JPS6176958A - Diagnostic testing method and kit used for said method - 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 The present invention relates to agglutination tests for the detection of ligands, and diagnostic test kits for use in the detection of ligands.

Diagnostic test methods that are based on the aggregation of an immunogen and an antibody, either the immunogen or the antibody being bound to a solid layer, are well known in the field of immunodiagnostic reagents. For example, U.S. Patent No. 3.088.875
The issue is that when a specimen contains antibodies against an antigen, the antibodies bind themselves to the antigen, thereby causing visible agglutination (a) of microspheres.
(lltltination) or aggregation (aggr
describes a technique in which antigen-coated plastic centrosomes are mixed with antibodies to produce antigen-coated antibodies.

The colored solid layer or particles provide auxiliary visualization (AI) of the agglomeration process.
d visualization). For example, beta-hemolytic streptococci (aeta haemolytic), where the solid layer stains sterile red or blue.
-lytic 5treptococci) are commercially available. Streptococcus A, B.

For detection and grouping of C and G, test kits containing four separate reagents, each containing a different coloration of latex particles, are also commercially available. By assigning a unique color for each test reagent, any confusion that may arise due to, for example, inaccurate labeling is avoided.

U.S. Pat. No. 4,419,453 describes a latex agglutination test in which the test reagents consist of antigen- or antibody-coated latex particles of one color and water-soluble, non-latex polymer particles that absorb dyes of a different color. It is listed.

When agglomeration occurs, the contrast between the color of the aggregate and the old color of the solution aids in visualization.

Diagnostic techniques such as latex agglutination tests are commonly used to augment initial diagnoses based on clinical symptoms exhibited by patients suffering from a particular disease. In the case of diseases characterized by very distinct symptoms, confirmation of the presence of the causative agent (eg bacteria or viruses) may involve relatively few tests. However, in diseases where symptoms can be attributed to any one of a large number of causative agents, more time and effort can be extended in carrying out tests for each of the possible causative agents. , and the amount of specimen required from the patient can be significant.

This can be a significant problem when it is only possible to take a small sample of biological material from a patient (eg, when the test fluid is neonatal brain cerebrospinal fluid (C3F)).

Heterogenous specific binding assays are known, in which multiple ligands can be determined simultaneously in one test sample, thereby reducing the number of tests required. Such combinatorial assays may be useful when the assay is of a screening nature, for example, for Rubella
), Cytomegalovirus
virus) and herpes simplex (He
It is believed to be particularly advantageous in the diagnosis of immunity to viruses and other antigens responsible for congenital malformations such as Rpes simplex (see, eg, British Patent Application No. 2.034.466A). Combination assays can indicate a patient's immunity to two or more antigens in a single test by detecting the presence of antibodies to each of the antigens. UK Patent Application No. 2.034.
No. 466A describes such an assay in which the ligands are differentiated through the use of multiple distinct and separable solid layers. Such a solid layer can be, for example, a plate coated with one immunologically active substance in a container whose walls are coated with another immunologically active substance. Following a suitable series of immunochemical reactions, the ligand-immunochemical complexes can be separated by simply removing the plate from the container. Furthermore, different specific binding substances may bind to particles of different sizes (UK Patent No. 1,561,042), or microscopically distinguishable rosettes may form between the ligand and the binding substances. Patent application No. 2,122,345
(U.S. Pat. No. 4,436,826), in which each specific binding substance binds to a latex particle that is distinguishably labeled with a radioactive substance or a moiety detectable, for example, by X-ray fluorescence spectroscopy. The test is known.

