JP2004012170A - Method for immunological measurement and measuring kit - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for immunologically measuring for clinically examining with high reliability by performing a reduction in a false positive and an improvement in idiosyncrasy by excluding nonspecific reaction by an antibody or a cross antibody for a substance of a constituents of an immunity reagent and to provide a measuring kit. <P>SOLUTION: The immunologically measuring method having a step of reacting an immunity reagent carried with a specific component in a human originating specimen with an insoluble carrier of a specific antigen or antibody to the specific component, before the specimen according to the human is reacted with the immunity reagent, has a step of previously reacting the antibody to the constituents of the immunity reagent or the antigen to the cross antibody existing in the specimen according to the human with the block reagent carried to the insoluble antibody. The combination of the immunity reagent and the block reagent is thus provided. <P>COPYRIGHT: (C)2004,JPO

Description

【００３８】
検体Ｎｏ．１〜９はＴＰ陰性検体であるが、Ｎｏ．７、８、９は検体中に牛血清アルブミン抗体を持った検体である。Ｎｏ．１０〜１３はＴＰ陽性検体である。比較例１では、牛血清アルブミン抗体を持った検体Ｎｏ．７、８、９において、牛血清アルブミン抗体による非特異的凝集反応が認められる。また、比較例２では、非特異的凝集反応の影響は軽減されたが、検体Ｎｏ．８および９は非特異的凝集反応の影響を受けていると考えられる。一方、本発明の試薬による測定は牛血清アルブミン抗体による非特異的凝集反応は観察されなかった。
【００３９】
実施例２
（１）ＴＰ抗原感作ラテックス試薬（第二試薬）の調製
実施例１と同様に調製した。
（２）ＴＰ測定用緩衝液（第一試薬）の調製
１％牛血清アルブミン−グリシン緩衝液（０．０１％Ｔｗｅｅｎ１００を含有）にブロック試薬を加えて、牛血清アルブミンを担持させたラテックスが０．０２５％、０．０５％、０．１０％、０．１６％含有するように調製した。また、対照法（比較例３）はブロック試薬を含まない他は同様な組成である。
（３）ＴＰ抗体の測定
実施例１と同様に測定した。その結果を表２に示す。
【００４０】
【表２】

【００４１】
検体Ｎｏ．２、３、４、５、８および９はＴＰ陰性検体であるが、Ｎｏ．８および９は検体中に牛血清アルブミン抗体を持った検体である。Ｎｏ．１０〜１３はＴＰ陽性検体である。対照方法（比較例３）は、牛血清アルブミン抗体を持った検体Ｎｏ．８および９において、牛血清アルブミン抗体による非特異的凝集反応が認められる。本発明の試薬による測定は第一試薬中のブロック試薬である牛血清アルブミンを担持させたラテックスが０．０２５％では、検体Ｎｏ．８は判定保留域に軽減でき、Ｎｏ．９は非特異的凝集反応を抑制することができた。同様に０．０５％以上では、牛血清アルブミン抗体による非特異的凝集反応は観察されなかった。
【００４２】
実施例３
（１）ペプシノーゲン ＩＩ 抗体感作ラテックス試薬（第二試薬）の調製
抗ペプシノーゲンＩＩモノクロナール抗体（Ａ）３ｍｇをリン酸ナトリウム緩衝液９．５ｍＬに溶解し、０．３μｍのポリスチレンラテックス（固形分１０重量％緩衝液）０．５ｍＬを加え、３７℃で２時間インキュベーションした後、これに２％ＢＳＡ−リン酸緩衝液２ｍＬを加え、３７℃で９０分間インキュベーションすることによりポストコーティングを行った。
次に遠心分離（１５，０００ｒｐｍにて１５分間遠心）を行い、上清を捨て、沈殿を牛血清アルブミン溶液（０．１Ｍグリシン、０．０５Ｍ塩化ナトリウム、０．０５％アジ化ナトリウム、０．２％ＢＳＡ）に懸濁させ、ペプシノーゲンＩＩ抗体感作ラテックス（Ａ）を調製した。同様に、抗ペプシノーゲンＩＩ抗体（Ｂ）を用いて、ペプシノーゲンＩＩ抗体感作ラテックス（Ｂ）を調製した。（Ａ）および（Ｂ）を混合して、ペプシノーゲンＩＩ抗体感作ラテックス試薬を調製した。
【００４３】
（２）ペプシノーゲン ＩＩ 測定用緩衝液（第一試薬）の調製
１％牛血清アルブミン−グリシン緩衝液にブロック試薬を加えて、牛血清アルブミンが担持させたラテックスが０．１６％含有するように調製した。また、対照法（比較例４）はブロック試薬を含まない他は同様な組成である。
【００４４】
（３）ペプシノーゲン ＩＩ の測定
実施例１のＴＰ抗体の測定と同様の操作で測定した。結果を表３に示す。表３中、ＲＩＡの欄の値は放射線免疫測定法による測定値である。
【００４５】
【表３】

【００４６】
検体Ｎｏ．１０は、血清検体中に牛血清アルブミン抗体を持った検体である。対照方法は、牛血清アルブミン抗体を持った検体Ｎｏ．１０は非特異的凝集反応により、正に乖離している。一方、本発明の試薬による測定は牛血清アルブミン抗体による非特異的凝集反応は観察されなかった。
【００４７】
【発明の効果】
