JPH07508102A - assay - 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] assay The present invention relates to immunoassays.

Body fluid samples obtained from living organisms, and solid samples such as 1f cells or ( diseases of various organisms, especially bacteria, viruses, parasites, and Tests for the presence of pathogenic (infectious) organisms are routinely performed. The inspection is mainly (these two) Implemented from the perspective of One is to diagnose a disease in a single individual and monitor the progress of the disease. 1. Sample collection and solidification of body fluids for the purpose of treatment monitoring and/or treatment. This is a body sample test. This type of test is often referred to as a "clinical" test. ), clinical laboratories typically perform tests for many different organisms1. As used herein, the term pathogen refers to an organism that causes a disease.

Another major type of test is to check whether blood and blood products are contaminated with pathogens. Screening of blood donations to maintain supply. Regulatory bodies in each country are testing power We have identified pathogens that should be addressed more closely. In most countries, HIV, Hf inflammation ( Screening for Cyto A non-B hepatitis), hepatitis B, and syphilis (required) Some countries also require testing for HTLV. most In this country, testing for both HIV-1 and HIV-2 is essential.

For patients undergoing or undergoing organ transplantation or immunologically compromised patients. Blood transfusions (used for this purpose) generally contain CMV (cytomegalovirus). The presence of new pathogens that pose a threat to the blood supply and As new subtypes of known pathogens are discovered, testing of blood donations will become mandatory. (This will expand to include those pathogens.)

The most widely used test for blood screening is the immunosomatic test. Ru. For most bloodborne viral pathogens, test blood for the presence of antigens. Obtaining the required sensitivity when testing for the presence of antibodies is more difficult than when testing for the presence of antibodies. Generally, testing for HIV, HTLV, HCV and CMV is difficult. This is a physical exam. There are two tests for hepatitis B. In other words, hepatitis B surface anti- Antigen assay for antigen (HBsAg) and hepatitis B core antigen (HBc) This is an antibody test that Currently, HBsAg testing is mandatory in most countries. type B liver Testing for inflammatory core antibodies is mandatory in some countries and will be introduced in more countries in the future. There are both antibody tests and antigen tests for syphilis, which is thought to be introduced. .

Blood screening is done on a large scale and results are quick because blood has a relatively short shelf life. To save both time and money, it is especially important to get it quickly. , assays have been proposed that can detect two or more pathogens in a single test.

Two subtypes of the same pathogen, specifically HIV-1 and HIV-2 (HIV- 1+2), and also measured HTLV-I and HTLV-I[(HTLV-I+I[). Antibody assays are commercially available to determine the However, homologous subtypes Considerable cross-reactivity is often observed between HIV-1 and HI The degree of immunological cross-reactivity between V-2 and was actually detecting a significantly large population of HIV-2-containing blood. should be kept in mind. Therefore, it is also stated in EP-A-0484787. to provide antibody assays for two subtypes of the same virus. antibodies against two completely different pathogens that show virtually no immunological cross-reactivity with There are quite significant technical differences between providing assays. here So, the term ``combination assay'' refers to a single test on a sample. Detects or measures two or more pathogenic organisms with virtually no immunological cross-reactivity It is used to mean an assay for Here, the term "different pathogens" Used to refer to pathogens that exhibit virtually no immunological cross-reactivity.

EP-A-0484787 covers viral pathogens that may be present in blood donations. Combination antibody assay for (blood viral pathogens) especially HIV A combination assay for HCV (hepatitis C virus) has been proposed. It is. The proposed heterogeneous phase assay involves coating a solid surface that captures the relevant antibodies. This method uses a variety of peptides that have been isolated. The obtained antigen-antibody complex is It can be detected by

Such combination assays, as well as currently commercially available HIV-1+2 The major drawback of the and HTLV-1+II combination blood assay is that the solid There is a need to co-coat multiple antigens on a surface. Antigen (peptide or Not only must the coating be uniform (coating and/or proteins), but also The pitope must be coated so that it is available for antibody binding; 1) There should be no interaction between various antigens, and the antigens should be kept in a satisfactory condition. The quality control tests that must be performed to ensure that .

Adding the cost of both the test itself and the cost of failing products Become. The problem gets worse and costs increase as more types of antigens are coated.

Addition of one antigen to be detected requires addition of at least one immobilized antigen become. For some pathogens, it is necessary to be able to detect multiple types of antibodies. parable For example, in the case of HCV, currently commercially available assays consist of four different antigens. It is. In the combination HIV-1+2/HCV assay, at least Six types of antigens are also required.

In addition to the co-coating issues in actual manufacturing, obtaining the required specificity is also 1 Extremely pure antigen with strong antigenicity is required (EP-A- 0484787).

Antibody/antigen combination assay for HIV and hepatitis B surface antigen Proposed. For example, EP-A-0286264 is capable of binding to HIV antibodies. Coating the solid phase with a peptide specific for HBsAg and coating the solid phase with an antibody specific for HBsAg. Coating is proposed. The two solid phases may be different and may also be The antibody and antibody may be coated on the same solid phase, as described in WO 91/10747. proposed an improvement to the type of assay described in EP-A-0286264. This is a peptide used for coating and detection of HIV antibodies. improvements in the properties of the detection system as well as in the properties of the detection system.

However, the only commercially available product based on the principles disclosed in EP-A-0286264 In HIV and HBsAg combination assays, co-coating Various problems arise when attaching HIV peptides to the internal surface of microtubes and in combination with the tubes. By coating anti-HBsAg antibodies on the beads used in It should be noted that this is avoided. Moreover, the sample is placed in a bead inside the tube. Remove beads after contact with beads and detect separately on beads and tubes As it involves several steps, it cannot be considered a true combination assay according to current understanding. Therefore, regarding the interaction between the labeled antibody and the labeled antigen used for 7 detection. Possible problems and during detection system (labeling) during detection of formed immune complexes potential problems have been avoided.

In the present invention, various types of antibodies are coated on a solid surface for a combination antibody assay. The problem of co-coating materials and the resulting consequences is that immunoglobulin It is simply and elegantly overcome by using the Brin capture format.

The immunoglobulin capture format itself has been known for many years. that In 1979, Duermeyer, W. et al. Regarding the detection of specific 1 gM antibody by ELISA, Journal of M It was described in edical Virology, 4:25-32. from humans The principle of the assay was described for the samples obtained.

Coat the wells of a solid phase, e.g., a microtiter plate, with anti-human IgM. Add the sample to the coated well and incubate the well. The antigen is then added to the wells and incubated, and finally the bound antigen is washed. is detected using an enzyme-labeled antibody. The authors of this paper, for example, .

Herpes simplex, CMV, EBV, I-xoplasma, hepatitis B core antigen or others proposed for use in assays against individual IgM antibodies such as antigens.

Assays that use immunoglobulin capture techniques for the detection of single pathogens in blood Rubella and hepatitis A assays have been used for several years.

WO38107680 describes antibodies in body fluids other than blood, especially saliva and urine. The application of immunoglobulin capture technology to measurements is disclosed. commercially available The saliva and floor HIV-1+2 assay uses the described IgG capture technique. Ru.

Although this technology has been available since 1979, it is difficult to capture antibodies. Compared to the many commercially available assays that use antigens for This is also evidenced by the fact that only a very limited number of assays are commercially available. There is a general prejudice against immunoglobulin capture technology in this technical field. It is considered that In particular, this technique is practical in terms of specificity or sensitivity.

There appears to be a general perception that it will not work effectively.

