JPS63145962A - High-sensitivity detection method of antibody displaying aids contact - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to the detection of antibodies against various viruses recognized to cause acquired immunodeficiency syndrome (AIDS). In particular, the invention relates to the analysis of body fluids for the above-mentioned antibodies as an indicator of contact with AIDS.

[Conventional technology]

Examples of recognized AIDS viruses include human T-cell leukemia virus [1 (HTLV-1), IJ7 node disease-associated virus (LAV), and acquired immunodeficiency syndrome-associated virus (ARV). As a result of seroepidemiological studies, tests have been developed to determine the presence of antibodies to these viruses, and the presence of detectable levels of these antibodies indicates that infectious virus is also present. In any case, the presence of such antibodies indicates previous contact with or infection with the virus and suggests the possible continued presence of the virus or viral genome.

[Problem that the invention seeks to solve]

The commonly used assay methods for detecting antibodies are:
In addition to being complicated and time-consuming to operate, it has the disadvantage of a high rate of false positives and false negatives. Therefore, there is a need for the development of highly reliable and highly sensitive assay methods and assay methods that can be completed in a shorter period of time.

[Means for solving problems]

In the assay according to the invention, a sample of body fluid (preferably serum or plasma) is contacted with an antigen of the AIDS virus, thereby allowing the AIDS antibodies in the body fluid to bind to the antigen and form an immune complex. Each complex is then coupled to a signal-generating system and the resulting signal level is measured as an indicator of the presence of antibody in the sample. Prior to this assay, the virus is generally lysed and separated by electrophoresis.

According to the present invention, an assay kit that can be used for multiple assay operations has been developed. The kit includes a series of solid supports to which viral antigens have been adsorbed prior to packaging. As adsorbed antigen, virus lysate (lysed
electrophoretic pattern of virus
Western plot (14est
ern blots) are preferred. The kit further includes a solution of a conjugate prepared by combining an anti-human Ig antibody and a signal generation system.

A signal generation system consists of two components, for example an enzyme and a substrate.
A component system is preferred, in which case the first component (enzyme)
is part of the conjugate and the second component (substrate) is assumed to be in a separate solution. To create a Western plot, a single rectangular plot can be cut perpendicular to the band into a series of similar strips. The plots used in such operations are those obtained by a single electrophoretic pattern that is elongated in the transverse direction, and thus consists of a series of long parallel bands. is the comparison pattern or its duplicate (
It is preferable that a photo, etc.) be included. These comparative patterns are generated by incubating identical supports with similar antigenic patterns with known positive and negative samples and appropriate expression using similar signal generation systems. Using such a method, a kit user can compare a test sample that he or she has assayed with a known, assayed sample, thereby making it possible to uniformly interpret the test results.

In another aspect of the invention, alkaline phosphatase and nitro blue tetrazolium chloride and 5-
Bromo-4-chloro-3-indolylphosphate (N
It has been found that a signal generation system comprising a combination of substrates consisting of BT/BCIP) exhibits excellent sensitivity when used in conjunction with a Western blot in an AIDS antibody assay. In a preferred embodiment, alkaline phosphatase is coupled to a horse or rabbit anti-human Ig capable of binding the antibody to be detected. To perform the assay, a Western plot of the electrophoretic pattern of the viral lysate is prepared, and the background is blocked with milk and detergent. This plot is then first injected into the test solution (preferably serum). sample, then with the conjugate solution, and finally with the NBT/BCIP?S solution.

Another aspect of the present invention provides a multiple tray for fully immersing the Western Plot strips in the assay solution and holding them one by one in the grooves.

The shape of the grooves is such that the liquid can be completely removed by suction without contact between the pieces and external substances that may cause dyeing. In a preferred embodiment,
The tray is equipped with a removable snug liner having a protrusion extending toward the groove. This liner is used to hold each strip in its respective groove during shipping and is removed from the tray prior to performing the verification operation.

During the incubation phase during the assay procedure, use a tight-fitting lid without protrusions toward the groove.

The AIDS virus used to produce the assay materials used in the present invention is publicly available from a variety of sources. For example, HTLV
-III, Organon Technic Force (Organon
Teknika), Bionetics Laboratory Products Division (Bionetics
Lab.

ratory Products Div, ) (Charleston, South Carolina); Protochick (
Prototech (St. Ball, MN);
Ortho Pharmaceuticals (Ortho
Pharmaceutical Co., Ltd., Raritan, New Jersey).