本発明によれば、免疫試薬の構成成分である物質に対する抗体あるいはその交叉抗体による非特異的反応を排除できるため、偽陽性の削減および特異性の向上が達成され、信頼性のより高い臨床検査が可能になる。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to an immunological measurement and a kit for measurement. More specifically, the present invention relates to an immunological measurement method and a measurement kit for suppressing a false reaction caused by an antibody against a substance such as a reagent material present in a human sample or a cross-antibody thereof causing an antigen-antibody reaction with the reagent material. .
[0002]
[Prior art]
An immunological measurement method, which is an in vitro assay utilizing a binding reaction between an antigen and an antibody, can be specifically detected or accurately measured for a trace component, and thus has been widely applied to clinical tests. They include an agglutination method (using blood cells, latex, etc. as a carrier), a radioimmunoassay (RIA), an enzyme immunoassay (EIA), and a fluorescence immunoassay depending on the method of labeling. Method (FIA), chemiluminescence immunoassay (CLIA), nephelometry and immunoturbidimetry.
[0003]
However, in the application of immunoassays to clinical tests, rheumatoid factor (RF), complement Clq, myeloma, HAMA, etc., which are present in biological fluids such as blood and urine collected from patients, do not contain such immunological assays. There was a problem that the specific agglutination reaction caused a false positive.
[0004]
Therefore, in order to suppress non-specific reactions due to the influence of RF or the like, a sample such as pretreatment of a sample with dithiothreitol or 2-mercaptoethanol or addition of an anti-IgM antibody, γ-globulin of various animals or a denatured product thereof is added. Improvement of immunoassay methods, such as pretreatment of E. coli, use of fragments such as Fab and F (ab ') _{2 of} an antibody, or use of an antibody in which an Fc site is blocked has been proposed.
[0005]
See, for example, Journal of Clinical Microbiology, Feb. 1976, p. 157-160 describes a pretreatment of a sample with dithiothreitol. Also, Medical Examination, Vol. 42, No. 9, 1993, p. Nos. 1504-1508 describe addition, inactivation, heat treatment, and 2-mercaptoethanol treatment of an anti-human IgM antibody or an anti-human IgG antibody. Japanese Patent Publication No. 61-942 discloses a method in which an unsensitized normal animal γ-globulin or a denatured product thereof is added and the antigen or antibody in the sample is immunologically measured in the presence thereof. .
[0006]
Regarding the use of antibody fragments, JP-B-63-38668 discloses the use of an F (ab ') ₂ fragment of an immunoglobulin cleaved as an antibody for measurement because the Fc site is involved in non-specific reactions. Disclosed immunoassays.
[0007]
In particular, in the agglutination method using latex, non-specific reaction by RF or the like is prevented by blending derivatives such as urea and formamide and di-lower alkyl sulfoxide (Japanese Patent Publication No. 60-4941), and Avoid interference such as RF by using latex particles sensitized with a mixture of a fragment (fragment) of F (ab ') ₂ of a globulin and an Fc fragment or a mixture of a Facb fragment and a pFc' fragment (Japanese Patent Publication No. 63-63863). And JP-B-63-41426).
