FR2601456A1 - HIGH SENSITIVITY DETECTION METHOD OF ANTIBODIES INDICATING EXPOSURE TO AIDS, NECESSARY AND ACCESSORIES FOR ITS IMPLEMENTATION - Google Patents

Abstract

ANTIBODIES SPECIFIC TO ANTIGENIC SITES ON AIDS-ASSOCIATED VIRUSES ARE DETECTED IN A BODY FLUID SAMPLE BY INCUBATING THE SAMPLE WITH ANTIGENS FROM AN AIDS VIRUS IMMOBILIZED ON A SOLID CARRIER. ANTIGEN-ANTIBODY COMPLEXES A SYSTEM PRODUCING A SIGNAL, AND DETERMINING THE LEVEL OF THE SIGNAL FROM THE ASSOCIATED COMPLEXES OBTAINED. A REQUIREMENT INCLUDING A SERIES OF SOLID BANDS ON WHICH THE VARIOUS PROTEINS ARE IMMOBILIZED IN AN ELECTROPHORETOGRAM, PLUS CONJUGATE SOLUTIONS, SUBSTRATE AND VAGEDILUTION AGENTS IS ALSO DESCRIBED, AS IS A PLATE 11 CONTAINING A MULTIPLE 12 SIMULTANEOUS DOSAGE SERIES. SYSTEMS 13, 14 PRESENT IN EACH TANK ALLOW THE INSERTION OF A SUCTION TUBE TO ELIMINATE THE LIQUID WHILE AVOIDING CONTACT OF THE TUBE WITH THE SURFACES OF THE BANDS.

Description

METHOD FOR DETECTION WITH HIGH SENSITIVITY OF ANTIBODIES INDICATING A

AIDS EXPOSURE, NECESSARY AND

ACCESSORIES FOR ITS IMPLEMENTATION.

  The present invention relates to detection

  antibodies against the various viruses known to give rise to Acquired Immune Deficiency Syndrome (AIDS). In particular, the invention relates to the analysis of body fluids to search for these antibodies as an indication of exposure to AIDS.

  Examples of viruses known to be associated with AIDS include human T-cell leukemia virus III (HTLV-III), lymphadenopathy-associated virus (LAV), and acquired immunodeficiency syndrome (ARV) related virus. Assays for the presence of antibodies against these viruses have been developed as a result of seroepidemiological studies which indicate that when detectable levels of these antibodies exist, an infectious virus may also exist. In any case, the presence of the antibodies is considered an indication of previous exposure to or infection with the virus, and suggests the possibility of

  permanent presence of the virus or viral genome.

  The analyzes commonly used for the detection of the antibody are afflicted with high percentages of false positive and false negative results, besides being tedious and slow to execute. An analysis with improved reliability and sensitivity is needed, as well as

  analysis that can be completed in a shorter time.

  The assays concerned by the present invention are those carried out by contacting a sample of body fluid (preferably serum or plasma) with antigens of an AIDS virus to cause the combination of the anti-AIDS antibodies present in the the fluid with the antigens and the formation of immunological complexes. Each complex is then associated with a signal producing system, and the signal level from the associated systems is measured as an indication of the presence of the antibodies in the sample. The virus is usually lysed and electrophoretically separated into

fractions before analysis.

  In accordance with the present invention, an analysis requirement useful for multiple trials has been developed. The kit contains a series of solid phase supports, on which the viral antigens are already adsorbed before packaging. The adsorbed antigens are preferably Western blots of electrophoretograms of the lysed virus. The kit further contains a solution of a conjugate formed by combining an anti-human Ig antibody with a signal producing system. The signal producing system is preferably a two-component system, such as an enzyme and a substrate, one of the constituents (the enzyme) being part of the conjugate and the second component (the substrate) being in a separate solution. Western blots can conveniently be prepared as a series of identical strips cut in a single rectangular spot in the direction perpendicular to the strips. The stain used in such a preparation may result from the transfer from a single electrophoretogram having an elongate transverse dimension, and thus consisting of a series of long parallel bands. Preferred kits also contain comparison diagrams, or reproductions thereof, such as photographs. These diagrams are the same supports with identical antigen adsorption diagrams, after incubation with positive and negative samples and appropriate development with the

260145K

  same signal generating system. This allows the user of the kit to compare the test samples analyzed by the user with known samples already analyzed and thus to interpret the test results in a uniform manner. In another aspect of the invention, it has been found that a signal producing system involving a combination of alkaline phosphatase and a substrate consisting of nitroblue tetrazolium chloride and 5-bromo-4-chloro phosphate 3-indolyl (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 to the antibody whose presence is to be detected. The assay can be performed by preparing a Western blot of an electrophoretogram of the lysed virus, the background being blocked by milk proteins and detergent. The stain 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 substrate.