Or Genetic Systems
5ysteIIls) (Seattle, Washington) to L
You can also handle AV manually.

Alternatively, tissue culture adapted viruses isolated from AIDS patients may be used. The virus used is preferably lysed, for example by
This can be done by conventional methods, such as using O3.

As the solid phase support, any inert solid phase material capable of immobilizing viral proteins may be used.

Preferred supports include, for example, nitrocellulose, aminobenzyloxymethyl (ABM) paper, 2-aminophenylthioether (APT) paper, nylon, charge-modified nylon, diazobenzyloxymethyl (DBM) paper, 2-diazo Plot membranes such as phenylthioether (DPT) paper and diethylaminoethyl (DEAE) cellulose, with nitrocellulose being particularly preferred.

The viral proteins are separated and immobilized on a support into discrete sites or bands that are spatially arranged according to a preselected pattern.

Proteins can be easily separated by electrophoretic fractionation in a plate gel such as agarose or polyacrylamide (preferably polyacrylamide). Therefore, in this case, the obtained electrophoretic pattern becomes a spatial arrangement. Immobilization of bands on a support may be achieved by direct transfer from the gel, using diffusion transfer (Southern blotting) or electrophoretic transfer (Western blotting). The latter method is preferred. Preferably, the immobilization is carried out by surface adsorption by contact and no intermediate binder is used.

To increase test-to-test uniformity and to minimize the occurrence of false-positive test results, it is preferred to have approximately equal concentrations of each viral protein in any single plot. Viruses themselves rarely provide such concentration levels. Therefore, during the band separation operation, it is preferable to reduce or increase specific bands. For this purpose, appropriate bands are concentrated or depleted using HPLC or affinity chromatography.

For example, if a virus sample contains an excess amount of p24 protein, the sample is passed through a lectin column that retains all proteins except p24. This selectively elutes bound proteins. The p24 is then added back into the mixture in the appropriate proportions; as another example, the p41
For samples lacking p41, pure p41 obtained using HPLC from other viral or protein sources can be used.
It is recommended to replenish at a predetermined concentration.

Blocking agents are generally used to prevent non-specific binding after immobilizing viral proteins. Milk proteins and detergents are particularly effective as blocking agents and are generally used in the form of an aqueous solution together with antifoaming agents. Typical milk protein concentrations range from about 1% to about 10% by weight.

The assay kit may contain various components (components) required to perform the several types of assay operations described above at the same time. A series of substantially identical strips of solid phase material are included on which viral proteins are immobilized in longitudinally spaced fashion. These strips may be cut from Western Blot membranes transferred from a single slab gel, so the strips are identical with respect to type and amount and location of viral proteins. By marking this strip, you can see which side the protein is bound to and the proper orientation.
It is preferable to ensure that all bands are in the same direction.

The second component of the kit is a conjugate of an antibody specific for the sample antibody that binds to the viral protein and a suitable enzyme, preferably alkaline phosphatase. Conjugate antibodies include anti-human immunoglobulin, such as anti-human IgG, IgM, IgA or other antibody class or subclass antibodies. Anti-history l-1gG is preferred.

The amount of conjugate to be used should be around the range of the total amount of sample antibodies expected in each assay procedure, so that all the sample antibodies on the support bind to form a complex. The concentration of conjugate in solution is determined by the sensitivity
). Generally, the lowest concentration that can give a visible result for the standard sample is used. In most embodiments, 1:5(11) to 1:3(
11) Use a dilution factor within the range of 0.

The third component of the kit is the substrate on which the enzyme acts. A combination of approximately equimolar amounts of NBT/BCIP is preferred. The amount of substrate used is such that a detectable change can be caused by the action of the enzyme. NBT/BC
For IP, this ranges from about 1XIO-' mole/E to about 10XIO-' mole/l for each component.

A cleaning solution containing a blocking agent may also be included in the kit. Also included are fully expressed comparison strips representing known positive and negative samples as an indicator to the operator. These strips may be included in the instructions in the form of photographs, or they may be included in person. Additionally, each test strip is immersed in sample, reagent and washing liquid for incubation and agitation, and is also provided with a receptacle for aspirating liquids during various stages of the assay procedure.
es) are also included in the kit.