[0008]
In JP-A-7-27764, the use of an antibody whose Fc site is blocked by a blocking reagent containing at least an antigen-binding site of an immunoglobulin that specifically binds to the Fc site of the antibody allows the use of substances such as RF. Methods for eliminating non-specific reactions are disclosed.
[0009]
In JP-A-2000-292424 or JP-A-2000-32279, in the case of collecting a blood sample, removal of an interfering substance due to a component derived from a blood collection tube dissolving into the sample, and a method of removing certain substances present in the sample, for example, Since proteins may be easily adsorbed on the surface of insoluble carrier particles, non-specific agglutination reactions may occur in particle agglutination reactions such as latex immunoturbidimetry. A method using a dummy particle is disclosed as a means for confirming a mistake during separation or the like.
[0010]
However, in recent years, a high sensitivity method has been required, and there has been a problem that a nonspecific reaction which cannot be eliminated by a conventionally known method rarely occurs.
[0011]
The present inventors have conducted intensive studies to solve the above-described problems, and as a result, an antibody against a substance such as a reagent material, which has not been known, or a cross antibody thereof is present in a human sample, and the antigen antibody The present inventors have found a method for eliminating a non-specific reaction by finding out that a false reaction is caused by the reaction, and preliminarily blocking the antibody present in a human sample, and have reached the present invention.
[0012]
[Problems to be solved by the invention]
An object of the present invention is to provide an immunological assay for suppressing a false reaction caused by an antibody or a cross antibody thereof to a substance which is present in a human sample and is a component of an immunoreagent, and causing an antigen-antibody reaction with the reagent material. To provide a kit.
[0013]
Another object of the present invention is to provide an immunoassay using the above-described immunoassay kit.
[0014]
It is still another object of the present invention to provide an immunological assay method capable of eliminating non-specific reaction of an antibody against a substance such as a reagent raw material or the like present in a human specimen with a cross antibody.
[0015]
Still other objects and advantages of the present invention will become apparent from the following description.
[0016]
[Means for Solving the Problems]
According to the present invention, the above objects and advantages of the present invention are, firstly, that a specific component in a human-derived specimen is reacted with an immunoreagent in which a specific antigen or antibody corresponding thereto is supported on an insoluble carrier. In the immunoassay comprising, before reacting a human-derived sample with an immunoreagent, an antibody present in the human-derived sample and presenting an antibody against a component of the immunoreagent or an antigen against a cross-antibody is carried on an insoluble carrier. This is achieved by an immunological assay characterized by reacting in advance with the resulting blocking reagent.
[0017]
According to the present invention, the above object and advantages of the present invention are, secondly, (1) an immunoreagent comprising a specific antigen or antibody against a specific component in a human-derived specimen supported on an insoluble carrier; 2) It is achieved by an immunoassay kit comprising a combination of a blocking reagent, which is present in the human-derived specimen and contains an antibody against a component of the immunoreagent or an antigen against a cross-antibody supported on an insoluble carrier. .
[0018]
Preferred Embodiment of the Invention
Hereinafter, the present invention will be described in detail. First, the immunoassay of the present invention will be described.
[0019]
In the present invention, the immunological measurement method is a measurement method utilizing an immune reaction (antigen-antibody reaction), and includes both a qualitative method and a quantitative method. Examples of the immunoassay include agglutination (agglutination using a carrier such as Lux and blood cells), radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescence immunoassay, and luminescence immunoassay. And a turbidimetric method based on an agglomeration method, a faze measuring method, a particle size distribution measuring method and the like.
[0020]
The antibody or the cross antibody thereof to the substance which is present in the human sample of the present invention and is a component of the immunoreagent is an antibody to a protein such as bovine serum albumin and casein which is a component of the immunoreagent.
[0021]
Proteins such as bovine serum albumin and casein have been used as stabilizers for immunological reagents.
