Inc
Priority date: 1986-07-08
Filing date: 1987-07-08
Publication date: 1988-02-03
Also published as: FR2601456A1; JPS63145962A; GB8716051D0; DE3722271A1

Description

SPECIFICATION

BACKGROUND OF THE INVENTION High sensitivity detection of antibodies indicative of aids exposure This invention relates to the detection of antibodies to the various viruses recognized as giving rise to Acquired Immune Deficiency Syndrome (AIDS). In particular, this invention relates to the analysis of bodily fluids for such antibodies as an indication of exposure to AIDS. Examples of recognized AIDS-associated viruses include Human T-cell Leukemia Virus-111 (HTLV-III), Lymphadenopathy Associated Virus (LAV) and Acquired Immune Deficiency Syndrome Related Virus (ARV). Tests for the presence of antibodies to these viruses have been developed as a result of seroepidemiological studies which indicate that where detectable levels of such antibodies exist, infectious virus may also exist. In any event, the presence of the antibodies is taken as an indication of prior exposure to or infection with the virus, and suggests a potential for the continued presence of the virus or the viral genome. Assays in common use for detection of the antibody are plagued by high percentages of false positives and false negatives, in addition to being cumbersome and time consuming. An assay having improved reliability and sensitivity is needed, as well as one which can be completed in a shorter period of time.

SUMMARY OF THE INVENTION Assays addressed by the present invention are those performed by contacting a sample of bodily fluid (preferably serum or plasma) which antigens of an AIDS virus to cause AIDS antibodies in the fluid to associate with the antigens and form immunological complexes. Each complex is then associated with a signal producing system and the signal level from the associated systems measured as an indication of the presence of the antibodies in the sample. The virus is generally lysed and separated electrophoretically into fractions prior to the assay. In accordance with the present invention, an assay kit useful for multiple assays has been developed. The kit contains a series of solid phase supports with the viral antigens already adsorbed thereon prior to packaging. The adsorbed antigens are preferably Western blots of electropherograms of the lysed virus. The kit further contains a solution of a conjugate formed by associating an anti-human Ig antibody with a signal producing system. Preferably, the signal producing system is a two-component system, such as an enzyme and substrate, one component (the enzyme) forming part of the conjugate and the second component (the substrate) in a separate solution. The Western blots may be conveniently prepared as a series of identical strips cut from a single rectangular blot in the direction perpendicular to the bands.The blot used in such a preparation may be the result of transfer from a single electropherogram having an elongated transverse dimension and thus consisting of a series of long parallel bands. Preferred kits also include comparison patterns or reproductions thereof, such as photographs. These patterns are the same supports with identical antigen adsorption patterns, after incubation with known positive and negative samples and appropriate development with the same signal producing system. These enable the kit user to compare the test samples assayed by the user with known samples already assayed and thereby apply a uniform interpretation of test results.In another aspect of the invention, it has been discovered that a signal producing system involving a combination of alkaline phosphatase and a substrate consisting of nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) is unusually sensitive when used in conjunction with a Western blot in an AIDS antibody assay. In preferred embodiments, the alkaline phosphatase is conjugated with goat or rabbit anti-human Ig which is capable of binding with the antibody whose presence is being detected. The assay may be done by preparing a Western blot of an electropherogram of the lysed virus, with the background blocked by milk proteins and detergent. The blot is then incubated first with a sample of the test fluid (preferably serum), then with a solution of the conjugate, and finally with a solution of the NBT/BCIP substrate. A further aspect of the invention resides in a, multi-trough tray for retaining the Western blot strips referred to above, one in each trough, fully immersed in the assay liquids. Features in each trough permit complete removal of the liquids by aspiration without contact between the strips and any external (and potentially contaminating) materials. In preferred embodiments, the tray is further equipped with a removable liner which fits snugly over the tray and has protrusions extending into each of the troughs. The purpose of the liner is to hold one strip in each trough for shipping purposes. The liner is removed from the tray prior to the performance of assays. A snug-fitting lid which lacks the protrusions which extend into the troughs is used during the assay in the incubation stages of the procedure.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a perspective view of a multi-receptacle tray designed to permit the performance of a number of assays simultaneously in accordance with the present invention. Figure 2 is a perspective view of a shipping liner for the tray of Fig. 1. Figure 3 is a perspective view of a lid for the tray of Fig. 1 for use during performance of an assay.