Significant levels of undesirable interactions are expected with immunoglobulin capture techniques. It is thought that this causes a lack of sensitivity. For example, many In WO 39/12231, which describes antibody capture assays for the detection of is a non-immunological protein-protein interaction between an antigen and an immunoglobulin Fc region. Coatings on solid surfaces do not raise concerns about the resulting lack of specificity. An immunoglobulin capture assay has been proposed in which the detected antibody is an anti-Fc antibody. 9 That is, immunoglobulins do not specifically capture the Fc region; Not used for non-specific protein-protein interactions.

Sensitivity is also important because, for example, immunoglobulins are not selectively captured and therefore the specific No enrichment of target antibody species occurs, which can be a problem in immunoglobulin capture assays is possible. whether the specific antibody species is present in an absolute sense or in a serum sample; given that they are present in very small quantities, both relative to the pond species present.

Detection of the antibodies under study is difficult and unreliable, especially as the assay lacks sensitivity. There is expected.

Unexpectedly and surprisingly, the inventors used immunoglobulin capture techniques to The ability to detect two or more different pathogens in the sample under study with excellent sensitivity and specificity. They discovered that they can be detected simultaneously.

The present invention provides a method for measuring antibodies against two or more different pathogens in a single test sample. Provided is the use of immunoglobulin capture techniques in.

That is, the present invention provides specific properties for two or more different pathogens in a liquid test sample. In a method for measuring foreign antibodies.

(i) Samples are tested for antibodies against immunoglobulins of ■ or two or more classes. 1 or 2 or more of each class present in the sample by contacting with a defined solid phase of immunoglobulin is captured on a solid phase.

(ii) examine the solid phase on which the immunoglobulins from the sample are captured; two or more species, each of which can selectively bind to an antibody specific to one of the pathogens different antigens, each of which produces a directly or indirectly detectable signal. and then simultaneously or sequentially contact the antigen being provided with the means to provide it.

(iii) Measuring the immunoglobulin-antigen complex formed on the solid phase Provides a method consisting of:

The immobilized antibodies may be directed against IgG, IgM or IgA.

It is also possible to use mixtures of antibodies against different classes of immunoglobulins. .

Particularly preferred are mixtures of anti-IgG and anti-IgM antibodies.

Assays of the invention [Combination assay J for antibody capture.

Since it uses a universal solid phase, it has infinite versatility. Users should consider Simply determine which antibodies and therefore which pathogens in a sample that are suitable for detection. can be selected by using a suitable antigen reagent. This is used to capture antibodies. coated with a different antigen for each combination of antibodies being investigated. This is in stark contrast to conventional assays, which require a solid phase that has been analysed.

The versatility of using a universal solid phase makes it ideal for multipathogen assays, especially blood screen tests. for both manufacturers and users of testing and clinical assays. This is a big advantage. As noted above, the universal antibody-coated solid phase , e.g. coated beads or coated microtiters - The wells of the plate will be used for detection of any particular antibody assay selectivity. of any and all combinations of pathogens, as determined by the combination of antigen reagents. It can be used for antibody testing.

This transferability makes it a universal antibody-coated solid phase for blood screening. For all combination antibody assays, whether for clinical or clinical testing. This is advantageous for assay manufacturers. At present, blood donation Mandatory requirements for physical examination vary from country to country. Therefore, different regions This requires a solid phase coated with a different antigen. for all regions Providing a universal antibody-coated solid phase significantly reduces manufacturing costs. do. In the case of clinical testing, each and every different combination antibody assay is For biochemistry, solid phases coated with different antigens are required. In this case too , manufacturing costs are significantly reduced by providing a universal antibody-coated solid phase. do.

A further advantage for manufacturers is the reduction in the number of components coated onto the solid phase. Therefore, manufacturing costs are reduced. antigen to capture antibodies The assay used allows for combinations of pathogens, even in the minimum case of two pathogens. Antibody assays require a wide variety of antigens. For example, combination HCV/H The IV-1+2 assay requires at least six different antigens. detection As more pathogens become available, more antigens must be coated onto the solid phase. However, according to the present invention, two additional pathogens can be detected. There is no need to immobilize the antibody on a solid phase; the presence of anti-1gG alone can inhibit various pathogens. It is possible to detect coatings, especially compared to the high purity antigens required for multi-antigen coatings. It is.

The transferability of the combination assay of the invention is also significant. For example, any pathogen Because specific combinatorial tests can be performed simply using the appropriate antigen reagent combinations, , which is also advantageous to the user. This allows the testing of any desired combination of pathogens to be What you can do by purchasing and storing one universal antibody-coated solid phase Therefore, it is particularly useful for clinical tests.

Blood screeners do not require knowledge of the nature of the contaminant for screening purposes. In case of infection, no matter if the blood sample is positive for any antibodies of the prohibited pathogen. , the blood is unsuitable for transfusion or for the production of blood products such as plasma or factor This is because it is true and must be discarded.

A single application for all circulating viral antibodies as defined by specific country regulations. The ability to test donated blood samples in a laboratory test is a major advance and will save blood banks time. 0 hours of savings, which provides very substantial savings in both time and expense. is not only important in saving technician time and therefore cost. It is also important because of the relatively short shelf life of whole blood.

The immunoglobulin capture assay of the present invention is a single-shot assay with excellent specificity and sensitivity. For a sample containing a mixture of antibodies of interest, each Substantial results obtained for corresponding serum or plasma samples containing only the species In the combination assay of the present invention we have obtained a total of 0 results.

Each of multiple antibodies directed against different organisms is treated as if it were a single antibody species. It was completely unexpected that the body would react as if it were present. Ru. Observation of additive effects for the large number of antibody species detected is unexpected offers significant practical advantages. Conventional assay format.

For example, the assay of the present invention, which can be performed using a microtiter plate, It was also unexpected that the sensitivity would be so high.

The assay of the present invention may be an IgG assay, a 1gM assay, or for special purposes. It can also be an IgA assay. Also, for different classes of immunoglobulins Mixtures of antibodies can also be used to coat the solid phase. Anti-IgG It is preferred to use a mixture of anti- and anti-1 gM antibodies.

compared to assays using coated antigen for antibody capture. A further important advantage of the assay of the present invention is that it uses immobilized anti-1gM This allows reliable detection of early infection. This is a blood screen. It is important in both reading and clinical examination.

IgM is the first class of immunoglobulins produced in response to infection.

Levels of specific IgM antibodies rise during the first few weeks after infection, then a little later. Specific 1gG is produced. IgG antibody responses are long-lasting, lasting for years and even It may last a lifetime. Theoretically, IgM can be used when the antibody of interest is coated with antigen. The captured antibody is detected using, for example, a labeled anti-IgM antibody. can be detected using conventional assays. However, in practice, like this A 1gM assay is generally performed in which the IgM is [sticky], i.e., the IgM is used for capture. They lack specificity because they tend to bind nonspecifically to antigens. antigen -The labeled anti-1gM antibody used to detect the IgM complex is specific for the antigen-1gM For this reason, it is not possible to distinguish between complexes and non-specifically bound IgM. Conventional assays using antigens generally use anti-IgG antibodies for detection and ultimately The assay is performed on IgG only.

In the assay of the invention, anti-1 gM antibodies can be coated onto a solid phase.

These antibodies then specifically captured 12M immunoglobulin from the sample.