The above methods for determining one or more ligands require complex instrumentation, or require mechanical separation of various types of solid layers, or require the use of immunogens, antibodies, and other It suffers from several drawbacks that make it inapplicable as a general means of detecting substances capable of specific binding. The above methods also generally take longer to perform than aggregation methods. Furthermore, those assays using radioactive or heavy metal components have the additional disadvantage of requiring special safety and waste disposal. Therefore, it is quick, safe, easy and
It is also widely applicable and significantly reduces the number of tests needed to identify the causative agent of an infection or disease, thereby reducing the number of biological materials that need to be collected from the patient to perform those tests. A need exists for technology that reduces capacity. Such a technology has now been discovered.

The invention therefore provides in a first aspect an aggregation method for the detection of a ligand or groups of ligands in a medium, the method comprising mixing the medium with a reagent containing two or more insoluble colored substances. each substance is suitable for forming distinct colored aggregates in the presence of a specific ligand or groups of ligands, and the presence of the ligand is determined by determining whether distinct colored aggregates are formed. It consists of making decisions.

The term distinctly colored means that the color of aggregates that form in the presence of a particular ligand or group of ligands is different from the color of any aggregates that form in the presence of other ligands or groups of ligands; means that they can be distinguished from the background color, ie, the color that reveals any non-agglomerated particles. The distinguishable colors of the aggregates are based on the fact that each insoluble substance differs in color from other insoluble substances.

It is desirable that the aggregates be visible to the naked eye.

The above agglutination test is carried out using a suitable solvent in which the colored substance is insoluble;
It is preferably carried out and washed in an aqueous solvent.

The term ligand as used in the context of the present invention refers to antigens, habtens, monoclonal and polyclonal antibodies, and other molecules that can be bound by specific binding substances. ! I
Encompasses quality. Such other substances include avidin, biotin, lectins, carbohydrates that can specifically bind to lectins, Protein A and Io.
It includes the FC fragment of G.

The medium can be a biological specimen, such as a body fluid taken from an animal, human or other, or it can be any other type of medium in which the ligand can be found. Such media include, for example, culture broths, suspensions from liquid or solid growth media, culture supernatants, tissue culture supernatants, enzymes or chemically extracted substances from bacteria and viruses [
For example, Lancefield Extract 1 for serological grouping of Streptococci
, food or environmental samples (eg, water samples from public supplies).

Biological specimens that can be collected from animals include brain, vertebral fluid, blood,
Includes urine, sputum, tissue extracts, sweat, tears, secretions, odor, mucus and synovial fluid.

The samples described above are not intended to be exhaustive, and one skilled in the art will recognize that other types of biological specimens may be collected and tested by the methods of the present invention. .

Insoluble@colored substances are colored aggregates that can be distinguished in the presence of a specific ligand or group of ligands by binding to or containing a specific binding substance or group of specific binding substances that can bind to the ligand or group of ligands. It is suitable for forming. A specific binding agent can be an immunological counterpart of a ligand; thus, if the ligand is an antibody, the specific binding agent is an antigen for that antibody, and vice versa.

It can also be substances such as avidin, biotin, lectins, carbohydrates that can specifically bind to lectins, protein A, and the FC fragment of IqG.

The insoluble colored substances are preferably microscopic particles. Particulate materials that can be colored or stained and are suitable for use in the above assays include invisible bacterial cells, alginate particles, sepharose beads, silica, alumina, red blood cells, polymer latices such as polystyrene latex, styrene-glycidyl methacrylate latex, and other polymer latexes such as those described in U.S. Pat. No. 4,419,453. Colored particles can be prepared using standard methods (e.g. U.S. Pat. No. 4.419.453 and German patent application DT-30).
00-483) or they may be purchased from suitable sources. Particularly suitable colors include red, yellow, blue, green, black, indigo, magenta and white.

The specific binding agent is attached to the particles by adsorption, by chemical coupling, by incorporation into the particles, or by any other method known in the art.

Adsorption or coating of the binding agent onto the particles is typically accomplished by incubation of the particles with a suitably buffered solution of the binding agent. Chemical coupling can be accomplished, for example, by the methods described in US Pat. No. 4,436,826, in which carbodiimide coupling reagents are used. A similar coupling method is also described in US Pat. No. 4,140,662.