DESCRIPTION OF THE PREFERRED AND SPECIFIC EMBODIMENTS AIDS viruses useful in preparing assay materials used in the present invention are publicly available from a variety of sources. One may obtain HTLV-III, for example, from Organon Teknika Corporation, Bionetics Laboratory Products Div., Charleston, South Carolina; Prototech Incorporated, St. Paul, Minnesota; or Ortho Pharmaceutical Corporation, Raritan, New Jersey. Alternatively, one may obtain LAV from Genetic Systems, Seattle, Washington. Still further, one may use a different isolate from an AIDS patient which has been adapted to survive in tissue culture and produced. It is preferred that the virus be used in lysed form. This may be achieved according to conventional methods, such as, for example, the use of SDS in a buffer solution. The solid phase support may be any inert solid phase material capable of immobilizing the viral proteins. Preferred supports are blotting membranes such as, for example, nitrocellulose, aminobenzyloxymethyl (ABM) paper, 2-aminophenylthioether (APT) paper, nylon, charge-modified nylon, diazobenzyloxymethyl (DBM) paper, 2-diazophenylthioether (DPT) paper, and diethylaminoethyl (DEAE) cellulose. Nitrocellulose is particularly preferred. The viral proteins are separated into discrete regions or bands spatially arranged in a preselected pattern for immobilization on the support. Separation is conveniently achieved by electrophoretic fractionation of the proteins in a slab gel such as agarose or polyacrylamide, polyacrylamide being preferred. The spatial arrangement is thus that of the resulting electropherogram. Immobilization of the bands on the support may be achieved by direct transfer from the gel. This may be achieved by diffusive transfer (Southern blotting) or by electrophoretic transfer (Western blotting). The latter is preferred. Immobilization is achieved by surface adsorption upon contact, preferably with no intermediate linking agent. To enhance uniformity from one test to the next and to minimize the occurrence of false positive test results, it is preferred that there be approximately equal concentrations of each viral protein in any single blot. It is rare that the viruses themselves will provide such concentration levels, and it is accordingly desirable that certain bands be either diminished or enhanced during the band separation procedure. This may be accomplished by using HPLC or affinity chromatography to concentrate or delete appropriate bands. If, for example, a virus sample contains an excessive amount of p24 protein, the sample may be passed through a lectin column which will retain all of the proteins except the p24. The bound proteins are then selectively eluted and the p24 is returned to the mixture in an appropriate proportion.As another example, virus samples deficient in p41 may be supplemented with pure p41 obtained by HPLC from another virus or source of the protein at a selected concentration. A blocking agent is generally included to prevent nonspecific binding once the immobilization of the viral proteins has occurred. Milk proteins and detergents are particularly effective blocking agents, generally applied in aqueous solution with an antifoaming agent. A typical milk protein concentration is within the range of about 1% to about 10% by weight. The kit of the present invention combines the various components needed to perform several assays of the above description simultaneously. Included among the kit components are a series of substantially identical strips of the solid phase material, each with the viral proteins immobilized on one side thereof and spaced at intervals along its length. The strips may be conveniently cut from a Western blot membrane transferred from a single slab gel. The strips are therefore identical in terms of the types, amounts and positions of the viral proteins. The strips are preferably marked to indicate the side to which the proteins are bound and to indicate the proper orientation, thereby assuring that all bands are presented in the same order. A second component of the kit is a solution of