JPH0718875B2 - Method for measuring trace substance content in blood or body fluids - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for measuring a trace substance contained in blood or body fluid, specifically, an antigen having a sugar chain (sugar chain-containing antigen). Is. More specifically, the content of a trace substance contained in blood or body fluid is determined by forming a complex of immobilized lectin, a sugar chain-containing antigen and a labeled antibody, and measuring the labeled amount in the complex. It relates to a method of measuring.

[Prior Art] Measurement of trace substances in blood was carried out by Barson in 1959.
And the radioimmunoassay (RIA) developed by Yarrow (NATURE, November21,1959, Vol.184, p1648-p16)
49). Even now, this technique is widely applied to the measurement of many hormones, blood drugs, enzymes, virus-specific antigens, trace protein components, cancer antigen-related substances and the like. However, this method has a limit in measurement sensitivity and specificity because a radioactive substance-labeled antigen reacts competitively with an antibody, and a considerably purified antigen is also required.

On the other hand, Miles et al. (1968) proposed an immunoradiometric assay.
(IRMA)) (NATURE, July13 1968, Vol.219,
p186-p189). This method uses two or more types of antibodies that recognize different portions of an antigen, one of which is immobilized on some solid phase system, and the other is labeled with a radioactive substance to sandwich the antigen in a sandwich-like manner. is there. This IRMA method does not necessarily require a purified antigen because multiple different antibodies can be used, and has excellent specificity and sensitivity. Many substances are currently measured by this method.

On the other hand, the substances to be measured by these methods are proteins to which a large number of amino acids are bound, but galactose, mannose, N-acetylglucosamine, sialic acid (N-acetylneuraminic acid), etc. The sugar chains of are bound to these. Recently,
It has been revealed that these sugar chains have important roles, but the role of sugar chains of each substance is currently being studied all over the world. It is presumed that the protein part is the basic skeleton and the sugar chain is the specific information for the receptor of the substance. In other words, if the protein of the tampon is the main body of the key, the sugar chains correspond to the hacks of the key.

Another cause of the attention of sugar chains is the relationship with carcinogenesis. Recent studies have observed changes in sugar chain structure due to canceration, suggesting that canceration can be diagnosed if the difference in sugar chains can be measured. However, the measurement of sugar chains is still in the research stage, and the methods currently used are such as separation by column chromatography, high performance liquid chromatography, etc. and cleavage of sugar chains by enzymes to see the difference in their reactivity. Yes, the testing efficiency is low and there are many problems in doing this in the clinical setting.

[Problems to be Solved by the Invention] Radioimmunoassay (RI
A) and immunoradiometric assay (IRMA) are considered to have high specificity because they utilize the binding reaction between antigen and antibody, but on the other hand, the antigen has various altered structures on its molecule. However, if the changed structure is a portion outside the recognition range of the antibody, no difference is observed in the measurement.
In addition, the development of antibodies that recognize only the altered structure on the molecule of the antigen is becoming possible by establishing the technology for producing monoclonal antibodies, but it is appropriate for antigens where the entire structure of the antigen, including substances with similar structures, is unknown. It is extremely difficult to search for specific antibodies. In addition, these antigens include protein-only antigens, protein antigens having sugar chains, and sugar-chain only antigens, and it is considered that the sugar chain structure is most likely to change due to various pathological conditions. Since it has a strong antigenicity, it is said that it is difficult to prepare an antibody that specifically recognizes a sugar chain.

As one means for solving such a problem, a method of measuring a sugar chain-containing antigen using a substance having a sugar chain-binding property, such as lectin, has been reported (JP-A-57-29949).
JP, JP-A-61-20867, etc.). However, the method described in JP-A-57-29949 is a method for measuring a sugar chain-containing antigen by a competitive reaction using a labeled lectin, and it is difficult to prepare the labeled lectin or it is a competitive reaction. There was a problem that the possible measurement sensitivity was not obtained.

Further, the method described in JP-A-61-20867 is a method that can improve the drawback of the measurement sensitivity of the method described in JP-A-57-29949 because it employs a sandwich assay, but it is still labeled. Problems with lectin production have not been solved. Furthermore, another big problem with this method is that
Since it is a method for measuring a sugar chain-containing antigen using a solid-phased antibody and a labeled lectin, nonspecific adsorption increases, and the background of the measurement system rises, making it impossible to assemble a practical measurement system. I can point. That is, even though the antibody used for immobilization has a plurality of sugar chains, it is difficult to easily cut these sugar chains by enzymatic treatment. Therefore, in the above-mentioned conventional method using a relatively large amount of immobilized antibody and labeled lectin, in addition to the sugar chain-containing antigen bound to the immobilized antibody as the target to which the labeled lectin binds, the immobilized antibody (the antigen is bound to Or
(Regardless of whether they are not combined)
The labeled lectin bound to the immobilized antibody raises the background, making accurate measurement of the antigen virtually impossible.