The present invention is particularly useful for detecting bacterial, viral or parasitic infections and for identifying antigens or antibodies in biological fluids. It's Haemophilus mening it
r d+ s ) and Streptococcus spp., it is particularly useful in the analysis of syneresis (e.g. neonates).An important advantage of the present invention is that, compared to more conventional agglutination testing methods, It is the reduced volume of syneresis that is used for analysis.

The invention also relates to serologically distinct bacterial strains, such as Streptococcus serogroups A, B, C, D, F and G1 Salmonella O or H antigens, and Neisseria meningitidis serogroups A, B,'
Useful for identification of C, Y, 29E and Z.

It will be appreciated that each insoluble colored substance will be suitable to form aggregates in the presence of a single ligand by containing or binding to a single specific binding substance. Methods and reagents using such adapted insoluble colorants represent one preferred embodiment of the invention.

In one particularly preferred embodiment of the invention, the reagent consists of a suspension of three colored antibody-coated latex particles, the color of each particle indicating the particular specific antibody with which it is coated. The overall appearance of the reagents before reaction is dull or grayish. Following mixing of the reagent with a medium containing an antigen that can be bound by one of the specific antibodies, the antigen reacts with the appropriate antibody resulting in the formation of aggregates. It will be clear from the color of the aggregate that the antibody was involved in the reaction and hence the body of the antibody will be revealed. Visualization of aggregates is enhanced by color contrast with the background color provided by non-agglomerated particles. Thus, for example, when the reagent contains red, blue and green particles, if agglomeration of the red particles occurs;
The background is a contrasting turquoise. If blue particles aggregate, they appear against an orange or yellow background, and if green particles aggregate, the background is purpleish.

For example, rheumatoid factor [Rheumatoid F]
(RF)] or protein Δ [found in many S. aureus bacteria], and protein A-like substances (found in some streptococci). Non-specific interactions tend to appear as dark clumps on a lighter background of similar hue.

Although the preferred embodiment of Section L is described with respect to the detection of a single antigen, the invention can be used to detect more than one antigen simultaneously. Thus, for example, in the three-color system described above, the simultaneous presence of two antigens can be demonstrated, for example, by the formation of aggregates of blue and green particles against a red front, or by aggregates of green and red particles against a blue background. In such cases, it can be readily appreciated that background color is particularly useful in allowing the results of immunochemical reactions to be interpreted.

It can also be recognized that each insoluble colored substance is likely to be adapted to form aggregates in the presence of a specific group of ligands. For example, each substance may contain or bind antibodies to two or more bacterial or viral antigens.

Accordingly, in another embodiment of the invention there is provided a method of aggregation substantially as described above, but the reagent contains at least two and preferably at least three insoluble colored substances, at least one of which and Desirably at least two each contain or are bound to at least two specific binding substances. The individual specific binding substances that make up the group can be combined with specific insoluble@color substances, or some of the binding substances can be common to two or more insoluble color substances.

In the former case, the agglutination method "localizes" the range of possible infectious agents to a small group of such agents.
Further testing is then required to accurately identify the infectious agent. are observed to be particularly useful in the diagnosis of diseases such as bacterial or viral diseases.

An example of the latter could be, for example, a reagent consisting of red, blue and green latex particles, the red particles being coated with antibodies against antigens A and B, the blue particles being coated with antibodies against antigens B and C, and the green particles being coated with antibodies against antigens B and C. Antigen △ and C
coated with antibodies against. Addition of a test specimen containing one antigen to which the reagent is sensitized results in co-aggregation of the two colored particles, and subsequent identification of the antigen is most easily made on the basis of the background color. Thus, for example, if the test specimen contains antigen A, the red and green particles will co-aggregate resulting in an easily recognizable blue background.