a conjugate of an antibody specific for the sample antibodies which bind to the viral proteins, the other component of the conjugate being an appropriate enzyme, preferably alkaline phosphatase. The conjugate antibody will be an antihuman immunoglobulin, such as anti-human IgG, IgM, IgA, or other antibody class- or subclassspecific antibody. Anti-human IgG is preferred. The quantity of conjugate used is an amount which is in excess of the expected range of the quantity of sample antibodies in each assay so'that all sample antibodies on the support become bound to form complexes. The concentration of the conjugate in the solution will be selected on the basis of sensitivity. The lowest concentration which will give visible results on a standard sample will generally be used. In most applications, this will fall between dilutions of 1:500 and 1:3000. A third component is a solution of the substrate upon which the enzyme acts, preferably the NBT/BCIP combination in approximately equimolar amounts. The quantity of substrate used is an amount sufficient to produce a detectable change due to the action of the enzyme. For NBT/BCIP, this will generally fall within the range of about 1 x 10-4 moles/liter to about 10 x 10-4 moles/liter for each component. A wash solution containing the blocking agent may also be included. In addition, fully developed comparison strips representing known positive and known negative samples are included as a guide to the operator. These strips may appear as photographs in an instruction manual or may be actual strips. Still further, receptacles are included for receiving individual test strips and immersing them in the samples, reagents and wash solutions, for purposes of incubation and agitation, while permitting aspiration of the liquids at various stages of the assay procedure. An illustrative embodiment of a multi-receptacle tray 11 useful in the practice of the present invention is shown in Fig. 1. The tray contains a series of elongated receptacles or troughs 12, arranged in parallel, each of sufficient length to accommodate one strip of solid phase support medium, and of sufficient width that the strip may lie face upward. At one end of each trough is a retaining barrier comprised of a pair of protuberances 13, 14 which hold the strip (not shown) away from one end wall 15 of each trough, thus defining a well 16 into which an aspirating tube (not shown) may be inserted to withdraw liquids without contacting the strip. The protuberances 13, 14 are separated by a gap 17 which, although not wide enough to permit passage of the strip, does permit liquid flow and thus drainage of all liquid in each trough into the well 16 for removal by aspiration. A liner 21 for the tray of Fig. 1 is shown in Fig. 2. The liner is of thin transparent material and has the same contour as the upper surface of the tray 11. It thus has a series of rectangular projections 22 extending downward, having the same shape as the troughs 12. In the embodiment shown, the projections 22 as well as the troughs 12 taper slightly toward the bottom so that the projections will fit readily into the troughs when the liner is placed over the tray. The liner serves a function during shipping, particularly when the tray is packaged with a test strip placed in each trough. The liner holds the strips in place during transport and handling of the tray, and is removed prior to performance of the assay. A lid 31 as shown in Fig. 3 may also be included in the test kit. The lid shown is of thin transparent material similar to the liner 21 of Fig. 2 but without the projections 22. The side walls 23, 32 of both the liner and lid, respectively, taper upward to facilitate the joining of all parts. The lid is used during the assay itself, and serves to retain test fluids and strips in the troughs during agitation that is typically practiced during the incubation stages of the assay. Agitation may consist of a gentle rocking of the tray. The following Example is offered for illustrative purposes and is intended neither to define nor limit the invention in any manner.