[Means for Solving the Problems] As a result of intensive studies to solve the problems of the above-mentioned conventional methods, the present inventors have found that the sandwich method using immobilized lectins and labeled antibodies causes the problems of the above-mentioned conventional methods. The inventors have solved the above problems all at once, and have found that a practical measurement system for sugar chain-containing antigens can be assembled, and have completed the present invention.

That is, the present invention provides a method for measuring a trace substance (sugar chain-containing antigen) in a measurement sample by the sandwich method,
The present invention relates to a method for measuring a trace substance, which comprises using a solid-phased lectin and a labeled antibody. More specifically, a complex of immobilized lectins and sugar chain-containing antigens is obtained by reacting a measurement sample with immobilized lectins, and further the complex is reacted with a labeled antibody to immobilize immobilized lectins. The present invention relates to a method for measuring a trace amount of a substance, which comprises obtaining a complex of a sugar chain-containing antigen and a labeled antibody and measuring the amount of the label in the complex.

The lectin used in the present invention is not particularly limited as long as it has a property of specifically recognizing and binding a certain sugar structure among sugar-binding proteins. In particular,
Concanavalin A (ConA, jack bean lectin), PNA
(Peanut lectin), RCA (castor lectin), LcH (lens bean lectin) and PHA (kidney bean lectin), WGA (wheat embryo lectin) and other plant lectins, L
Animal lectins such as FA (slug lectin) can be used.

The solid phase to which the lectin is bound is not particularly limited as long as it can bind the lectin. Specifically, carriers used for chromatography such as agar and agarose gel, and water-insoluble carriers such as polystyrene beads can be used. The lectin and the carrier can be bound by a conventional method.

The labeled antibody used in the present invention is an antibody specific to the antigen to be measured (particularly the protein portion of the antigen) labeled with an appropriate marker (radioactive substance, enzyme, fluorescent substance, chemiluminescent substance, etc.). is there. Such a labeled antibody can also be prepared by a conventional method.

The measurement sample used in the present invention includes blood, serum, plasma,
It may be any body fluid (saliva, ascites, pleural effusion, urine, bile, etc.).

In the method of the present invention, an immobilized lectin is reacted with a measurement sample to pick up an antigen that binds to the lectin, and then this antigen is sandwiched with a labeled antibody, and the labeled amount of the complex is measured. Specifically, the lectin immunoradiometric assay (LIRMA) will be described with reference to the drawings. First
In the figure, 1) Put a small amount of a measurement sample 2 such as serum or body fluid in a test tube 1. The measurement sample 2 is composed of a lectin-bound antigen 3 containing an antibody-bound antigen 4 and an antibody-unbound antigen 5 and a lectin-unbound antigen 6. Next, a small amount of lectin-bound agarose beads 7 is put. Depending on the case, a buffer solution may be added at this time. At this time, the standard solution having the determined antigen concentration is added to the measurement system in the same manner as the sample. It is desirable that the standard antigen has an elucidated sugar chain structure.

2) The test tube 1 is fixed on a shaker, and the reaction between the lectin and the antigen is performed while shaking to obtain a lectin agarose bead-antigen conjugate 8. This is because the agarose beads are very likely to precipitate. Since this reaction is fast, it can be completed in 1 to 2 hours.

3) A large amount of buffer solution is added to the test tube 1 in which the reaction has been completed, the agarose beads are floated, and then centrifuged. Agarose beads precipitate easily. At this time, remove the supernatant with 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 with a radioisotope to the precipitated agarose beads. At this time, a buffer solution may be added at the same time. If I-125 is used as the radioisotope, the widely used IRMA I-125 labeled antibody can be used.

5) The reaction between the antigen and the antibody is carried out with shaking to obtain the lectin agarose beads-antigen-labeled antibody conjugate 10. Stable results can be obtained by performing at low temperature for a long time.

6) The same washing operation as in 3) above is performed to remove the unreacted bound substance 11 of the radioisotope-labeled antibody.

