JPS63503241A - Improved laboratory diagnostic methods especially for infections caused by HTLV3 viruses - 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 provides improved laboratory diagnostics for infections caused by HTLV I [virus]. More specifically, it has greater specificity than known methods, and In the first step, it is possible to reveal the presence of typical IgM class antibodies. provide a method to enable

Currently used serological diagnostic methods detect certain infections by testing blood samples. It is based on the investigation of specific antibodies.

Antibodies are immunoglobulins, meaning molecules that are perfectly matched to the antigen they act on. It is well known that it is a structural protein.

Various antibodies (specific for antigenic determinants) have similar specificities, but the abbreviations: IgG, IgA, IgD, IgE.

Based on the division into five classes distinguished by IgM, different immune systems Consists of immunoglobulins with chemical properties.

Thus, there are different classes of antibodies with similar specificities; therefore, IgM and Antibodies of the HTLV and IgG classes can coexist, and both It acts against.

Also, each class of antibodies causes the formation of antibodies of the same class when injected into animals. It is also known that

For example, when human) IgG immunoglobulins are injected into animals, they become human) Whatever the specificity of IgG, H) for and against IgG. Anti-antibody effects occur only when antibodies are used.

As mentioned above, serological diagnosis of the infection also depends on the presence of relevant antibodies in the patient's blood. It consists of an examination of the body.

One method currently used is to prepare a compound containing a known antigen and to a solid medium, such as a small polystyrene sphere. .

Blood samples taken from patients in the study are then reacted with the antigen-coated globules. Ru.

During this stage, when specific antibodies for a definite infection are present in the blood, They are captured by the antigen present on and immobilized on.

At this point, the globules are coated with a conjugated anti-antibody (i.e. usually obtained from an immunized goat, Anti-antibodies conjugated to specific enzymes such as peroxidases, phosphatases, etc. or anti-immunoglobulin); the relevant medium Subsequent addition causes the development of a color indicating the presence of antibodies and thus the status of infection.

A specific antibody is captured by its own antigen and its presence is then detected by an anti-antibody. This serological diagnosis is based on determining specific antibodies by revealing The method has very good sensitivity compared to other methods, but specificity is insufficient .

More specifically, HTLV I [In the case of viruses, I which characterizes the first stage of infection] The greatest nonspecificity occurs in studies with gM antibodies. At present, A.I.D. In the case of S virus, I (TLV II), the diagnostic method is based on the relevant IgG class antibodies. Based on research on One method of technology that is widespread today is described below.

In such cases, purified antigens are immobilized on the surface of 6 mm diameter polystyrene spheres. and place it in a steam heating container.

After diluting up to 400 times to avoid non-specific reactions, the test serum was Place it in a container where it will react with the reaction mixture.

At this stage the water bath temperature is maintained at 40°.

At the end of the incubation, a suction-and-up pump device of known type is used. Therefore, the globules are washed and then, after pre-binding with peroxidase, a special Add different IgG group antibodies.

At the end of the second incubation, wash one more time and remove the anti-antibody, if present. Add a medium to act on the conjugate.

The presence of anti-antibody conjugate, if present, is indicated by color development, which indicates infection status.

With this method, even if the test serum is significantly diluted, some Ig Due to nonspecific absorption of G immunoglobulin, false positive judgments are obtained in exactly the same way. .

In addition, nonspecific positive readings are often obtained with 1 gM immunoglobulin as well. Depending on this type of diagnosis, only the presence of this class of immunoglobulin can be detected. can be revealed.

Only 1 gM immunoglobulin was found to be effective against HTLV I [open viral infection (which lasted for several months)]. Currently known diagnostic methods do not detect the presence of such an infection during this first stage. It does not make it possible to detect the presence of

Therefore, in order to prevent contagion (as in the case of blood donors) and Ensure that this type of sensation is detected as early as possible in the first stage in order to treat it in a timely manner. There is a great need for diagnostic methods that allow the determination of infection.

To this end, the invention provides the following steps: a) Antibiotics that specifically attack HTLV I b) separating the antibody by binding the antibody with the relevant antigen in a solid phase; to elute it hot; C) coated with the same type of antigen or partially coated with the same type of antigen subjecting the thus purified antibody on the second globule to a second incubation; d) A third incubation with anti-antibodies of the selected class, followed by perform detection HTLV I [Provides an improved method for laboratory diagnosis of viral infection] characterized by It is something to do.