The inventors were surprised to find that captured IgM is specifically detected by labeled antigen. Surprisingly, this system is not "sticky" and requires conventional antigen- They found that there were no specificity problems with capture assays. as mentioned above To coat the solid phase, a mixture of anti-IgG and anti-IgM antibodies was used. It is particularly preferred to use the presence of anti-IgM antibodies on the solid phase in the sample. Detection of initial antibodies present in Detection of sexual antibodies is guaranteed.

The antibodies immobilized on the solid phase are polyclonal antibodies, e.g. all immunoglobulins. Can be polyclonal anti-human antibodies, including antibodies against the phosphorus class. Ru. Also, polyclonal stakes and trees directed against specific classes of immunoglobulins For example, using polyclonal anti-IgG and polyclonal anti-1gM antibodies You can also do that. For example, polyclonal antibodies can be produced using affinity chromatography. It can be purified by Monoclonal antibodies can also be used if desired. anti-immunity Epiglobulins are IgG, IgM, and IgA immunoglobulin gamma.

Specific for μ and α chains.

Polyclonal and monoclonal antibodies used to coat solid phases are This can be manufactured by methods known per se. Various anti-immunoglobulins, such as rabbit 1 Sheep and goat polyclonal anti-1gG and anti-1gM immunoglobulins.

and mouse monoclonal anti-1gG and anti-1gM immunoglobulins are commercially available. It's on sale. When a mixture of different antibodies is used for coating, The ratio of different antibodies can be varied as desired9. It is not limited to the examination of samples, and veterinary applications are also possible. However, Therefore, the antibodies used in the coating are immunospecific to the species from which the sample being investigated is obtained. In the case of samples from humans, which must be tested against epidemic globulin. For example, the coating antibody must be an anti-human antibody.

As mentioned above, one particularly advantageous feature of the assay of the present invention is that it Universal antibody-coated solid phase means can be used regardless of the That is. What antibodies are used as coating antibodies? or 2 or more classes Antibodies against immunoglobulins, especially IgG and TgM, that are isolated from a sample Each class of immunoglobulin is captured in its indicated proportion. specific By selecting a specific combination of antigens, the assay user can The food can be tested for pathogens. Antigen reagents are provided pre-mixed. It is also possible for the user to use individual antigen reagents in any desired combination. When used individually, solids with captured immunoglobulin The surface is contacted with antigen reagents simultaneously or sequentially in any order. desired mixture of reagents. It is generally more convenient to add the compounds in one step.

The antigen used in the assay of the present invention is an antigen that selectively interacts with the antibody under investigation. can be any antigenic entity. For example, an antigen can be a peptide or polypeptide. Whether it is a peptide, a synthetic or recombinant antigen, or a virus, for example, from cultured cells. In some cases, for example, for HCV, the antigen may be purified from a lysate.

Using a recombinant fusion polypeptide consisting of two or more antigenic regions of a specific organism It is particularly useful to

The antigen itself allows for the detection of immunoglobulin-antigen complexes, either directly or indirectly. can be labeled by any means capable of providing a detectable signal that enables the labeling.

The labeled antigen is called an [antigen conjugate].

The detectable signal can be an optical, radioactive or physicochemical signal For example, dye 1 colored particle directly, the radiation source! The active species 5 magnetic resonance species or fluorescence By labeling the antigen with an antigen or indirectly, it is possible to induce any kind of measurable change provided by labeling with the enzyme itself, which can be activated. alternatively The signal is due to aggregation, diffraction or birefringence effects involving the antigen conjugate. It can be anything.

In other embodiments, the means capable of indirectly providing a detectable signal is an antigen. It consists of antibodies that bind to. As mentioned above regarding the antigen, the antibody (Abl) can provide a direct or indirect means of labeling (single-antibody detection system).

It also binds to Abl, itself directly or as described above in relation to antigens. or can be indirectly labeled using a further antibody (Ab2). (dual antibody detection system), single or Dual antibody systems are particularly useful when production of suitable antigen conjugates is difficult. part For example, some antigen conjugates have insufficient stability to be commercially available, and some The original substance may lose its antigenicity upon conjugation.

Single-antibody detection systems have the advantage of requiring fewer washing and incubation steps. On the other hand, it is necessary to manufacture antibody conjugates (labeled antibody Ab+) for each antigen to be detected. It has the disadvantage of being necessary. A double antibody detection system uses appropriate selection of Abl and Ab2. Depending on the selection, one antibody conjugate is used for detection of all captured antibodies as described below. (labeled antibody Ab2). Each antibody (Abl) When excited in the same animal species, the second antibody (Ab2) is an anti-one species antibody. This is because it can be done. For example, if each antibody Abl is a sheep antibody A labeled anti-sheep antibody can be used as Ab2. Testing of all test substances The benefit to the user is that only one antibody conjugate is required for production, especially in automated systems. extra incubation steps and extra washing steps are required when using This is enough to compensate for the shortcomings. Advantages of only needing one antibody conjugate also provides other significant benefits to manufacturers.

Particularly preferred labeling systems for antigen or antibody conjugates are enzyme labeling systems, especially antigen or antibody conjugates. The antibody is conjugated to an enzyme that catalyzes a detectable color change in the presence of an appropriate substrate. It is a system that has been developed. This format, enzyme-linked immunoassay or ELISA , widely used in commercial products, such as microtiter plates, proprietary product “beads” or “rlMX” (trademark) system, or hollow rods. Automated equipment is used to perform such assays using pipette tips. possible, and computer software can be used to process the results obtained. 6 The enzyme systems used are well known per se, e.g. ELISA and 0therSolid Phase Immunoassays, Theor ``Etical and Practical Aspects'' @D, M.

& Challacombe S, J, II.

Examples of desirable enzyme systems are alkaline phosphatase, β-galactosidase.

using urease or peroxidase such as horseradish peroxidase. There is a system that

The solid phase on which the antibody is captured may be, for example, beads or a microtiter plate plate. well or cup, and other forms such as solid or hollow rod or pipette, or e.g. Particles of 1 μm to 5 mm ( Such particles, regardless of the material from which they are made, can be classified as "latex" (often called particles).

The solid phase is made of plastic or polymeric material, e.g. nitrocellulose, polyvinylvinyl Nyl chloride, polystyrene, polyamide, polyvinylidene fluoride or others The particles may also be made of natural polymers, such as rubber. It can be made of tex or protein. microtiter plate and Beads are used extensively in both blood screening and clinical testing, and are commercially available. is widely available.

Other solid phases that can be used include, for example, porous fibrous or hygroscopic materials, such as iron, polyvinyl chloride or other synthetic polymers, natural polymers e.g. Cellulose, derivatives of natural polymers such as cellulose acetate or nitrate membranes, sheets and strips made of cellulose or glass fibers are included. Paper such as diazotized paper can also be used. For example fibrous or hygroscopic materials Films and coatings of e.g. the materials mentioned above may also be used as solid phases. Can be done.

The above-mentioned examples of solid phases are given for illustrative purposes only; The present invention is not limited to use in immunoassays. It can be carried out on any solid phase.

Solid phases such as membranes, sheets, strips, films or coatings can be or, more commonly, loaded into a device for the measurement of a single sample, here The term ``assay device'' as used in ``assay device'' refers to a solid phase, typically a layered solid phase.

e.g. membranes, sheets, strips, coatings, films or other layered means on which antibodies against one or more classes of immunoglobulins are immobilized. means a means for carrying out an immunoassay consisting of a solid phase that is .

