JPS63315953A - Method for measuring trace substance content in blood or body fluids - 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] [Industrial Application Field] The present invention is applicable to trace substances contained in blood or body fluids, such as hormones, drugs, rfJf elements, virus-specific antigens, trace protein components, cancer antigen-related substances, etc. The market relates to methods for measuring the content of

More specifically, a conjugate of lectin-bound agarose, antigen, and labeled antibody was prepared, and its activity was measured in the immunoradiomeric assay F Lect I.
n-immunorad Iometer Ic aSS
This invention relates to a method for measuring the content of trace substances contained in blood or body fluids (da fluid, ascites, N water, urine, bile, etc.).

[Prior art] The measurement of trace substances in blood was developed by Barson in 1959.
radioimmunoassay (Rad 10: mmun) developed by
oasay (RIA>) has made dramatic progress (
NATURE, November 21, 1959
．． Vol, t84. p1648-p1649). Even today, this technology is widely applied to many hormones, blood drugs, enzymes, virus-specific antigens, trace protein components, and cancer antigen-related substances. But the method of mouth (, 1 antibody, 1 radiation, 1 beef purchase)! F. In order to avoid a competitive reaction with the recognized antigen, there is a limit to the measurement sensitivity and the raw material, and a considerably purified antigen is required.

In response to this, in 1968 Miles et al.
diometric aSSay (IR~IA)
) was developed (NA Ding IJRE, July 13.196
8. VOl, 219. (p. 186-p. 189), this method uses two or more antibodies that recognize different parts of the antigen, or immobilizes one antibody in some kind of homologous system, and labels the other with radioactivity [biological material] to bind the antigen in a sandwich-like manner. This is a method of inserting the This IRM7 method uses multiple different antibodies, does not require purified antigen, and has excellent specificity and sensitivity.

Many substances are currently measured using this method.

On the other hand, the substances that these methods measure are proteins with many amino acids bound together, but as the molecules become relatively high, galax, mannose, N-acetylglucosamine, and sial-M (N-acetylglucosamine) Sugar chains such as lamic acid) are attached to these. It has recently been discovered that these sugar chains account for an important 190% of the body's production, and research is currently underway around the world to determine the role of each sugar chain in each substance. A common assumption is that the protein part consists of a basic skeleton, and that the sugar chains provide specific information for the receptor of that substance. In other words, if the protein is the main body of the key, the sugar chain corresponds to the notches of the key. Another reason why sugar chains are attracting attention is their relationship with canceration. According to a recent 6/1 study, changes in the sugar chain structure have been observed due to canceration, and it has been suggested that it may be possible to diagnose canceration if differences in sugar chains can be measured. However: Measurement of the Ui chain is still in the research stage, and methods include column chromatography, high-performance liquid chromatography, or cutting with enzymes to see differences in reactivity. There are many.

[Problems to be solved by the invention] Radioimmunoassay (R
IA) and immunoradiometric assay (IRM△)
It is thought that specificity is high because it utilizes the binding reaction between antigen and antibody, but on the other hand, even if the antigen has various structural changes on its molecule, it is difficult to detect structures that are outside the recognition range of antibodies. If this is the case, there will be no difference in measurement. In addition, the development of antibodies that only recognize the changed molecular structure of antigens
Although this is becoming possible with the establishment of techniques for producing monoclonal antibodies, it is extremely difficult to search for suitable antibodies for antigens whose entire +M structure is unknown, including substances with similar structures. In addition, these antigens include anti-I-K, which is a protein only, a protein antigen with a sugar chain, and an antigen with only a sugar chain.It is thought that the sugar chain structure is most likely to change depending on various pathological conditions, but in general It is said that it is difficult to create antibodies that specifically recognize sugar chains because the protein trading portion has strong antigenicity.