When one or more specific binding substances are common to more than one insoluble coloring substance, by using appropriate substitutions of insoluble coloring substances and specific binding substances, the number of insoluble coloring substances used is The ability to specifically detect up to 2 -2 Gantts can be determined by a skilled person. Therefore, three color reagents can be used to detect and identify six ligands. For example, such a reagent may consist of red, green and blue particles, with the red particles containing ligands A, C
sensitized to J5 and D, green particles are sensitized to ligands B, E
115 and D, and the blue particles are sensitized to ligands C, F and E.

The results of each addition of ligands A-F are shown below.

Ligand Aggregate color Background color (aggregated particles)
(Non-aggregated particles) △ Red Blue + Green B Green Blue + Red C Red + Blue Green D Red + Green Blue E Blue + Green Red F Blue Red + Green As shown, the color of the aggregates is based on the ligand A1B view and F. The background color is most useful in the detection and identification of ligands C, D and E, while the background color is most useful in the detection and identification of ligands C, D and E.

In each of the above methods according to the invention, it is in some cases desirable to pre-treat the medium before the test; methods of such pre-treatment include treatment with acids, or enzymatic extraction, and also filtration, centrifugation. , dilution, concentration and heating.

For example, it is possible to inactivate or significantly reduce the activity of the interfering substances by heating.

In conventional agglutination techniques, the antigen under test or the animal immunoglobulin fraction (hereinafter referred to as control antiserum) of the same type but with different specificity used on the test latex is coated with It is common to use a control latex which is a suspension of latex particles. The agglutination of the control latex in the presence of the test analyte is
Indicates a non-specific interaction.

A disadvantage of using control latex reagents in conventional methods is that at least one additional portion of biological test fluid is required for each test or series of tests, and this additional portion is subsequently not useful with respect to the identification of infectious agents. generate information.

A technique has now been discovered in which the unfavorable requirements of separate control latex reagents are overcome. Accordingly, in another aspect of the invention, a direct agglutination test for the presence of a ligand in a medium is provided, in which the medium is coated with (1) an antibody capable of binding the ligand, and (ii) a control antibody. the antibody is insolubilized by binding to the particles of the first color; observing whether agglutination has formed; and determining the color of the agglutination. It is something that

For example, in a typical test method, a test specimen is mixed with a reagent containing blue latex particles coated with antibodies against the antigen to be detected and red latex particles coated with a control serum. If the suspected antigen is present and there are no non-specific interactions, blue agglutination will occur against a red background. If a non-specific interaction occurs, both red and blue particles aggregate to produce a purplish mass. Although red and blue particles are described as examples, it will be clear to one skilled in the art that any two contrasting colors can be used in their place.

In yet other embodiments, the invention provides a kit for use in detecting a ligand, the kit comprising a reagent containing two or more insoluble colored substances, each substance binding to a specific ligand. and has its own distinctive color.

In still other embodiments, the reagent comprises an antibody capable of binding the m ligand, and (ii) particles of a second color coated with a control serum, wherein the antibody binds to the particles of a first color. It is insolubilized.

Also provided are kits comprising reagents containing two or more insoluble colored substances, at least one and preferably at least two of which each contain or bind at least two specific binding substances. .

Others that may be usefully included in the kits are infusion cards, mixing sticks, positive control antigens for each antigen to be tested, negative control antigens (e.g., saline solutions), and test kit use. Contains a set of instructions for

The invention will now be illustrated by examples. The examples should not be construed as imposing limitations on the scope of the invention.

Example 1 Production of sensitized latex ■ Production of antibody Immunoglobulin G was mixed with Steinbach (5tein
buch) and Audran>a
nd Biophys, ), 134.279-28
Octanoic acid [B.D.H. Chemicals Co., Ltd.
1q by processing at DH Chemicals Ltd, )'].