A multi-container tray 11 useful in the practice of the present invention is shown in FIG. 1 and is equipped with a series of elongated containers or e12 arranged in parallel, each carrying a solid support medium. It has a length that can accommodate one piece of paper, and a width that allows the piece to be inserted upwards. At one end of the groove there is a retention barrier consisting of a pair of projections 13,14. These protrusions form a well (
When a suction tube (not shown) is inserted into this well, liquid can be aspirated without coming into contact with the strips. Protrusion 13.1
4 are separated by a gap 17. This gap is not wide enough to allow a small piece to pass through, but liquid can flow through it, so all the liquid in each chamber is removed from the well 16.
and can be removed by suction.

A liner 21 for the tray shown in FIG. 1 is shown in FIG.

This liner is made of a thin, transparent material and has the same contour as the upper surface of the tray 11, so it is equipped with a series of downwardly projecting rectangular protrusions 22 that are identical to the grooves 12. In the illustrated embodiment, both the protrusions 22 and the grooves 12 taper toward the bottom, so that when the liner is stacked on the tray, the protrusions easily fit into the grooves. This liner serves this purpose during shipping, particularly when packaging the tray with a test strip in each groove.

This liner keeps the strips in place during the tray transportation and handling process and is removed before performing the assay operation.

The above test kit may include a lid 31 shown in FIG. This lid is liner 21 in Figure 2.
Similarly, it is made of a thin and transparent material, but the protrusion 22
I don't have it. Both the liner side wall 23 and the lid side wall 32 taper upwardly to facilitate joining of all parts. This lid is used in the process of the assay itself, and has the role of holding the test solution and strips in the groove during stirring, which is generally performed during the culture stage of the assay. Agitation may be achieved by gently shaking the tray.

The following examples are presented for illustrative purposes only, and the invention is not defined or limited thereby.

Example 2: Preparation of electrophoretic pattern on unitocellulose HTLV-[11 virus is obtained from Organon Techni-Rikisha. This virus is extracted from H9 cells infected with it by cell lysis, and then inactivated using a 0.5% detergent.

The virus is lysed and diluted in buffer to a final solution concentration of 10.150- and fractionated by electrophoresis on a 10% polyacrylamide plate gel in the presence of sodium dodecyl sulfate. The protein bands thus generated on the gel are transferred onto a nitrocellulose sheet by electrophoresis.

Next, mark the side of the sheet where the protein bands are marked by drawing a line along the edge that corresponds to the top of the electrophoretic pattern. The sheet was then treated with 0.3% Twee in phosphate buffered saline.
n) Wash repeatedly with 0.20 solution and 0.1% sodium azide solution in deionized water and cut into 32 strips of 11 caXo, 34 cm in size.

The two strips at both ends are used for quality control. The presence of viral proteins is confirmed by performing standard immunoassays on these end strips using positive and negative control sera.

■Preparation of reagent By mixing the following components in deionized water,
Prepare wash/dilution solution (final volume should be 241).

Tris 580.9 g Non-fat dry milk 1.2 Pupil antifoaming agent A
7.7 mTween 20 720 d NaCl 1120 gNaN3
24 g MCI Enough to bring the pH to 7.5 Bio-Rad Lab.

Chem
Using an anti-human IgG alkaline phosphatase conjugate obtained from Cal.
An enzyme conjugate solution is prepared by adding a predetermined amount of the conjugate described above that can give a detectable result, along with 8.45 parts by volume of deionized water. The amount of conjugate is determined by serial dilutions.
Select in advance based on the end point measurement method using .

3.12 parts by weight of 5-bromo-4-chloro-3-indolyl phosphate and 6.25 parts by weight of nitroblue tetrazolium chloride were mixed with O, lM Tris buffer, sodium azide, dimethylformamide and deionized water. A substrate solution is prepared by combining and adjusting the pH to 9.5±0.1 using hydrochloric acid.