EXAMPLE I. Preparation of Electropherogram on Nitrocellulose HTLV-III virus is obtained from Organon Teknika. The virus has been deactivated with 0.5% detergent after having been extracted by cell lysis from H9 cells which had been infected with the virus. The virus is diluted and lysed in a buffer solution at 10 mg per 50 ml final solution, and fractionated by electrophoresis on a 10% polyacrylamide slab gel in the presence of sodium dodecyl sulfate. The protein bands resulting on the gel are electrophoretically transferred to a sheet of nitrocellulose. The side of the sheet bearing the protein bands is then marked with a line along the edge corresponding to the top of the electropherogram. The sheet is then washed repeatedly in 0.3% Tween 20 in phosphate-buffered saline and 0.1% sodium azide in deionized water, then cut into 32 strips measuring 11 cm by 0.34 cm. The two end strips are retained for quality control. Standard immunoassays are performed on these end strips using positive and negative control sera to confirm the presence of virus proteins. II. Reagent Preparation A wash/diluent solution is prepared by combining the following ingredients in deionized water (based on a final volume of 24 liters):

An enzyme conjugate solution is prepared using anti-human IgG alkaline phosphatase conjugate obtained from Bio-Rad Laboratories, Inc., Chemical Division, Richmond, California, by combining 2.05 parts by volume of the above wash/diluent solution with 18.45 parts by volume of deionized water, and adding conjugate in a preselected amount sufficient to provide detectable results. The amount may be preselected on the basis of an end point determination using serial dilutions. A substrate solution is prepared by combining 3.12 parts by weight of 5-bromo-4-chloro-3indolyl phosphate and 6.25 parts by weight of nitroblue tetrazolium chloride with 0.1 M TRIS buffer, sodium azide, dimethylformamide and deionized water, adjusting the pH to 9.5 0.1 with hydrochloric acid. III. Assay A tray containing ten troughs as in the drawing is used. One strip is placed in each trough with the line markings facing up and all to one side. Wash/diluent solution is then added at an amount of 3ml to each trough, saturating each strip. One patient sample added to each trough at 30 microliters per sample, and the tray is rocked gently (incubated) for thirty minutes at room temperature. The troughs are then vacuum aspirated, and 5ml of wash/diluent solution is added to each, followed by incubation for ten minutes. Aspiration and washing are then repeated. After aspiration, 3ml of enzyme conjugate solution is added to each trough and incubation is resumed for thirty minutes. The troughs are aspirated, and 5ml of wash/diluent solution is added to each followed by ten minutes' incubation. The troughs are aspirated and the wash repeated. Substrate solution is then added at 3ml per trough, followed by incubation for ten minutes and aspiration. Each trough is then washed with 5ml of deionized water twice with 5 minutes incubation each time, then aspirated and the strips examined. Colored bands appear in a pattern determined by the electropherogram to indicate the presence of antibodies to AIDS-associated virus in each sample. The foregoing description is intended primarily for purposes of illustration. It will be readily apparent to those skilled in the art that numerous variations and modifications of the above may

Claims

be made without departing from the spirit and scope of the invention, as defined by the claims which follow. CLAIMS

1. A method for determining the presence of antibodies having specificity for antigenic sites on an AIDS-associated virus in a sample of bodily fluid which comprises: (a) contacting said sample with antigens selected from the group consisting of said AIDSassociated virus and fractions thereof to form complexes of said antibodies with said antigens; (b) associating with each of said complexes a signal producing system comprised of: (i) a conjugate of alkaline phosphatase with a second antibody having affinity for said complexes, and (ii) a mixture of nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate in amounts sufficient to produce a color change upon contact with alkaline phosphatase; and (c) determining the signal level from said associated signal producing systems as an indication of the presence of said antibodies in said sample.

2. A method in accordance with claim 1 in which said second antibody is anti-human Ig.

3. A method in accordance with claim 1 in which said nitroblue tetrazolium chloride and said 5-bromo-4-chloro-3-indolyl phosphate are in approximately equimolar amounts.

4. A method in accordance with claim 1 in which said antigens are a lysate of an AIDSassociated virus.

5. A method in accordance with claim 1 in which said antigens are a lysate of HTLV-III.

6. A method in accordance with claim 1 in which said antigens are a lysate of LAV.

7. A method in accordance with claim 1 in which said antigens are a lysate of ARV.

8. A method in accordance with claim 4 in which said antigens are electrophoretically fractionated and transferred to a solid phase support medium.

9. A method in accordance with claim 8 in which said solid phase support medium is nitrocellulose.

10. A method for determining the presence of antibodies having specificity for antigenic sites on an AIDS-associated virus in a sample of human serum which comprises: (a) immersing in said sample a solid phase support having adsorbed thereon an electropherogram of protein fractions of a lysed AIDS-associated virus to form immobilized complexes of said antibodies with said protein fractions; (b) incubating said immobilized complexes with a solution of an excess of conjugate of antihuman Ig and alkaline phosphatase to bind said conjugate to said immobilized complexes; (c) recovering said conjugate-bound immobilized complexes from remaining conjugate in said solution; (d) incubating said recovered conjugate-bound immobilized complexes with a solution of nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate to produce a detectable color change indicating the presence of said antibodies in said sample.