The immobilized antibody preferably has a defined region, herein referred to as the "antigen capture zone". exists in

Assay devices introduce a solid phase within a rigid support or enclosure, which also It consists of some or all of the reagents needed to perform the assay. sample is common sample, e.g., onto the assay device at a predetermined sample application area. inject or drop into the area or immerse the relevant part of the device into the sample. Applied by soaking. Sample application area is different from antibody capture area In some cases, the device generally allows antibodies in the sample to migrate to the antibody capture zone. The sea urchins are placed in The necessary reagents are then applied in the appropriate order to a given application castle. . This area may or may not be the same as the sample application area; again, the reagent If the application area is at a different site than the antibody capture area, the device will generally The body is positioned to move to the capture area. Here, the antigen-antibody complex formed Detected. All or some of the reagents needed for the immunoassay can be liquid or dry. introduced into the device in the form of In this case, the device generally Different devices that happen when done automatically or by the user of the device Interactions between sites that cause various reagents to interact with each other in the case of an immunoassay being performed Arranged to make contact in the correct order.

A wide range of assay devices have been described in the immunoassay literature. Examples of this are described in U.S. Pat. ing. Due to their design and speed of operation, some assay devices Some devices are also called "rapid assay" devices. It is called. “Rapid assay” devices typically take 10 minutes from sample application (regular microtiter plate or bead assays give results within Requires an incubation step and generally requires at least 1 incubation step before yielding results. therefore, the assay device is a microtiter or Although more expensive than other format assays, it can be used, for example, if results are needed quickly. It has particular application in clinical examinations, for example in the case of emergency surgery.

Assay devices do not require sophisticated laboratory equipment or require no laboratory equipment at all. It has the special advantage of being usable even when So these are e.g.

In emergency wards, at doctors' surgeries, in pharmacies and sometimes for home testing.

Can be used as an “on-site” test, these are useful in areas with little laboratory equipment. It is especially useful.

As mentioned above, the combination assay of the present invention is useful for screening blood donations. It is especially useful. That is, the assay is performed on HIV, such as HIV-1, HrV- 2 and HIV subtypes such as HIV-1 subtab-r; HCV; hepatitis B; inflammation (antibodies against core antigens); HTLVs such as HTLV-I and HTLV- 1[:CMV; EBV (Epstein-Perle virus); and optionally Can be used to test for antibodies against at least two viruses selected from syphilis .

,! =　<　　<　i:HI　Vtatoeba) IIV-1+2　;HCV, HTLV and ,? -1fHTLV-I+[; and hepatitis B core (HBc); e.g. and HC’J: HIV and HTLV; HTLV and HCV; HIV, HCV and HB c: HIV, HCV.

A combination of two or more selected from HTLV and HBc is preferred, and the above-mentioned syphilis An important combination is the regulation of a particular country. In some cases, blood donations may be contaminated with other pathogens. You can also be tested for rubella.

In addition, the demand for blood screening increases as new pathogens (new organisms and New subtypes of known organisms have been re-examined to cover these pathogens. Essential assays will be expanded to For example, now in most countries Testing for both HIV-1 and HIV-2 is required. Recent HrV-1 The discovery of subtype 0 led to the requirement that this subtype also be detected. I'm there. In other words, it is unlikely that the combination of pathogens that need to be detected will expand in the future. Certainly, the present invention also encompasses assays for such combinations of pathogens.

Further convertibility can be introduced using a two-component tube/bead system. for example After contacting the tube and beads with the sample, separate the beads and is contacted with antigen(s) against pathogens of two or more different species, and The tube can be contacted with different antigens or combinations of antigens.

Other variations of the tube/bead two-component assay format include ■ or two or more A class of anti-immune globulin is immobilized on one component, and an anti-hepatitis B surface antibody is immobilized on one component. A format in which one component (anti-HBsAg) is immobilized on the other component, e.g.

Anti-1gG and optionally anti-1gM were added to the inner surface of the small tube with anti-HBsAg. There are funa mats that are coated onto beads and vice versa. beads and Next, Part 1: In the assay combining HBsAg/multiple antibodies hand.

and <l: HBsAg, HIV, HCV, and optionally hepatitis B (:ff7) .

HTLV and one or more additional test substances selected from syphilis. It can be used to say.

Peptides and polypeptides that specifically interact with HIV-1 and HIV-2 is well known and is described, for example, in EP-A-0307148. 9 The present inventors blocked the sulfhydryl group of the cysteine residue of 1 synthetic HIV peptide. Blocking with a detergent (see EP-A-0307149) is particularly useful.

The resulting labeled peptides, especially enzyme-labeled peptides, can be used in assays with equivalent unblocked peptides. It was found that the peptide has higher antigenicity than the truncated peptide.

The antigen used to detect hepatitis B core antibodies is the core antigen. Various types of hepatitis B The DNA and predicted protein sequence of Taib are described, for example, in Ono et al. (198 3).

Nucl, Acjds Res, 11.1747, and this report suitable peptide and polypeptide cores, both synthetic and recombinant, from Antigens can be induced.

It is currently clear that hepatitis C has one immunologically dominant epitope. However, it is preferable to test antibodies against two or more regions. For example, as an antigen, two or more epitopes, such as CB-A-2,239,24 5, for example at least one structural region and at least Using a fusion protein composed of amino acid sequences from one nonstructural region is advantageous. Antigens from the core and envelope regions are preferred structural antigens. Ru. Nonstructural antigens include, for example, NS3. You can choose from the NS4 and NS5 areas. I can do it.

The HTLV antigen may be, for example, p21e or gp46 recombinant protein, or p21e or gp46 recombinant protein, or p 21e or gp46 (e.g. , U.S. Pat. No. 4,743.678), v4 p24e is manufactured by Cambridge eBiotech Corporation, 1600 East Gude Drive, Rockville, IJD 20850-5R00. USA mosquito et al., gp46 is from Repligen Corporation, I. Kendal 5 square, Building 700°Cambridge, Ma 0 2139. Available from USA.

For detection of CMV antibodies, purified cultured CMV core protein, such as p66, or can use recombinant pptso that is dominant in Western blots. FBV antibody test Capsids or early antigens can be used for production. Purified syphilis is used to detect syphilis antibodies. Pyrochete antigens can be used.

The combination assay of the present invention can be used in clinical diagnosis to detect the reserve of several pathogens. Can be used for clinical screening tests. If the test is positive, then test for each pathogen. Separate tests can be performed with If the result is negative, further investigation is required. Overall, two hours and money are saved. Examples of such combinations include:

It is used in the so-called rTORcH screening of pregnant women, which is carried out in many countries. There are some combinations. The combination of pathogens considered depends on the blood screening requirements. Similarly, it varies depending on the country, but in general, rubella, somatosoplasma, CMV, and and herpes. Mixture of immobilized anti-1gG and anti-1gM The use of is particularly advantageous since there is a guarantee that recent infections will be detected.

Assay device formats, especially [provided in Rapid Assay J format] The combination assay of the present invention can be used in situations where screening results are urgently needed. This is particularly useful in cases such as emergency surgery. For example, two or more different blood medium viral pathogens, such as HIV-1+2 and HBc, as mentioned above. A rapid assay for It becomes.

The assay device combined with the pathogen of interest can be used, for example, at a doctor's surgery. Useful for [on-site] testing to aid diagnosis, clinical laboratory testing and return to surgery. Both of these avoidances can save time and money overall. moreover Assay devices can be used when laboratory equipment is not readily available, for example in remote areas. It is useful for testing in developing countries. Examples of pathogen combinations include CMV and and HIV, TB and HJV.