(2) Test scraping A tray with 10 grooves as shown in Figure 1 is used. Place one strip into each groove so that the marks are all facing in the same direction. Next, add 3 wash/dilute solutions to the valleys to saturate each strip. Add 30 μl of each test sample to the groove, and gently shake the tray at room temperature for 30 minutes (incubation).

Next, vacuum the valleys, add 51i washing/dilution solution to each, and incubate for 10 minutes. Then, suction and washing are repeated. After suction, the enzyme conjugate solution of 3- is added to the groove, and the culture is restarted and continued for 30 minutes.

After suctioning the valley groove and adding 51111 wash/diluent,
Incubate for 10 minutes. Vacuum the grooves and repeat cleaning.

Next, three substrate solutions were added to the valley groove, cultured for 10 minutes, and then aspirated. The valleys are then washed twice with 5N1 deionized water, incubated for 5 minutes each time, and the strips are examined after aspiration. If colored bands are seen according to a pattern determined by electrophoresis,
The presence of antibodies to AIDS-related viruses in each sample is indicated.

The operations described above are provided for illustrative purposes only. It will be apparent to those skilled in the art that numerous further modifications, changes and substitutions may be made in addition to those described herein without departing from the spirit and scope of the invention as defined in the claims. Dew.

〔Effect of the invention〕

According to the detection method of the present invention, antibodies indicative of AIDS exposure can be easily detected with high reliability and sensitivity, and in a short period of time.

[Brief explanation of the drawing]

FIG. 1 is a perspective view of a multi-container tray designed to perform multiple assay operations simultaneously in accordance with the present invention;
2 is a perspective view of a shipping liner for use with the tray of FIG. 1, and FIG. 3 is a perspective view of a lid used during a verification operation for the tray of FIG. 11...Multi-container tray, 12...Container (groove) 13
．． 14...Protrusion, 15...Wall, 16...Well,
17...Gap, 21...Liner 22...Protrusion, 23...Side wall, 31...Lid 32...Side wall Patent applicant Bioland Laboratories, Inc. 2, Name of invention Indicates AIDS contact Highly Sensitive Detection Method for Antibodies 3, Relationship with Amendment Cases Patent Applicant: Bio-Rad Laboratories, Inc. 4, Agent: Pacific Nogizaka 6 (19), 6-29 Akasaka 9-chome, Minato-ku, Tokyo 1986 September 22nd (shipment date) 6. Application to be amended, documents and drawings proving authority of representation. 7. Contents of correction

Claims (27)