7) Finally, the lectin-conjugated agarose beads, the antigen, and the conjugate 10 of the radioisotope-labeled antibody that have been precipitated are applied to a radioactivity analyzer to measure the radioactivity.

As an example, a standard curve is drawn using the radioactivity und obtained from the standard concentration as shown in FIG. 2, and the radioactivity und of the unknown sample is applied to the standard curve to obtain the concentration of the sample. Alternatively, the radioactivity und of the unknown sample is calculated as a ratio to the radioactivity und of the specified standard concentration, and this is quantitatively interpreted.

[Example] Example 1 WGA (wheat embryo lectin) and LcH (lens bean lectin)
LIRMA of alpha-feto protein (AFP), which is currently the best test item for liver cancer, using agarose beads from EY Laboratoyies
Was done.

In order to match the amount of sugar chain antigen binding of lectin, dilute standard solution and serum sample to 1/10 with buffer, add 50 μl of WGA or LcH agarose beads per original concentration, and shake horizontally at 25 ° C for 2 hours. While incubating,
1 ml of phosphate buffer (including Triton-X 100) was added to the reaction solution, and the mixture was centrifuged at 3000 rpm, 4 ° C. for 5 minutes, the supernatant was removed, and phosphate buffer was added again, and the same operation was repeated 4 times. By these operations, the antigen not reacted with WGA or LcH is completely removed.

50 μl of I-125 labeled anti-AFP antibody is added to the washed agarose beads and incubated at 4 ° C. for 48 hours with shaking. After completion of the reaction, the agarose beads are washed in the same manner as above to remove unreacted I-125 labeled anti-AFP antibody, and the bound radioactivity of the precipitated agarose beads is measured by a gamma counter.

A standard curve is drawn using the measurement results of the standard solution, and the antigen concentration of the unknown sample is determined from this. Alternatively, determine the ratio of the radioactivity of the unknown sample to the specified standard. This is suitable for interpreting the measured values when the standard sugar chain structure is not volume-responsive to lectins.

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

When compared with the measurement result of the IRMA method, the measurement with the LcH lectin is similar to the measurement result with the IRMA method. This is because the LcH lectin recognizes the fucose structure and thus reacts well with the standard derived from liver cancer and the serum of liver cancer.
However, since WGA recognizes the bisecting N-acetylglucosamine (bisecting GlcNAc) structure, the ratio of 1000 ng / ml to the standard is small. In addition, when quantitatively compared, the concentration of liver cancer by IRMA is D, C, B, A, E in descending order, but it is D, C, B, A, E in WGA and B, D, C, in LcH. A and E. This reveals that Sample B has more Fucose structures.

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

[Effects of the Invention] As described above, the present invention is to measure an antibody-recognizing antigen only from a lectin-binding antigen.
Unlike the above, it does not measure the antigen recognized only by the antibody.
Therefore, by changing the lectin, it is possible to observe the quantitative difference due to the sugar chain structure even in the sugar chain antigen, which is a completely new measurement method that has never been seen before.

Further, since the method uses a solid-phased lectin and a labeled antibody, it is possible to overcome the drawbacks found in the conventional methods using the solid-phased antibody and the labeled lectin.

[Brief description of drawings]

FIG. 1 is an explanatory view of a lectin immunoradiometric assay (LIRMA) according to the present invention, and FIG. 2 shows a standard curve using a radioactivity unit (CPM) obtained from a standard concentration (ng / ml). It is shown. 1 ... test tube, 2 ... measurement sample such as serum or body fluid, 3 ... lectin-binding antigen, 4 ... antibody-binding antigen, 5
...... Antibody non-binding antigen, 6 …… Lectin non-binding antigen, 7 ・ ・ ・
… Lectin-bound agarose beads, 8 …… Lectin-agarose beads-antigen conjugate, 9 …… Radioisotope-conjugated antibody, 10 …… Lectin-agarose beads-antigen-labeled antibody conjugate, 11 …… Unbound Labeled antibody

Claims (2)

[Claims] 1. A method for measuring a trace substance (a sugar chain-containing antigen) in a measurement sample, which comprises using a solid-phased lectin and a labeled antibody in a method for measuring a trace substance (sugar chain-containing antigen) by a sandwich method. 2. A complex of immobilized lectins and sugar chain-containing antigens is obtained by reacting a measurement sample with immobilized lectins, and further the complex is reacted with a labeled antibody to immobilize immobilized lectins.
The method according to claim 1, wherein a complex of the sugar chain-containing antigen and the labeled antibody is obtained, and the amount of the label in the complex is measured.