Reversible use of a universal antibody-coated solid phase for detection of multiple antigens Gender can also be used in other ways in clinical testing. In clinical tests, the purpose is generally the identification of pathogens in a sample. Nowadays, many samples cells in various formats using conventional microtiter plate or bead formats. an aliquot of each sample must be tested for the presence of pathogens. It is customary to prepare a series of assays for each different pathogen. be. This is because conventional assays are coated with antigen to capture pathogen-specific antibodies. This is because it uses a method that has been Therefore, for each pathogen.

Means coated with different antigens, typically beads or microtiters - plate must be used. Now, for a particular sample The amount of time it takes to obtain results increases, and the time it takes to obtain results increases. There is also the risk of aliquots being misplaced or mixed up.

Moreover, the antigen-coated means generally contain other reagents, e.g. Supplied in kit form along with negative controls, wash solutions and diluents. Therefore Therefore, the user must have many different kits.

Using the assays of the invention, universal antibody coating means, e.g. Microtiter plates can be used for simultaneous but separate measurements of each of multiple pathogens. can be used simply by using the appropriate antigen reagent. a series of different Samples can be tested simultaneously, partially for one pathogen and partially for other pathogens. I can do it. However, aliquots of a single sample can be tested simultaneously for different pathogens. Test each aliquot separately, for example in a separate microtiter plate. It is particularly advantageous to test for different pathogens in several wells. this is.

Saves time and money and risks failure due to lost or mixed-up samples also decreases.

Therefore, the present invention also has the following aspects. More than 2f11 different pathogens in liquid test samples In a method for simultaneously but separately measuring specific antibodies against.

(i) test each sample against one or more classes of immunoglobulins, e.g. Each unit is a single module consisting of a solid phase on which an antibody is immobilized. of each of the l or more units present in each sample. In this case, the immobilized antibody is captured on a solid phase. All units in the module are of the same class or group of classes.

(ii) each unit already in contact with the sample is specific to one of the pathogens under consideration; An antigen that can selectively bind to a specific antibody, each directly or indirectly by contacting the antigen with a means to provide a detectable signal, and then in.

(Mountain) Measuring the immunoglobulin-antigen complex obtained on the solid phase.

Provides a method consisting of

The number of different units used, and therefore the number of antigens used, depends on the disease being considered. It depends on the number of original bodies. Each unit is, for example, a microtiter plate. It can be one of the wells of a plate or a container containing the beads.

Alternatively, each unit may be ,! I, a defined area on a strip, sheet, film or coating It is also possible to do this. predetermined for the capture of components from the sample being studied. Devices containing such areas are known per se. In its simplest form, the invention Assay strip devices for assays performed simultaneously but separately.

For example, referred to herein as an antibody capture region. Capture exemption across the width of the strip Can be constructed from a strip of hygroscopic material with a band of epiglobulin Ru. Bands are separated by areas not coated with captured immunoglobulin. The immobilized immunoglobulins in each section are preferably separated into sections. Brin can be thought of as a unit antibody capture region.

Assay devices for simultaneous but separate assays can be used, e.g. As described above in connection with the nation assay.

Assays of the invention for simultaneous but separate assays for different pathogens The transferability of the device is! 2. Great benefits for both business owners and users provide. The main advantage is that a single device with multiple defined antibody capture regions can be used. with multiple antigen reagents for simultaneous testing of many different antibodies in a sample. It is something that can be supplied. As before, the manufacturer is required to provide information on all test substances. The advantage is that only one device needs to be created. Users also need has the advantage of having only one device and, in addition, This has the advantage that combinations can be freely selected.

The assay can be used to examine any specific combination of pathogens in a sample. It can be implemented. For example, a sample from a patient suffering from hepatitis.

Can simultaneously test for hepatitis A, hepatitis B anti-core antibody and HCV. Wear. Samples from patients with non-specific urethritis were tested for gonorrhea, chlamydia and candida. can be inspected for.

In a simultaneous but separate assay variant of the invention, the same class or on one or more units in place of each unit containing the immobilized antibodies of the ras group. may be immobilized with different classes of immunoglobulins, e.g. A series of specific antibodies carried out over an extended period of time using two-unit sets such as Simultaneous assays are useful for detecting early infection and tracking the course of disease, e.g. It is particularly useful for tracking seroconversion of HCV or HCV. A set of 3 types of units.

One unit is immobilized with anti-IgG, one is immobilized with anti-1gM, and the third unit is immobilized with anti-IgG. The set has anti-IgA immobilized on it to determine if the newborn is infected with HIV. This is especially useful in deciding whether A newborn's blood comes from the newborn's own antibodies and the mother's body. antibodies, and are therefore not an indicator of infection using conventional tests. Determining whether a newborn has his or her HIV antibodies is difficult.

Other examples where antibodies of more than one class are used include the procedure used in pregnant women in many countries. There is a case of the so-called "rTORcH" test. Pathogens are rubella, toxoplasmosis, and C. selected from MV and herpes. This combination varies depending on the group. rubella The test includes a 1gM assay to detect recent infections that are likely to be dangerous. Therefore, rTORcH including rubella must be included. At least one unit in the set used is coated with anti-1gM. must be rated.

Two or more types of units immobilized with antibodies against different classes of immunoglobulins The module consisting of two parts is itself part of the invention. Therefore, the present invention is , two or more species used in simultaneous but separate assays of different pathogens. It is a module consisting of units, and the unit is a fixed unit for immunoglobulins. in at least one unit thereof, in the other unit. Antibodies directed against immunoglobulins of a different class than immobilized immunoglobulins If the body is immobilized, for example, one unit contains immobilized anti-1 gM; or two or more units provide a module with immobilized anti-IgG. Ru. In other implementations, the module consists of three units and one The unit has immobilized anti-IgG and one unit has immobilized anti-1 gM and the third unit has immobilized anti-IgA.

A module consisting of two or more units may be used in an assay device, e.g. Provided as an arrangement of antibody capture zones above, such as an IgG capture zone and a 1 gM capture zone. It will be done. Such assay devices can perform simultaneous but separate assays for different pathogens. For example, it can be used for any of the specific applications mentioned above.

Alternatively, a module may consist of two or more sets of units, each set containing identical units. A set of different units, which may consist of provided as a module. For example, a set of units is Such strips, which may be provided as trips, can be used for two or more units of can be assembled as desired into a module consisting of a set of for example , the module consists of two strips of microwells, one containing anti-I One can be coated with gG and one can be coated with anti-IgM.

A third strip coated with anti-IgA can also be added. Ma Icrowell strips, i.e. sets of units, can be used, if desired, in conventional Can be assembled in the form of a microtiter plate. For example, plate A column or cover of microwells coated with anti-1gM and anti-IgG rams may be placed alternately, for example for tracking seroconversion, in which case Also, especially with microwell strips or similar sets of units, There are advantages in construction costs and in versatility of use.

Unless otherwise indicated, the following description refers to the combination assay of the present invention and Applies to both simultaneous but separate assays.

In clinical tests, the liquid test sample can be any body fluid, e.g. Plasma, serum, saliva, urine, brain, cerebrospinal fluid, milk, lymph, or lachrymal fluid. Also, and For example, a solid sample of cells or tissue may be in liquid form for testing, e.g. a tissue exudate. It can be done.

For blood screening (which is advantageously performed as a combination assay) ), the pathogens of interest are so-called blood viral pathogens (HIV, hepatitis B and Hepatitis C and optionally HTLV, CMV and EBV), and also syphilis.

Laboratory tests involve a much wider range of pathogens, including the organisms listed above and other viruses. viruses, bacteria as well as other types of pathogenic organisms. To give a non-limiting example: It is as follows.