NBT / BCIP.

  Another aspect of the invention resides in a multi-pan tray for maintaining the Western blot bands discussed above, one in each pan, fully immersed in the test liquids. Systems in each cuvette 30 allow complete removal of liquids by non-contacting suction between the strips, or any external (and potentially contaminating) material. In preferred embodiments, the tray is further provided with a removable liner which fits soft friction on the tray and has protuberances extending into each of the cuvettes. The purpose of the liner is to maintain a strip in each bowl for shipping. The liner is removed from the board before running the scans. A soft friction-fit cover, free of the protuberances that extend into the cuvettes, is used during

  analysis in the incubation stages of the process.

  FIG. 1 is a perspective view of a multi-receptacle tray designed to allow the simultaneous execution of a number of analyzes in accordance with

the present invention.

  FIG. 2 is a perspective view of a

  shipping liner for the tray of Figure 1.

  FIG. 3 is a perspective view of a neck circle for the plate of FIG. 1, intended to be

  used during the execution of an analysis.

  AIDS viruses useful for preparing the analytical materials used in the present invention are made available to the public by various sources. For example, HTLV-III can be obtained from

  Organon Teknika Corporation, Bionetics Laboratory Products Div., Charleston, South Carolina; Prototech Incorporated, St. Paul, Minnesota; or Ortho Pharmaceutical Corporation, Raritan, New Jersey. LAV can also be obtained from Genetic Systems, Seattle, Washington.

  A different isolate from an AIDS patient that has been adapted to survival in tissue culture and put into production can still be used. It is preferred that the virus be used in lysed form. This can be achieved by conventional methods, such as for example the use of SDS (dodecyl sulfate).

sodium) in a buffer solution.

  The solid phase support may be any inert solid phase material capable of immobilizing the viral proteins. Preferred carriers are absorbent membranes such as, for example, nitrocellulose, aminobenzyloxymethyl (ABM) paper, 2-aminophenylthioether (APT) treated paper, nylon, charge modified nylon, benzyloxymethyl diazo5 paper (DBM) , paper treated with 2-diazophenylthioether (DPT), and diethylaminoethyl cellulose (DEAE). Nitrocellulose is particularly preferred. The viral proteins are separated into separate regions or bands, arranged spatially in a predetermined disposition for immobilization on the support. Separation is conveniently accomplished by electrophoretic fractionation of proteins in a plate gel such as agarose or polyacrylamide, with polyacrylamide being preferred. The spatial arrangement

  is thus that of the electrophoretogram obtained. The immobilization of the strips on the support can be carried out by direct transfer from the gel. This can be done by diffusion blotting (Southern blotting) or by electrophoretic transfer (Western blotting).

  The latter is preferred. The immobilization is carried out by superficial adsorption by contact, preferably

  without intermediate linker.

  To improve the uniformity of a subsequent test and to minimize false-positive test results, it is preferred that there be approximately equal concentrations of each viral protein in each isolated spot. It is rare for the viruses themselves to provide these levels of concentration, and it is therefore desirable that some bands be decreased or increased during the band separation process. This can be done using HPLC (High Performance Liquid Chromatography) or affinity chromatography to concentrate or remove appropriate bands. If, for example, a virus sample contains an excessive amount of

  p24 protein, the sample can be passed through a lectin column which will retain all proteins except p24. The bound proteins are then selectively eluted, and the p24 is returned to the mixture in an appropriate proportion. Another example is that of p41-deficient virus samples which can be supplemented with pure p41 obtained by HPLC from another virus or other source of the protein at a selected concentration.

  A blocking agent is generally included to prevent nonspecific binding as soon as 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 in the range of about 1% to about 10% by weight.

  The kit of the present invention assembles the various components necessary to perform several assays simultaneously as described above. Among the constituents of the necessary

  There is a series of substantially identical bands of the solid phase material, each with the viral proteins immobilized on one of its faces and spaced at intervals along its length. The strips can conveniently be cut in a Western blot membrane transferred from a single plate gel. The bands are therefore identical with respect to types, amounts and positions of proteins

  viral. The strips are preferably marked to indicate the side to which the proteins are bound and to indicate the proper orientation, thus ensuring 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 antibodies in the sample that bind to the viral proteins, the other constituent of the conjugate being an appropriate enzyme, preferably an alkaline phosphatase.