11. A diagnostic test kit for analyzing samples of bodily fluid for the presence of antibodies having specificity for antigenic sites on an AIDS-associated virus, said diagnostic test kit comprising: a plurality of solid phase supports each having adsorbed thereon antigens of said AIDSassociated virus in discrete regions arranged in a preselected pattern, whereby both said preselected pattern and the density of each adsorbed antigen is substantially identical from one said solid phase support to the next; a solution of a conjugate of the first component of a two-component signal producing system with a second antibody having binding affinity for complexes of said antigens with said antibodies, in an amount sufficient for all of said solid phase supports; and a solution of the second component of said two-component signal producing system, in an amount sufficient for all of said solid phase supports.

12. A diagnostic test kit in accordance with claim 11 in which said two-component signal producing system is a system which produces a visually observable change when said two components are combined.

13. A diagnostic test kit in accordance with claim 11 in which said first component is an enzyme capable of acting upon said second component to produce a detectable signal.

14. A diagnostic test kit in accordance with claim 11 in which said first component is alkaline phosphatase and said second component is a substrate capable of undergoing a detectable change upon contact with alkaline phosphatase.

15. A diagnostic test kit in accordance with claim 14 in which said second component is a mixture of nitroblue tetrazolium choride and 5-bromo-4-chloro-3-indolyl phosphate in amounts sufficient to produce a color change upon contact with alkaline phosphatase.

16. A diagnostic test kit in accordance with claim 11 further comprising a solution of a blocking agent to reduce nonspecific binding on said solid phase support.

17. A diagnostic test kit in accordance with claim 16 in which said blocking agent is comprised of milk proteins.

18. A diagnostic test kit in accordance with claim 11 in which said preselected pattern is an electropherogram transferred to said solid phase support from an electrophoretic separation medium.

19. A diagnostic test kit in accordance with claim 11 in which said solid phase support is nitrocellulose.

20. A diagnostic test kit in accordance with claim 11 in which said preselected pattern is a Western blot of an electropherogram of said antigens.

21. A diagnostic test kit in accordance with claim 10 further comprising a receptacle for retaining said solid phase support immersed in any of said sample and said solutions.

22. A diagnostic test kit for analyzing a plurality of samples of human bodily fluid for the presence of IgG antibodies having specificity for antigenic sites on an AIDS-associated virus, said diagnostic test kit comprising: a plurality of strips cut from a solid support having adsorbed thereon by Western blotting an electropherogram of antigens of said AIDS-associated virus in elongated bands transverse to said strips; a solution of a conjugate of anti-human IgG and alkaline phosphatase; a solution of nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate in approximately equimolar amounts; a solution of milk proteins; and a tray containing a plurality of liquid-retaining compartments each of sufficient size to receive one of said solid supports fully immersed in liquid.

23. A tray for incubating strips of solid phase material in reagent liquids, said tray comprising: a plurality of elongate liquid-retaining compartments each sized to receive one said strip fully immersed in liquid; and a barrier in each said elongate liquid-retaining compartment to retain one said strip at a distance away from one end wall thereof while permitting liquid flow there-through.

24. A tray in accordance with claim 23 in which said barrier is comprised of a protuberance extending upward from the base of each of said elongate liquid-retaining compartments.

25. A tray in accordance with claim 23 in which said barrier is comprised of a pair of protuberances extending upward from the base of each said elongate liquid-retaining compartments, said pair separated by a gap narrower than the width of each said strip.

26. Apparatus for retaining strips of solid phase material to be incubated in reagent liquids, said apparatus comprising: a tray containing a plurality of elongate liquid-retaining compartments each sized to receive one said strip fully immersed in liquid, each said compartment containing a barrier positioned to retain one said strip at a distance away from one end wall thereof while permitting liquid flow therethrough; and a removable liner contoured to fit over the upper surface of said tray with projections extending into each said elongate liquid-retaining compartment to stabilize the position of each said strip.

27. A method for determining the presence of antibodies having specificity for antigenic sites on an AIDS-associated virus in a sample as claimed in Claim 1 substantially as herein described with reference to the Example.

28. A diagnostic kit as claimed in Claim 11 substantially as herein described with reference to the Example.

29. A tray for incubating strips of solid phase material as claimed in Claim 23 substantially as herein described with reference to the accompanying drawings.

30. Apparatus for retaining strips of solid phase material to be incubated in reagent liquids as claimed in Claim 26 substantially as herein described with reference to the accompanying drawings.