Rubella, measles, herpes (simple and genital), chlamydia, gonorrhea, hepatitis A, chickenpox , mumps, human parvovirus, Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacterium avian tuberculosis, M. aureus Ucoccococci, Listeria monocytosis, Bacillus anthracis (antigens/toxins), Actinomycetes (e.g. Treptomysis, Nocardia, Rhodococcoccus), Salmonella typhi, Yersiniaen Terocoriphi, Helicobacter pylori, Campylobacter jejuni, Horse nose variola, Pseudomonas aeruginosa, Legionella pneumonia and congenerous species, F. tularensis, Prussame ritensis, Mycoplasma pneumoniae, Lebutospira terogans, Borrelia spp. caused by species, Treponema pallidum, Candida albicans, as well as protozoal pathogens. Diseases caused by diseases such as amoebiasis, babesiosis, Chagas disease, leishma anti-immune globules immobilized with pathogens of niosis, malaria and toxoplasmosis Phosphorus has two or more species from two or more classes, namely IgG, IgM and IgA. can do. As mentioned above, IgG is the most abundant immunoglobulin in serum. igM is the first immunoglobulin produced in response to infection, but provides an earlier indicator of infection than IgG.

Blood (plasma or serum) tests generally use IgG, depending on the purpose of the assay. or 1 gM alone is used or a mixture of anti-IgG and anti-1 gM is used. used in one unit of the combination assay or anti-IgG or and anti-1gM are provided in separate units in simultaneous but separate assays. .

It is also preferred to capture IgG and/or IgM in saliva assays.

For urine assays, the use of anti-IgG is preferred, optionally with IgM and/or Or IgA can be combined.

In an assay format of the invention that uses a two-component bead/tube format.

Immobilization of 1 or 2 classes of anti-immunoglobulin on the inner surface of the plastic tube one or two different classes of anti-immunoglobulin are used in the tube. The suitability of the assay of the present invention can be further enhanced by immobilization on beads that can be raised. For example, anti-IgG can be coated onto one of the two components. and can be coated with anti-TgM on top of the other.

Furthermore, using a combination bead/tube format, tube and After contacting the beads with the sample, the beads are separated and contacted with one antigen. When the tube is exposed to other antigens, two antibody assays can be performed on a single sample. It becomes possible to do the following.

Although both blood screening and laboratory tests have been described above, the assays used are There is no difference in the principle or type of materials used in blood screening.

Regulatory agencies in certain countries limit the test substances to a specific range and the tests are simply the same. Tends to be more fully automated than clinical testing due to the large number of tests performed It is in. However, throughout this specification, unless otherwise specified, all statements are generally relevant to the assays of the invention and are performed in an assay device. Assays performed on microtiter plates and beads, e.g. Includes assays that can be performed.

Assays of the invention can be performed in a conventional manner (e.g. "ELISA"). andOther 5 solid Phase [mmunoassays, Th eoretical and practical aspect sword high jeni enY.

(see D. & L. & Chat Iacombe, S. J., eds.), Immunoglobulin For example, the appropriate concentration of immunoglobulin and and pH 1, for example, in the range 7 to 11, especially in the range 9 to 1 O. It can be immobilized by this. Buffers, e.g. sodium carbonate/ The use of sodium bicarbonate buffers is preferred; as noted above, suitable immunization Globulin products are commercially available, and the appropriate dilution of commercially available antibody solutions is For example, I:200-1:4000v/v.

Labeled antigen reagents (conjugates) can be produced by any of a variety of methods (e.g. , l (see 6mely & Challacombe supra), antigen-enzyme conjugate is commonly used.

Samples can be diluted. For example, a blood or plasma sample is For example, a 50 μm sample is diluted v/v, and a 50 μm sample is diluted in a microwell. diluted solution. The sample diluent used may contain 1 or 2 It should be noted that it must not contain human immunoglobulins of the above classes. Ru.

, microtiter plate or bead format assays. The sample is then generally incubated with a solid phase. Incubation temperature and time are interdependent, requiring longer times at lower temperatures 1 Normal ink The fermentation conditions were 37°C for 1 hour. After incubation, remove the plate or beads should be thoroughly washed prior to incubation (conjugation) with antigen reagents. Typical incubation conditions for the mating step are 30 min at 37°C. It is 0 minutes. After incubation with the conjugate, a further washing step is performed and EIj In the case of SA, the enzyme is incubated with the substrate, again in this case, 3 steps. The usual incubation conditions are 30 minutes at 7°C. The stop solution is generally Added at the end of incubation. For ELISA, the results are generally It is obtained by reading the absorption of the nits with a spectrophotometer. Other labeling systems can be used If the method is modified accordingly, e.g. or in the case of a fluorescent immunoassay, the results are In case of 1 colored particles obtained after incubation, positive or negative result can also be measured with the naked eye.

For assay devices used outside the laboratory, with minimal reaction steps.

Because positive or negative results can be measured with the naked eye, colored particles are used as labels. For more sensitive assays, the EIjSA system can be used, which is particularly preferred.

The present invention also includes:

(a) Antibodies against immunoglobulins of I or more classes, especially anti-I A solid phase, especially a plastic substrate, on which a mixture of gG and anti-1 gM antibody is immobilized is used. microtiter plate.

(b) each capable of selectively binding to antibodies specific for one of the pathogens under consideration 2 The above antigen reagents contain a signal that can detect each antigen directly or indirectly. antigen reagents, and optionally the following: 1 or 2 or more L (C) Positive and negative controls, wash solutions and dilution solutions.

We provide a kit consisting of:

Kits may be provided as is (as well as antibody-coated components and and antigen reagent components may be provided separately.

The present invention also includes: A mixture of anti-IgG and anti-1gM antibodies was immobilized. im Provide a suitable solid phase for the assay. The mixture of antibodies further includes an anti-IgA antibody. The antibody is in particular an anti-human antibody. The solid phase is e.g. , any solid phase mentioned above, such as microtiter plates and beads.

Also included are solid phases suitable for use in assay devices.

Both "detection" and "measurement" candy, as used herein, refer to Qualitative, quantitative and semi-quantitative assays are meant.

Determination of antibodies against multiple pathogens in different samples (combination assay) assay”) or the simultaneous use of multiple different antibodies in aliquots of the same sample. The use of the immunoglobulin capture format for separate measurements is 1 hour and It is advantageous in terms of both costs. A large number of pathogens may be present in one unit or in a series of parallel units. The potential transferability of the format can be measured simply by the selection of antigen reagents within the kit. had not been previously evaluated. Universal antibody for all antibody assays Coating means, e.g. antibody coated microtiter plates, antibodies Enables use of immobilized antibodies in coated beads and assay devices The extremely practical and economic benefits of this should not be underestimated.

It should also be understood, of course, that the invention is not limited to the detection of pathogens. The present invention provides that whatever the nature of the antigen, it may be an antibody, such as a non-pathogen-related antibody. It can be applied to detect any antibody of interest, no matter what excites it. Such antibodies Examples of excited states include autoimmune diseases and allergies, e.g. autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus and rheumatoid arthritis Chi fever and organ-specific autoimmune diseases and any autoimmune associations have been considered. diseases such as thyroid autoimmune disease, autoimmune hemolytic anemia multiple disseminated sclerosis □ Includes aphthous ulcers, pernicious anemia and ulcerative colitis. all you need is interest An entity that can specifically bind to an antibody.