  The antibody of the conjugate will be a human anti-immunoglobulin, such as human anti-IgG, anti-IgM or anti-IgA or a specific antibody of another class or subclass of antibodies. Human anti-IgG is preferred.

  The amount of conjugate used is an amount that exceeds the range expected for the amount of antibody in the sample in each assay, such that all of the sample antibodies on the support bind to form complexes. The concentration of the conjugate in the solution will be chosen on the basis of sensitivity. The lowest concentration that will give readable results on a standard sample will generally be used. In most applications, this one is

  will be between the 1: 500 and 1: 3000 dilutions.

  A third component is a solution of the substrate on which the enzyme acts, preferably the NBT / BCIP combination in approximately equimolar amounts. The amount of substrate used is a

  sufficient to produce a detectable change resulting from the action of the enzyme. For NBT / BCIP, this amount will generally be in the range of about 1 x 10-mole / liter to about 10 x 10 mole / liter for each component.

  A wash solution containing the blocking agent may also be incorporated. In addition, fully developed comparison bands representing known positive and negative positive samples are included as a guide for the operator. These tapes may be in the form of photographs in an instruction manual or may be actual tapes. In addition, receptacles are included to receive the individual test strips and immerse them in the samples, reagents, and wash solutions for incubation and agitation, while permitting the aspiration of liquids. at various stages of the analysis process. An exemplary embodiment of a multi-receptacle tray 11 usable in the practice of the present invention is shown in FIG. 1. The tray contains a series of elongated receptacles or cuvettes 12, arranged in parallel each being of sufficient length to house a solid phase support medium strip and of sufficient width for the strip to rest with its face upward. At one end of each cuvette is a retention barrier comprising a pair of protuberances 13, 14, which hold the band (not shown) remote from an end wall 15 of each cuvette, thereby defining a well 16 in which a suction tube (not shown) can be inserted to withdraw liquid without coming into contact with the strip. The protuberances 13, 14 are separated by a gap 17, which, although insufficiently wide to allow the passage of the band, allows the flow of the liquid and therefore the drainage of all the liquid present in each of the cups in the bowl. well 16 for

aspiration elimination.

  A liner 21 for the tray of FIG. 1 is shown in FIG. 2. The liner is made of a 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 downwardly, having the same shape as the cups 12. In the embodiment shown, the projections 22 and the cups 12 are slightly tapered downwards, so that the projections fit easily in the bowls. when the liner is placed on the bowl. The liner plays a role in shipping, particularly when the tray is packaged with a test strip placed in each bowl. The liner holds the tapes in place during transportation and handling of the

  tray, and is removed before running the scan.

  A cover 31 as shown in FIG. 3 may also be included in the test kit.

  The cover shown is a cover 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 the cover, respectively, taper upwards to facilitate the joining of all parts. The lid is used during the analysis itself, and it serves to retain the test fluids and strips in the cuvettes during the agitation which is generally performed during the incubation stages of the incubator. 'analysis. agitation

  may consist of a slight rocking of the plate.

  The following example is given by way of illustration and is not intended to define or limit the scope of the invention in any way. EXAMPLE I Preparation of an ElectroDhoroaramel on Nitrocellulose The HTLV-III virus is provided by Organon Teknika. The virus was inactivated with 0.5% detergent, after being cell lysed out of H9 cells that had been infected with

the virus.

  The virus is diluted and lysed in a 10 mg buffer solution for 50 ml of the final solution, and fractionated by electrophoresis on a gel plate

  10% polyacrylamide in the presence of dodecysul-

260 1,456

  sodium fate. The protein bands obtained on the gel are electrophoretically transferred to a nitrocellulose sheet. The side of the sheet carrying the protein bands is then marked with a line along the edge corresponding to the top of the electrophoretogram. The sheet is then washed several times in 0.3% Tween 20 in phosphate buffered saline and 0.1% sodium azide in deionized water and then cut into 32 bands measuring 11%. cm x 0.34 cm. Both end bands are retained for quality control. Standard immunoassays are performed on these end bands using positive and negative control sera to confirm the presence of

viral proteins.