[0022]
For example, in the agglutination method, an immunoreagent is prepared by carrying an antigen or an antibody on an insoluble carrier such as latex particles or blood cells.However, in order to stabilize the immunoreagent, post-immunization is performed using a protein such as bovine serum albumin or casein. The coating has been applied. When an antibody against bovine serum albumin or casein that has been subjected to the post-coating treatment is present in a human sample, there is a problem that an agglutination reaction (non-specific agglutination reaction) due to an antigen-antibody reaction is induced. The present invention is intended to solve this problem by blocking the antibody which causes non-specificity in a human sample by a blocking reagent in which the same material as the protein used in the post-coating is bound to an insoluble carrier. is there.
[0023]
Examples of use of block reagents are mainly applicable to immunoassays using general-purpose automatic analyzers and special-purpose machines. In the reaction process where two or more types of reagents are mixed with a sample, the antigen or antibody is carried. In a step before the used latex reagent is dispensed, the blocking reagent blocks the antibody that causes non-specificity in the human specimen by mixing the specimen and the blocking reagent in advance. After this step, a latex reagent carrying an antigen or an antibody is dispensed to suppress non-specific agglutination with an antibody against a substance such as a reagent material present in a human sample or its cross-antibody. Can be realized.
[0024]
As an example of this blocking reagent, when bovine serum albumin is used for post-coating, the same reagent is prepared by supporting the same on an insoluble carrier.
[0025]
As the insoluble carrier used for the blocking reagent, latex particles generally used for immunological agglutination can be used, of course, and the precursor thereof may be used, or liposomes, gold colloid, or the like may be used. be able to. In any case, the size of the latex particles is desirably smaller than that of the latex particles carrying the antigen or antibody which is the immunological measurement reagent, and is applied within a range that does not hinder the immunological agglutination reaction to be measured and the measurement. For example, when the insoluble carrier is latex particles, those having a particle size of 0.5 μm or less are preferably used, and more preferably 0.2 μm or less. More preferably, it is 0.1 μm or less.
[0026]
When applied to the latex agglutination turbidimetric method, it is desirable to mix the blocking reagent with the first reagent which is a buffer solution beforehand, or to mix them before use. When the immunological measurement reagent is a two-reagent system comprising a first reagent containing a blocking reagent and a second reagent which is a latex reagent carrying an antigen or an antibody, the latex concentration of the blocking reagent contained in the first reagent (weight basis) As), the higher the content, the more effective it is, preferably from 0.0001 to 5%, more preferably from 0.005 to 1%, even more preferably from 0.03 to 0.3%. If the amount is too large, the reagent absorbance blank becomes high, which is not preferable. The reagent absorbance blank of the first reagent is preferably 1 or less. More preferably, it is 0.005 to 0.5. The weight ratio of the insoluble carrier as the raw material of the blocking reagent in the first reagent to the latex of the second reagent is preferably 0.2 to 50 times, more preferably 1 to 5 times. When the insoluble carrier as a raw material of the blocking reagent is latex particles, the number is preferably 1 to 5,000 times, more preferably 20 to 200 times the number of latex particles of the second reagent.
[0027]
In the immunological assay of the present invention, the sample is not particularly limited, and the present invention can be applied to a target sample in a normal immunological assay. Samples include those of human origin, particularly human blood (serum, plasma, whole blood), urine, feces, and the like. In particular, it is advantageous for a specimen having a high antibody content such as blood.
[0028]
The substance to be measured in the immunoassay of the present invention is not particularly limited, and those measured by a usual immunoassay can be measured. Examples include tumor markers such as CEA, AFP, CA19-9, pepsinogen I and II, ferritin, coagulation / fibrinolysis markers such as protein C, protein S, ATIII, FDP, D-dimer, HBs antigen, HCV antibody, syphilis Examples include infectious disease markers such as antibodies, hormones such as TSH, prolactin and insulin, and tissue components such as myoglobin and myosin.
[0029]
According to the present invention, there is conveniently provided an immunological kit comprising a combination of the above immunological reagent and a blocking reagent, which is used in the immunological assay of the present invention. Each of these reagents is stored in a separate container, and the combination provides the immunological kit of the present invention.
[0030]
【Example】
The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the following examples.
[0031]
Preparation Example 1
Preparation of blocking reagent (bovine serum albumin antibody absorption reagent) To 10 mL of 1% bovine serum albumin-glycine buffer, add 10 mL of 0.055 μm polystyrene latex (solid content 10%) 1% suspended glycine buffer, and mix well with a mixer. After that, incubation was performed at 37 ° C. for 90 minutes or at 56 ° C. for 60 minutes to prepare a blocking reagent carrying bovine serum albumin.