In contrast, the present invention uses a lectin and an antibody that recognize only a specific sugar chain (14 structures), which has the most variation in the structure of the antigen: sugar chains + 14 structures, by changing the type of lectin used. It has characteristics that can be observed in

[Means for Solving the Problems 1] The present invention deals with trace substances in 1m fluids and body fluids, such as hormones, blood mulberries, enzymes, virus-specific antigens, trace protein components,
In a method for measuring the content of cancer antigen-related substances, etc.
The lectin-bound agarose beads are reacted with serum or body fluid to obtain a conjugate of the lectin-bound agarose beads and the antigen, and then the conjugate is reacted with a radioactive substance-labeled antibody against a specific antigen to label the lectin-bound agarose beads and the antigen. This is a method for measuring the content of trace substances in blood or body fluids, which is characterized by obtaining a conjugate with an antibody and measuring its radioactivity.

Lectins, which are sugar-binding proteins, have the property of specifically recognizing and binding to certain sugar structures.

Concanavalin △ (C○nA) which can be raised by 1 from Bean H plants
, Jack bean lectin), PNA (Bean lectin), RCA (Hima lectin), LcH (lentil lectin), and PI-(A (Phaseol lectin)), as well as animal lectins such as IFA (Slug lectin). More than a dozen species have been discovered.

Since lectins recognize only specific sugar chain structures depending on their type, they are currently of constant importance in the study of sugar chain structures.

This is evidenced by the fact that affinity glu chromatography using 7-callose gel bound with cutin has become an important means for sugar chain research.

Agarose is often used instead of agar in electrophoresis, especially for the migration of high molecular weight substances.

The LIRMA developed by the present inventors picks out the antigen recognized by the lectin by reacting this lectin-bound agarose beads with serum or body fluid, and then irradiates the specific antigen [sandwiching this antigen with biologically labeled antibodies]. , a method of measuring radioactivity.

[Function] The lectin immunoradiometric assay (LIRMA) according to the present invention will be explained according to the drawings.

In FIG. 1, 1> A trace amount of a measurement sample 2 such as serum or body fluid is placed in a test tube 1. The measurement sample 2 consists of an antibody-bound antigen 4, a lectin-bound antigen 3 containing an antibody-unbound antigen 5, and a lectin-bound antigen 6. Next, add a small amount of lectin-bound 7 callose beads 7. In some cases, this 'r Xi dr
Liquids may also leak. Also, at this time, the standards 't and fl of the determined antigen temperature are added to the measurement system in the same manner as in 'Test 4'. It is desirable that the standard antigen be one whose sugar chain structure has been elucidated.

2) Fix the test tube 1 on a cinnabar machine and react the lectin with the antigen while shaking to obtain 1 q of lectin agarose beads and antigen conjugate 8. This is because agarose beads are very easy to precipitate. This reaction is quick, so 1-2
You can finish it in time.

3) Pour buffer solution into test tube 1 where the reaction has been completed.

After floating the agarose beads, place them in a centrifuge. Agarose beads precipitate easily. Remove the supernatant at this time using an aspirator. By repeating this operation several times, the lectin and unreacted antigen can be completely removed.

4> Add a small amount of antibody 9 bound to a radioactive isotope to the precipitated agarose beads. At this time, a buffer solution or the like may be added at the same time. Also, as a radioactive isotope, 1-125
By using this, the l-125-labeled antibody of IRMA, which is widely used, can be used.

5) A reaction between the antigen and the antibody is carried out while adding atomization to obtain a conjugate 10 of lectin agarobese, antigen, and labeled antibody. Stable results can be obtained if the process is carried out at low temperatures for long periods of time.

6> Perform the same washing operation as in 3) above to remove unreacted radioisotope-labeled antibody conjugate 11.

7) Finally, the precipitated conjugate of lectin-bound agarose beads, antigen, and radioisotope-labeled antibody 10 is subjected to a radioactivity assay to measure radioactivity.

- As an example, draw a standard curve using the radioactivity counts obtained from the standard concentration as shown in Figure 2, and then fit the radioactivity counts of the unknown sample into the quasi-curve a to determine the degree of preparation of the sample. Alternatively, the radioactivity count of an unknown sample to the radioactivity count of a specific target concentration is determined as a ratio, and this is interpreted quantitatively. This method is effective in cases where it may be difficult to obtain a standard curve when a lectin with low antigen binding is used.