  II. REAGENT PREPARATION A wash / dilute solution is prepared by combining the following ingredients in deionized water (based on a final volume of 24 liters): TRIS 580.9 g Dry skim milk 1.2 kg Antifoam A 7.7 ml Tween 20 720 ml NaCl 1120 g NaN3 24 g HCl to adjust the pH to 7.5 An enzyme conjugate solution is prepared using a conjugate of alkaline phosphatase and anti-human IgG obtained from Bio-Rad Laboratories, Inc., Chemical Division, Richmond, California, by combining 2.05 parts by volume of the above wash / dilution solution with 18.45 parts by volume of deionized water. and adding conjugate in a pre-selected amount sufficient to give detectable results. The quantity can be chosen in advance on the basis of a point determination

  final using serial dilutions.

  A substrate solution is prepared by combining 3.12 parts by weight of 5-bromo4-chloro-3-indolyl phosphate and 6.25 parts by weight of nitroblue tetrazolium chloride with 0.1M TRIS buffer. sodium azide, dimethylformamide and deionized water, by adjusting the pH to

  9.5 0.1 with hydrochloric acid.

III. Analysis.

  A tray containing ten cuvettes is used as in the drawing. A strip is placed in each of the bowls, with the lines marking upwards, and all on one side. The wash / dilute solution is then added in an amount of 3 ml to each cuvette, saturating

  each band. A sample from one patient is added to each cuvette at a rate of 30 microliters per sample, and the cuvette is gently swung (incubated) for thirty minutes at room temperature.

  Vacuum cuvettes are then aspirated and 5 ml of wash / dilute solution are added to each, followed by incubation for 10 minutes. Suction and washing are then repeated. After aspiration, 3 ml of enzyme conjugate solution is added to each cuvette and the incubation is resumed for thirty minutes. The cuvettes are aspirated, and 5 ml of wash / dilution solution are added to each, followed by incubation for ten minutes. The bowls are sucked up and the washing is repeated. Then add the solution

  Substrate amount of 3 ml per cuvette, then incubate for ten minutes and aspirate. Each cuvette is then washed with 5 ml of deionized water, twice with 5 minutes of incubation each time, then aspirated and examined. Colored bands appear in a pattern determined by the electrophoretogram, indicating the presence of antibodies to an AIDS-associated virus in each of the samples. 10 The foregoing detailed description is

  given primarily for the purpose of illustration.

  It is obvious to those skilled in the art that many additional modifications, variations and substitutions, not mentioned here, could be made without departing from the spirit.

and the scope of the invention.

Claims (27)