■ Binding of antibody to 0 colored latex Colored latex [EStapOr, K2S, 0.2
μ, polystyrene, black, red, blue, yellow or green, lawn-
Rhone-Poulenc, Collar Revised Application No. 8-016 "Glycine salt solution pH 8 for cows"
,2 [0,85% NaCj! 0.1M glycine in l
] It was adjusted to 8.2 with NaOH] 600 ug of antibody in 11 nl was added. Latex and antibodies at 56℃
was heated for 30 minutes. After cooling to room temperature, bovine albumin [Miles Lab
oratories Ltd, )] and 1% (W/
V) Obtained 111 degrees.

■ Production of multivalent latex Three different colored latexes, each sensitized with antibodies of different specificity, e.g. red latex coated with antibodies against Salmonella serogroup A, blue latex coated with antibodies against serogroup B and green latex coated with antibodies against serogroup C were mixed together in equal proportions. The latex produced was brown in color.

Example 2 Use of polyvalent latex A latex agglutination test was performed using a white card [5yracard-R], Wellcome Diagnostics [1ia].
lnO3tics Ltd)]. Equal volumes of test specimen and multivalent latex (usually 20μ) were mixed together in a 2 cm diameter circle on a white card. The card was shaken for 3 minutes, after which agglutination of the specimen was confirmed. The color of the aggregates was identified as red, blue or green, for example, while the color of the unagglomerated latex changed from brown to a combination of the colors of the two particles that remained unagglomerated in suspension.

@ Bacterial Colony Identification A single colony of bacteria is removed from the solid growth medium and its
．． , To, 85% Saline 200μ
emulsified in l. The bacterial suspension was mixed with a polyvalent latex as described above.

Antigen Result 1) Saline only Brown homogeneous solution 2) Salmonella serogroup A Red in blue-green solution, e.g. Salmonella aggregates 3) Salmonella serogroup B In orange solution e.g. Salmonella blue aggregates 4) Salmonella For example, Salmonella aggregates (S, nevport) (C) Antigen detection in biological fluids W cerebrospinal fluid collected from a patient with meningitis was collected from a patient with Salmonella polyvalent serogroup C as described above. Tested against latex.

produced aggregations of red latex particles supporting the presence of antigens from Salmonella serogroup B organisms in the cerebrospinal fluid. CSF from uninfected individuals did not produce latex agglutination.

Claims (8)

[Claims] (1) an agglutination method for the detection of a ligand or groups of ligands in a medium, comprising mixing the medium with a reagent containing two or more insoluble colored substances;
Each substance is compatible with the formation of distinct colored aggregates in the presence of a specific ligand or group of ligands, and the presence of the ligand is determined by determining whether distinct colored aggregates have formed. Method. (2) The aggregation method of claim 1, wherein each insoluble colored substance is adapted to form an aggregate in the presence of a single ligand. (3) The aggregation method according to claim 2, wherein the reagent consists of latex particles of three colored substances. (4) The agglutination method according to claim 1, wherein the reagent contains at least two insoluble colored substances, at least one of which contains or binds to at least two specific binding substances. (5) A kit for use in detecting a ligand or a group of ligands according to the method of claim 1, comprising a reagent containing two or more insoluble colored substances, each substance being a specific ligand or ligand. The kit is adapted to form distinct colored aggregates in the presence of groups. (6) A kit for use in detecting a ligand according to the method of claim 3, wherein the reagents contain latex particles of three colors. (7) In a direct agglutination test method for the presence of a ligand, the medium is combined with (i) an antibody capable of binding the ligand and (
ii) mixing with a reagent containing particles of a second coated with a control serum; observing whether agglutination has formed; and determining the coloration of the agglutination; A method in which the substance is insolubilized by adhesion to particles. (8) A kit for use in a test as defined in claim 7, comprising: (i) an antibody capable of binding the ligand; and (ii) particles of a second color coated with a control serum. A kit comprising a reagent containing the antibody, the antibody being insolubilized by adhesion to particles of a first color.