[Example] Example 1 Agarose beads (EY Labora) of WGA (Combin embryo lectin) and LCI-1 (lentil lectin)
alpha-fetoprotein [Alf], which is currently the best test item for liver disease.
ha-reto protein (AFP>
]'s LIRMA was deleted.

In order to match the sugar chain antigen binding ω of lectin, the standard solution and serum sample were diluted 1/10 with buffer, and 50 μL of WGA or LCH agarose beads per original concentration was added to this solution at 25°C for 2 hours. Incubate with a horizontal camera, and add 1 ml phosphate buffer (Trit) to the reaction solution.
on-X (containing 100), add 3000 rl) m,
Centrifuge at 4°C for 5 minutes, remove rγ, add phosphate buffer again, and repeat the same procedure 4 times. These operations will completely remove antigens that do not react with VG△ or LCH. Add 50 μL of l-125,1-recognizing Alpha-feto protein antibody to the washed agarose beads.
Add and incubate at 4'C for 48 hours without shaking. After the reaction is completed, the agarose beads are washed in the same manner as above to remove unreacted l-125 labeled AFP antibody, and the precipitated agarose beads are bound to 6X Q'l.
Measure the performance using a gamma counter.

) Draw a standard curve using the measurement results of the standard liquid, and use this to determine the antigen concentration of the unknown sample. Alternatively, the ratio of the radioactivity of an unknown sample to a specific standard is determined.

It is suitable for interpreting measurements that are not capacitance-responsive to standard glycan structures or lectins.

The results of the above measurements are shown in the table below.

! When compared with the measurement results of the RMA method, the measurements with LCH Requin are similar to the results of the IRMA method. This is because LCH lectin recognizes fucose (FUCOse > 4f4 structure) and reacts well with liver cancer-derived standards and liver cancer serum.
cNAc) structure, the ratio to the standard of 11000 n/ml is small. Also, when comparing quantitatively, IRMA
The concentration of liver cancer is ranked in descending order, with C, B, A, and E having the highest concentration, but WGA having the highest concentration.

C, B, A, E, and B, D, C in LCH.

A and E are required. From this, sample B is Fucose
+m'? > is found to be more common.

In this way, the LIiMA method has the great advantage that different information can be obtained from the same sample depending on the sugar chain structure, and quantitative information and qualitative information can be observed simultaneously.

; Effects of the Invention] As described above, the present invention measures the antigen recognized by an antibody only from the lectin-bound antigen, which is different from the conventional RIA and I
Unlike RMA, it does not measure the W2 GA antigen using only antibodies. Therefore, by changing the lectin, it is possible to observe quantitative differences depending on the sugar chain structure of sugar chain antigens, which is a completely new measurement method that has never been seen before.

[Brief explanation of drawings]

Figure 1 is an explanatory diagram of the lectin immunoradiometric assay (LIRMA) according to the present invention, and Figure 2 is a standard curve using radioactivity counts (CPM) subtracted by 1 from the standard concentration (ng/ml). This shows that. 1... Test tube, 2... Measurement sample such as serum or body fluid, 3... Lectin-bound antigen, 4... Antibody-bound antigen, 5... Antibody-unbound antigen, 6... Lectin-unbound antigen, 7... Lectin-bound agarose beads, 8...
・Lectin agarose beads and antigen conjugate, 9...
Antibody conjugated with radioisotope, 10... Conjugation of lectin agarose beads, antigen and labeled antibody, 11...
Unbound labeled antibody.

Claims (1)

[Claims] In a method for measuring the content of trace substances in blood or body fluids, such as hormones, blood drugs, enzymes, virus-specific antigens, trace protein components, cancer antigen-related substances, etc., lectin-bound agarose beads are reacted with serum or body fluids. , a conjugate of lectin-bound agarose beads and an antigen is obtained, and the conjugate is reacted with a radioactive substance-labeled antibody against a specific antigen to obtain a conjugate of lectin-bound agarose beads, an antigen, and a labeled antibody, and this is radioactively A method for measuring the content of trace substances in blood or body fluids.