  A method of determining the presence of antibodies having specificity for antigenic sites on an AIDS-associated virus in a body fluid sample, characterized in that it comprises the steps of: sample in contact with antigens selected from the group consisting of this AIDS-associated virus and fractions thereof to form complexes of these antibodies with these antigens; (b) associating with each of these complexes a signal producing system comprising: (i) an alkaline phosphatase conjugate with a second antibody having an affinity for these complexes, and (ii) a mixture of tetrazolium blue nitroxide chloride and 5bromo-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 these associated signal generating systems as an indication of the presence of   these antibodies in this sample.   2. Process according to claim 1, characterized in that the second antibody is a anti-human Ig.   3. Process according to claim 1 or 2, in   wherein the nitroblue tetrazolium chloride and the 5-bromo4-chloro-3-indolyl phosphate are in approximately equimolar amounts.   4. Method according to one of claims 1 to   3, characterized in that the antigens are a lysate 260 1,456 of a virus associated with AIDS.   5. Process according to claim 4, characterized in that the antigens are a lysate of HTLV-III.   6. Process according to claim 4, characterized in that the antigens are an LAV lysate. 7. The process of claim 4   characterized in that the antigens are an ARV lysate.   8. Process according to one of Claims 4 to   7, characterized in that the antigens are fractionated electrophoretically and transferred to a medium of solid phase support.   9. Process according to claim 8, characterized in that the support medium in phase solid is nitrocellulose.   10. A method for determining the presence of antibodies having specificity for antigenic sites on an AIDS-associated virus in a human serum sample, characterized in that it comprises the steps of: (a) diving into this sample a solid phase support on which a tropho-trophogram of protein fractions of a lysed AIDS-associated virus is adsorbed to form immobilized complexes of these antibodies with these protein fractions; (b) incubating these immobilized complexes with a solution of an excess of anti-human Ig and alkaline phosphatase conjugate to bind this conjugate to these immobilized complexes; (c) separating these immobilized complexes bound to the conjugate of the remaining conjugate in this solution; (d) incubating these separated conjugated immobilized immobilized complexes with a solution of nitroblue tetrazolium chloride and 5-bromo-4-chloro-3indolyl chloride to produce a detectable staining change indicating the presence of these antibodies in this sample. 11. A diagnostic test kit for the analysis of body fluid samples 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 on which are adsorbed antigens of this AIDS-associated virus in separate regions placed in a predetermined disposition, this arrangement being chosen in advance and the density of each adsorbed antigen being both are substantially identical from one of these supports in solid phase to the next; a solution of a conjugate of the first component of a two-component signal-producing system with a second antibody having a binding affinity for complexes of these analogs with these antibodies in an amount sufficient for all such solid-phase supports ; and   a solution of the second component of this two-component signal production system in an amount sufficient for all such solid phase supports.   Diagnostic test kit according to Claim 11, characterized in that the two-component signal generation system is a system which produces a visually observable modification.   when the two components are combined.   Diagnostic test kit according to Claim 11 or 12, characterized in that the first constituent is an enzyme capable of acting on the   second component to produce a detectable signal.   14. Necessary diagnostic test kit following one   Claims 11 to 13, characterized in that the first component is an alkaline phosphatase and the second component is a substrate capable of undergoing detectable modification by contact with alkaline phosphatase.   A diagnostic test kit according to claim 14, characterized in that the second component is a mixture of nitroblue tetrazolium chloride and 5-bromo-4-chloro-315 indolyl phosphate in amounts sufficient to produce a modification of staining by contact with the alkaline phosphatase.   16. Necessary diagnostic test kit following one   Claims 11 to 16, characterized in that it further comprises a solution of a blocking agent for   reduce nonspecific binding on the medium by solid phase.   17. Necessary diagnostic test kit following   Claim 16, characterized in that the blocking agent consists of milk proteins.   18. Necessary diagnostic test kit following   Claims 11 to 17, characterized in that the pre-selected distribution is an electrophoretogram transferred to the solid phase support from an electrophoretic separation medium.   19. Necessary diagnostic test kit following one   claims 11 to 18, wherein the support in   solid phase is nitrocellulose.   20. Diagnostic test kit according to one of claims 11 to 19, characterized in that the   chosen layout & advance is a western blot of a 2 6 0 1 4 5 6   electrophoretogram of said antigens.   21. Required diagnostic test following one   Claims 11 to 20, characterized in that it further comprises a receptacle for holding the solid phase support immersed in the sample or in the solutions.   22. A diagnostic test kit for analyzing multiple human body fluid samples for the presence of IgG antibodies having specificity for antigenic sites on an AIDS-associated virus, said diagnostic test kit comprising: strips cut in a solid support on which is adsorbed by "Western blot" an electrophoretogram of antigens of this AIDS-associated virus in elongated strips transverse to said bands; a solution of a conjugate of anti-human IgG and alkaline phosphatase; a solution of nitroblue tetrazolium chloride and 5-bromo-4-chloro-3indolyl phosphate in approximately equimolar amounts; a milk protein solution; and a tray containing a plurality of liquid holding compartments, each of sufficient size to receive one of these carriers   solids completely immersed in liquid.   23. A tray for incubating solid phase membranes in reactive liquids, the tray comprising: a plurality of elongated liquid-retaining compartments, each sized to receive a strip completely immersed in liquid; and a barrier in each of these elongate compartments holding the liquids to retain a 260 1,456   strip of this type at a distance from an end wall thereof, while allowing   the flow of liquid through it.   24. Tray according to claim 23, characterized in that the barrier comprises a protuberance extending upwardly from the base of each of the elongate compartments holding liquid. The tray of claim 23, characterized in that the barrier comprises a pair of protuberances extending upwardly from the base of each of the elongated liquid-retaining compartments, said pair being separated by a gap   narrower than the width of each band.   Apparatus for retaining bands of solid phase material to be incubated in reactive liquids, characterized in that it comprises: a tray containing a plurality of elongate liquid-retaining compartments, each of which is dimensioned to receive a strip completely immersed in liquid liquid, each compartment containing a barrier arranged to retain a strip away from an end wall thereof, while permitting liquid flow therethrough; and a removable liner whose contours   fit to the upper surface of the tray with projections extending into each of the elongate compartments holding liquid to stabilize the position of each of the strips.   27. Apparatus according to claim 26, further characterized by a lid whose contours are such that it closes each of the elongate compartments retaining the liquids leaving sufficient space   for stirring the reactive liquids placed therein.