The invention will be described in more detail below.

Example Test serum 2 was placed in a container that also contained spheres whose surfaces were coated with purified antigen of HTLV I. 00μ! or more.

Unlike the known methods, in this case the serum is not diluted and the incubation cycle is used. The process was carried out at a temperature of 40°, first for 45 minutes under stirring at 180 rpm, then for 15 minutes in a water bath.

At the end of the incubation, the spheres are washed with normal wash solution and mixed with an amount of goat serum. Add 250 μl of a diluent consisting of phosphate buffer to the solution.

The water bath temperature is between 50° and 58' for 30 minutes. The optimum temperature is 5 for 30 minutes. It turned out to be 6°. At such temperatures, various specific immunoglobulins are separated from each of those antigens, and eluates of various specific antibodies as well as non-specific antibodies are Obtained in buffer liquid.

At the end of the elution phase, the temperature inside the container at which the antibody is reabsorbed by the antigen present on the ridge Lift up liquid quickly to prevent drops.

The collected liquid containing purified antibodies is then coated with the same antigen as in the first one. into a second container containing a second rounded ball.

Then a third incubation is started, always at 40° for 1 hour, but after that During this period, only immunoglobulin acts specifically against HTLV III on the ridge. .

At the end of this step, the bulbs are washed and 200 μl of conjugate is placed in the container. next Further incubation was carried out for 2 hours and 10 minutes at 40', then Wash the bulb and place it in a test tube.

Then add 300 μl of 0PD and incubate the whole again for 30 minutes at room temperature. do.

Then 1 d of IN sulfuric acid is added.

By measuring the optical density of the sample thus obtained, the desired result can be obtained. can.

By this method the presence of antibodies of class IgG and IgM can be detected, thereby Reliably diagnose HTLV III virus infection almost at the early stage of the disease. becomes possible.

Here, the detection system consisting of peroxidase is phosphatase or β-galactosylate. can be based on other enzymes such as Dase; alternatively, anti-antibodies can be It can be combined with a radioisotope used as a protector.

56 A method consisting of eluting antibodies by heating in a isolate the antibodies that are attacked and then purify the antibodies in the eluate to assess specificity. immunology to determine which blood systems or groups they act on. Let us further point out what is already known in the field of epidemiological hematology.

In contrast, in the present invention, a test antibody captured by a known antigen is and other purified antigens of the same type, in order to demonstrate identity with maximum specificity. The method is applied in reverse so that they are eluted in order to elute them.

Therefore, a feature of the present invention is that antigen dissolution of antibodies captured by the same related antigen At the point of separation and prior to the addition of each conjugate, the thus purified antibody is subsequently incubated with the antigen. It is fixed to the second layer.

Those skilled in the art will be able to provide modifications and variations, if desired, which do not fall within the scope of the invention. It is within the range.

quiet! First trial report ANNEX To ＾KE INTERNAτ 工○NAL 5EARCHREP ORT Oyo1

Claims (7)

[Claims] 1. The following steps: a) selecting by incubation serum antibodies captured by the antigen; b) separating the thus purified antibodies by thermal elution; c) separating the antibodies of the same type as above; subjected to a second incubation with the antigen; d) a third incubation with an anti-antibody of the selected class, followed by HTLVIII virus infection characterized by detection by addition of each conjugate diagnostic method. 2. the first ink, partly by stirring and partly by heating; 1. The method of item 1 above for carrying out an evolution. 3. Prepare purified antibodies for each antibody by elution at temperatures ranging from 50° to 58°. The method of item 1 above, in which the antigen is separated from the antigen and then immobilized on the second layer of an antigen similar to the antigen. Law. 4. The following steps: a) Separation of specific antibodies from undiluted serum by incubation death; b) adding diluent and further elution at a temperature of about 56°; c) separating the eluate while keeping it at the same temperature; d) further similar to the above; selecting antibodies with similar antigens by incubation; e) Add conjugated (or radioisotope-labeled) antibody and ink f) adding a suitable substance to develop a color in the presence of the antibody; or if the antibody used is labeled with a radioisotope, include a gamma ray counter indicator. A method for diagnosing HTLVIII infection, the method comprising: 5. 4. The method of item 4 above, wherein the elution is carried out at 56°. 6. The first incubation is carried out partly by stirring and partly by heating. The method according to item 4 above. 7. 10. The method of one or more of the preceding claims for the diagnosis of viral infection syndromes.