[Claims] (1) A method for testing the presence of antibodies specific for antigenic sites of AIDS-related viruses in a body fluid sample, the method comprising: (a) selecting the sample from the group consisting of AIDS-related viruses and fractions thereof; (b) each complex and (i) a second antibody having an affinity for alkaline phosphatase and said complex; and (ii) nitro blue tetrazolium chloride in an amount capable of producing a color change on contact with alkaline phosphatase and 5-
(c) the signal level obtained from said coupled signal generating system is indicative of the presence of said antibody in said sample. Measuring as an indicator; a method characterized by steps. (2) The method according to claim (1), wherein the second antibody is an anti-human Ig. (3) The method according to claim (1), characterized in that the nitroblue tetrazolium chloride and the 5-bromo-4-chloro-3-indolyl phosphate are used in approximately equimolar amounts. (4) The antigen is a lysate of an AIDS-related virus.
Claim No. (1) characterized in that
) Method described in section. (5) The method according to claim (1), wherein the antigen is a lysate of HTLV-III. (6) The method according to claim (1), wherein the antigen is a lysate of LAV. (7) The method according to claim (1), wherein the antigen is a lysate of ARV. (8) Claim (4) characterized in that the antigen is fractionated by electrophoresis and transferred onto a solid support medium.
) Method described in section. (9) The method according to claim (8), wherein the solid support medium is nitrocellulose. (10) A method for testing the presence of antibodies specific for antigenic sites of AIDS-related viruses in a human serum sample, the method comprising: (a) detecting the electrophoretic pattern of a protein fraction of an AIDS-related virus lysate; (b) forming an immobilized complex of the antigen and the protein fraction by immersing the adsorbed solid phase support in the sample; (b) adding the immobilized complex to anti-human Ig and alkaline phosphatase; (c) from the conjugate remaining in the solution, the immobilized complex bound to the conjugate is removed from the conjugate remaining in the solution; (d) The immobilized complex bound to the conjugate recovered in (c) above is treated with nitro blue tetrazolium chloride and 5-
A method comprising the steps of: incubating with a solution of bromo-4-chloro-3-indolyl phosphate to produce a detectable color change indicating the presence of the antibody in the sample. (11) A diagnostic test kit for analyzing a body fluid sample for the presence of antibodies having specificity for an antigenic site of an AIDS-related virus, the kit comprising: a diagnostic test kit for analyzing a body fluid sample for the presence of antibodies having specificity for an antigenic site of an AIDS-related virus; A plurality of solid phase supports adsorbed with antigens of related viruses (provided that the predetermined pattern and the density of each adsorbed antigen are substantially the same for each solid phase support); all of the solid phase supports a solution of the first component of the two-component signal generation system and a second antibody having binding affinity for the complex of said antigen and said antibody in an amount sufficient for the body; and all said solid phases. A diagnostic test kit comprising: a solution of the second component of the two-component signal generation system in an amount sufficient for the support. (12) The diagnostic test kit according to claim (11), wherein the two-component signal generation system is a system that produces a visible change when the two components are combined. (13) The diagnostic test kit according to claim (11), wherein the first component is an enzyme that can act on the second component to generate a detectable signal. (14) The first component is alkaline phosphatase, and the second component is a substrate capable of producing a detectable change upon contact with alkaline phosphatase. Diagnostic test kit. (15) A claim characterized in that the second component is a mixture of nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate in an amount that can cause discoloration upon contact with alkaline phosphatase. A diagnostic test kit according to item (14). (16) The diagnostic test kit according to claim (11), further comprising a solution of a blocking agent for reducing non-specific binding on the solid support. (17) The diagnostic test kit according to claim (16), wherein the blocking agent contains milk protein. (18) The diagnostic test kit according to claim (11), wherein the predetermined pattern is an electrophoresis pattern transferred from an electrophoresis medium to the solid support. . (19) The diagnostic test kit according to claim (11), wherein the solid phase support is nitrocellulose. (20) The diagnostic test kit according to claim (11), wherein the predetermined pattern is a Western plot of the electrophoretic pattern of the antigen. (21) The diagnostic test kit according to claim (11), further comprising a container for holding the solid support immersed in either the sample or the solution. (22) A diagnostic test kit for analyzing multiple samples of human body fluids for the presence of IgG antibodies having specificity for the antigenic site of an AIDS-related virus, the kit comprising: a long band-like antigen of the AIDS-related virus; A plurality of strips obtained by cutting the adsorbed solid support in a direction intersecting the band by Western plotting the electrophoretic pattern; A solution of a conjugate of anti-human IgG and alkaline phosphatase; Nitro blue Tetrazolium chloride and 5-bromo-4
- a solution containing approximately equimolar amounts of chloro-3-indolyl phosphate; a milk protein solution; and a plurality of liquid-retaining properties, each of a size capable of receiving one piece of the solid support fully immersed in the liquid. A diagnostic test kit comprising: a tray with parts; (23) A tray for incubating strips of solid phase material in a reagent solution, one of the strips fully immersed in each liquid.
a plurality of elongated liquid-retaining sections large enough to receive strips; and a barrier provided within the liquid-retaining sections separating the strips from one end wall and allowing liquid to flow therethrough. A tray featuring. (24) The tray according to claim (23), wherein the barrier is a protrusion that projects upward from the base of each liquid-retentive region. (25) The barrier is a pair of protrusions that protrude upward from the base of each liquid-retaining region, and the protrusions are separated by a gap narrower than the width of each piece. A tray according to claim (23). (26) A device for holding strips of solid phase material to be cultured in a reagent solution, the device comprising a plurality of devices, each of which is large enough to receive one of the strips fully immersed in the liquid. a long liquid-retaining region (within each liquid-retaining region, a barrier is provided that separates the strip from one end wall and allows liquid to flow therethrough);
and a removable liner that can be fitted onto the top surface of the tray and has protrusions projecting toward each liquid-retentive area and is used to maintain the position of each strip. . (27) The device according to claim (26), characterized in that a lid is wrapped to cover each of the liquid retaining portions while leaving room for stirring the reagent solution.