Therefore, the teachings herein apply to antibodies against non-pathogens as well as to antibodies against pathogens. This also applies to the detection of body-associated antibodies,9 and the present invention covers all such embodiments. It should be understood that it includes all.

The invention can be used for the detection and measurement of human and animal pathogens.

It has been found to have both human and veterinary applications, and is useful in the meat industry. It also includes applications.

The following non-limiting examples illustrate the invention.

The following reagents were used in the assay described below.

l, solid phase: polyclonal anti-human IgG (DAK and polyclonal 96-well plate coated with a mixture of anti-human I gM (DAKO) Micro titer plate (Nunc), Nunc products are L Tec hnologies, POBox 35゜Washington Road, Abbotts Inch Industrial Estate, Pa1s ley, Renfr ■ Next Tang ■ Amani ■ B TA34EF, 5cot-1and, DAKO products are DAKo, 16M anor Courtyard, Hugjenden Av■Shibu■B )iigh Wycombe, Bucks RP35RE, England Available from.

2. Conjugated with HRP (horseradish bereoxodase). CB of HIV-1 Core and envelope antigens from L-1 isolate [5attentau, Q, J, et al.

(1986), HIV-1 recombinant consisting of 5science 234.11201 protein (HIV-1 conjugate) 3.) (HIV-2 peptide consisting of gp36 envelope antigen conjugated to RP) (HIV-2 conjugate) 4. Hepatitis core antigen conjugated to HRP 5. Positive sample: a) HIV-1, internal control No. 4034, diluted with HIV-1 negative serum b) HIV-2, internal pair 11 uo, 91/+74, HI V-2 negative serum! interpretation c) Anti-HBc core positive sample 6, negative sample diluted with HBc negative serum: Normal human serum alternation obtained from blood donation A 100 μm diluted sample (10 μm sample and 90 μm sample diluent) was Add to wells of microtiter plate and incubate for 60 min at 37°C under humidified conditions. Cubated. The wells were then thoroughly washed five times with wash solution. Each cleaning process Remove the contents of each well by aspiration, wash solution (T*een-containing glycine borate) (salt buffer) and soaking for 30 seconds. Final cleaning process later.

Remove the contents of the well and pat dry on a paper towel or tissue. I let it happen. association in HEPES buffer containing bovine serum albumin and surfactant. A 50 μm working concentration solution of the coalesce(es) was added to a single solution as described below. or in combination to the wells and the plate incubated for 30 minutes at 37°C under humidified conditions. Incubated. After further washing steps as described above, TMB (3 ゜3', 5. Substrate solution containing 5'-tetramethylbenzidine) and hydrogen peroxide Add 100 μl of the solution to each well and incubate the plate for 30 minutes at 37°C under humidified conditions. The reaction was stopped using incubation and then 50 μM of 2M sulfuric acid. Well's Absorption was recorded at 450 nm with 690 nm as a reference wavelength.

Example 1 antibodies of interest (anti-HIV-1, anti-HIV-2 and anti-HBC), respectively (7) Dilute positive samples containing one of the above-mentioned HIV-1, HIV-2 and H Bc conjugates were used alone or in various combinations as shown in the table below to produce the above-mentioned proteins. Tested according to protocol. HIV-1 samples were I/40. I/80° A dilution series of 1/160 and 1/320 was used. For HIV-2 samples.

Diluted to 1/32.1/64.1/128 and 1/256. many different HBc-positive samples were diluted 1/2.

(i) HIV-1-positive samples were tested at increasing dilutions for the following conjugates: HIV- 1; HIV-1+HTV-2; HTV-1+HBc; HIV-1+HIV -2+HBc, respectively. The results are shown in Table 1.

Dilution >3 2.897 >3)3 increase 2. θ Wataru 5 1.905 2. 0+1 1.8471.282 1.148 1.214 1.129 ・0. 694 0.663 0.695 0.6720.41 0.378 0.41 2 0.3780.232 0.230 0.282 0.235 Negative 0.0 58 0.06+ 0.068 0.076 conjugate HIV-I HIV-1+ HIV-1+ HIV-1+HIV-2HIV-2HIV-2+ Bc (ii) HIV-2 positive samples are tested at increasing dilutions to -2: HIV-2+HIV-1; HIV-2+HBc: HIV-1+HI Each was tested with V-2+HBC. The results are shown in Table 2.

Table 2 Sample → HIV-2HIV-2HIV-2HIV-2 dilution of 0.099 1.045 1.009 1.002 increase 0.588 0.635 0.6 19 0.6190.386 0.397 0.386 0.3830.232 0.252 0.249 0.2510, +58 0.167 0.171 0.1830.111 0.124 0.126 0.135 Negative 0.053 0.060 0.061 0.072 Negative 0.053 0.062 0.0 63 0.075 conjugate HIV-28IV-2+ HIV-2+ HIV-2 +HIV-I HIV-I HIV-1+ (source) 6 different HBc positive samples conjugates of the following: HBc; HBc+HIV-1; HBc+HIV- 2: Tested with HBc+HIV-1+HIV-2. The results are shown in Table 3.

0.956 1.072 0.970 0.9260.980 1.073 0 ．． 986 0.9130.994 1.053 0.966 0.0321.0 72 1.189 1.058 1.0301.062 1.177 1.06 6 1.0251.058 1.156 1.062 1.005 Negative 0.0 56 0.070 0.061 0.074 Negative 0.060 0.069 0 ．． 063 0.073 zygote HB c HB c + HB c + HB c +HIV-I HIV-2HIV-1+ The results listed in Tables 1 to 3 show that the various antigen conjugates were used in combination as well as when used alone. This clearly shows that each antibody can be detected efficiently even when two zygotes Almost no interference was observed even when three zygotes were used; A slight increase in background was observed in negative samples when using zygotes. Only.

Example 2 Series of samples A-D containing HIV-1, HIV-2 and HBc antibodies.

The original single sample was diluted and prepared as shown in Table 4.

A I/40 1/32 1/2 B 1/80 1/64 1/2 CI/160 1/12B +/2 D I/320 1/256 1/2 Samples A-D below <7) Conjugate: HIV-1; HIV-2; HBc; HT V-1+HIV-2; HIV-1+HBc; HIV-2+HBc; HIV- 1+HIV-2+HBc. The results are shown in Table 5.

Table 5 A 1.3+4 0.961 0.361 1.602 1.415 1.18 5 1.689B 0.840' 0.619 0.386 +, 232 1. 075 0.848 1.482CO, 5190, 3680, 40+ 0.80 4 0.803 0.650 1.102D O, 2980, 2160, 402 0,4650,6+4 0.526 0.828-ve' 0.062 0.0 49 0.052 0.059 0.059 0.054 0.068-ve O,0550,0490,0500,0540,0570,0520,064- ve　O,0540,0470,0490,0540,0570,0510,0 62-ve　O,0530,0460,0500,0520,0570,052 0,063 conjugate H[VI HIV-2HBc H[VI HIV-1] 11V-2HIV-IHIV-2+1 (Bc +HBc HIV-2 These connections The result is that each antibody can be selectively detected even in the presence of other antibodies, i.e. This clearly shows that the presence of unrelated antibodies does not affect the detection of related antibodies. difference Furthermore, the results obtained at low dilutions qualitatively illustrate the presence of three specific antibodies, indicating the presence of rare At higher dilutions the results obtained are substantially additive.

The results obtained showed that specificity was compromised even when detecting antibodies against two pathogens. The results show that the sensitivity of the fumbination assay is extremely high. There is.

Continuation of front page (81) Designated countries EP (AT, BE, CH, DE.

DK, ES, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE) , 0A (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE, SN.

TD, TG), AT, AU, BB, BG, BR, BY.

CA, CH, CN, CZ, DE, DK, ES, FI, GB, GE, HU, JP, KG, KP, KR, KZ, LK, LU, LV, MD, MG, MN, MW, NL, No.

NZ, PL, PT, R○, RU, SD, SE, SI, SK, TJ, TT , UA, US, UZ, VN (72) Inventor Beckford, Ursuzuraigiri Sri Lanka DAI 15 L.R. Kent, Dartford, Temple Hill, Central Road (no street address), Murex Diagnostics Limited Notice

Claims (1)

[Claims] 1. Measuring specific antibodies against two or more different pathogens in a liquid test sample In the method of (i) the sample has been immobilized with antibodies against one or more classes of immunoglobulin; one or more of each class present in the sample by contacting with a defined solid phase (ii) immunoglobulins from the sample are captured on a solid phase; is captured on the solid phase against antibodies specific for one of the pathogens under study. two or more different antigens, each of which can selectively bind an antigen that has been provided with a means of directly or indirectly providing a signal that can be the immunity obtained by contacting simultaneously or sequentially and then (iii) forming on a solid phase. A method comprising measuring globulin-antigen complexes 2. The immobilized antibody is an anti-IgG, anti-IgM or anti-IgA antibody, or 3. The method according to claim 1, wherein is a mixture of two or more thereof. Anti-IgG and anti-IgM is immobilized on a solid phase. 4. Antibodies to be immobilized may be affinity-purified polyclonal or monoclonal. 5. The method according to any one of claims 1 to 3, wherein the antibody is a polyantibody. Each antigen is detectable Any of claims 1 to 4, which is directly labeled by a means capable of providing a signal. Method 6. A means capable of providing a detectable signal to an antigen is: (i) its provides a means by which it can bind to antigens and provide a detectable signal of its own consisting of the antibody Abl, (ii) an antibody Abl capable of binding to the antigen, and an antibody Abl capable of binding to the antigen; A second antibody, Ab2, provides a means by which a detectable signal can be provided. The method described in any one of "Claims 1 to 4" 7. The sample to be examined is a donated blood sample, as described in any one of "Claims 1 to 6" the method of 8. The two or more pathogens to be considered are HIV, hepatitis B virus, and hepatitis C virus. , HTLV, cytomegalovirus, Epstein-Barr virus and syphilis? 9. The method according to any one of claims 1 to 7, selected from Two or more diseases to be considered The original substance is rubella, measles, herpes (simple and genital), chlamydia, gonorrhea, and liver type A. inflammation, chickenpox, mumps, human parvovirus, Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacterium avian tuberculosis, Staphylococcus aureus, Listeria monocytosis, Bacillus anthrax (antigen/toxin), Actinomycetes, Ti Clostridium fusemia, Yersinia enterocorifica, Helicobacter pylori, Campyloba jejuni, Bacillus equina, Pseudomonas aeruginosa, Legionella pneumoniae, and conspecific species. , F. tularensis , Prussella melitensis , Mycoplasma pneumoniae , Leptospira inte rogans, Borrelia species, Treponema pallidum, Candida albicans, and Claims 1 to 7 selected from pathogens of diseases caused by protozoan pathogens. ” Method 10. The two or more pathogens to be considered are rubella and toxopra. ``Claim 1'' selected from Zumiasis, cytomegalovirus, and herpesvirus. The method described in any of 8. 11. Simultaneous detection of specific antibodies against two or more different pathogens in liquid test samples However, in a method in which each sample is measured separately, (i) each sample is Each unit consists of a solid phase on which an antibody against a class of immunoglobulin is immobilized. into each sample by contacting one of the units into a single module. One or more of the immunoglobulins of each class present are captured on a solid phase, and this In this case, the immobilized antibody has the same cluster in all units in the module. (ii) each unit of the module that has already been in contact with the sample; nits with an antibody that can selectively bind to an antibody specific to one of the pathogens under consideration. means for providing a detectable signal directly or indirectly is brought into contact with the given antigen, and then (iii) measuring the immunoglobulin-antigen complex obtained on the solid phase; how to become 12. At least one unit comprises a solid phase on which an anti-IgG antibody is immobilized. , at least one unit consists of a solid phase on which an anti-IgM antibody is immobilized, and Optionally, at least one unit consists of a solid phase on which an anti-IgA antibody is immobilized. The method according to “Claim 11” 13. The sample under consideration is an aliquot of a single sample "claim 11 or 12” 14. The solid phase can be beads, microtiter plate wells or cups, medium consisting of solid or hollow rods or pipettes, or particles, or , membranes, sheets, strips, films of porous, fibrous or hygroscopic materials or coating, optionally incorporated into the assay device. 15. Method according to any one of Items 1 to 13. Simultaneous but distinct of different pathogens A module consisting of two or more units used in an assay. Nit is composed of immobilized antibodies against immunoglobulins, at least one of which The unit contains a class of immunoglobulin that is different from the immunoglobulins immobilized on other units. A module on which antibodies against immunoglobulins are immobilized 16. One unit contains one or more units with immobilized anti-IgM. Module 17. of claim 15, wherein the module 17. has immobilized anti-IgG. Consists of three units, one with immobilized anti-IgG and one with immobilized anti-IgG. One unit had immobilized anti-IgM and the third unit had immobilized anti-IgM. 18. Module according to claim 15 having anti-IgA. anti-IgG and Suitable for use in immunoassays where a mixture of anti-IgM antibodies is immobilized solid phase 19. 20. The solid phase according to claim 18, wherein an anti-IgA antibody is also immobilized. B wells, microtiter plate wells or cups, solid or consisting of hollow rods or pipettes, or particles, or porous, fibrous or from membranes, sheets, strips, films or coatings of hygroscopic material. and optionally incorporated into the assay device. Solid phase described in 21. Measures specific antibodies against two or more different pathogens in liquid test samples In the method of (i) the sample has been immobilized with antibodies against one or more classes of immunoglobulin; one or more of each class present in the sample by contacting with a defined solid phase (ii) immunoglobulins from the sample are captured on a solid phase; is captured on the solid phase against antibodies specific for one of the pathogens under study. two or more different antigens, each of which can selectively bind an antigen that has been provided with a means of directly or indirectly providing a signal that can be the immunity obtained by contacting simultaneously or sequentially and then (iii) forming on a solid phase. A method comprising measuring globulin-antigen complexes 22. Simultaneous detection of specific antibodies against two or more different pathogens in liquid test samples However, in a method in which each sample is measured separately, (i) each sample is Each unit consists of a solid phase on which an antibody against a class of immunoglobulin is immobilized. into each sample by contacting one of the units into a single module. One or more of the immunoglobulins of each class present are captured on a solid phase, and this In this case, the immobilized antibody has the same cluster in all units in the module. and (iii) each of the modules that have already been in contact with the sample. The unit can bind selectively to antibodies specific to one of the pathogens studied antigens, each of which provides a detectable signal directly or indirectly The step is brought into contact with the given antigen, and then (iii) measuring the immunoglobulin-antigen complex obtained on the solid phase; how to become 23. According to "claim 21 or 22", the antibody to be investigated is a non-pathogen-related antibody. the method of 24. The antibodies to be considered are autoimmune antibodies or allergy-related antibodies. 25. Method according to claim 21 or 22. "Claims 2 to 6, 11 and 12" Claims 21 to 24 having the parameters defined in any of Method described in any of the above