[0032]
Example 1
(1) Preparation of TP antigen-sensitized latex reagent (second reagent) 0.3 μm polystyrene latex (10% solid content) in 0.5 mL TP recombinant antigen solution (60 μg / mL) 2% suspension phosphate buffer After adding 5 mL and mixing well with a mixer, the mixture was incubated at 37 ° C. for 60 minutes. To this, 4 mL of 1% BSA-phosphate buffer was added, and the mixture was incubated at 37 ° C. for 90 minutes to perform post-coating. Next, centrifugation (centrifugation at 15,000 rpm for 15 minutes) was performed, and the precipitate was redispersed in a phosphate buffer containing 0.2% bovine serum albumin to prepare 10 mL of a TP antigen-sensitized latex reagent (No. 1). Two reagents).
[0033]
(2) Preparation of buffer for TP measurement (first reagent) A blocking reagent was added to a 1% bovine serum albumin-glycine buffer (containing 0.01% Tween 100), and the latex carrying bovine serum albumin was reduced to 0%. .16%.
[0034]
(3) Measurement of TP antibody Model 7170 (HITACHI) was used as an automatic analyzer. The first reagent and the second reagent were set in the apparatus, and the measurement was performed by a usual method. First, 10 μL of the sample and 160 μL of the first reagent were dispensed into a reaction cell and incubated at 37 ° C. for 4.5 minutes. After that, the second reagent is dispensed in an amount of 80 μL, whereby the syphilis antibody present in the sample and the latex carrying the TP antigen in the second reagent undergo aggregation due to an antigen-antibody reaction. By measuring, a measured value was output from this result. Table 1 shows the results.
[0035]
Comparative Example 1
Example 1 except that a 1% bovine serum albumin-glycine buffer (containing 0.01% Tween 100) was used so that the blocking reagent was not added thereto, and the final reagent had the same composition as the first reagent. Performed similarly to 1. Table 1 shows the results.
[0036]
Comparative Example 2
The same reagent as in Comparative Example 1 was used except that heat-denatured bovine serum albumin incubated at 56 ° C. for 30 minutes was used as the first reagent. Others were performed similarly to Example 1. Table 1 shows the results.
[0037]
[Table 1]

[0038]
Sample No. Nos. 1 to 9 are TP negative samples. Samples 7, 8, and 9 are samples having bovine serum albumin antibody in the sample. No. 10 to 13 are TP positive samples. In Comparative Example 1, Sample No. 1 having bovine serum albumin antibody was used. In 7, 8, and 9, nonspecific agglutination by bovine serum albumin antibody is observed. In Comparative Example 2, the influence of non-specific agglutination was reduced, but Sample No. 8 and 9 are thought to be affected by non-specific agglutination. On the other hand, in the measurement with the reagent of the present invention, non-specific agglutination by the bovine serum albumin antibody was not observed.
[0039]
Example 2
(1) Preparation of TP antigen-sensitized latex reagent (second reagent) Prepared in the same manner as in Example 1.
(2) Preparation of buffer for TP measurement (first reagent) A blocking reagent was added to a 1% bovine serum albumin-glycine buffer (containing 0.01% Tween 100), and the latex carrying bovine serum albumin was reduced to 0%. It was prepared to contain 0.025%, 0.05%, 0.10%, and 0.16%. The control method (Comparative Example 3) has the same composition except that no blocking reagent is contained.
(3) Measurement of TP antibody The measurement was performed in the same manner as in Example 1. Table 2 shows the results.
[0040]
[Table 2]

[0041]
Sample No. Nos. 2, 3, 4, 5, 8 and 9 are TP negative samples, Samples 8 and 9 have bovine serum albumin antibodies in the sample. No. 10 to 13 are TP positive samples. In the control method (Comparative Example 3), the sample No. having the bovine serum albumin antibody was used. In Nos. 8 and 9, non-specific agglutination by bovine serum albumin antibody is observed. In the measurement using the reagent of the present invention, when the latex carrying bovine serum albumin as a blocking reagent in the first reagent was 0.025%, the sample No. No. 8 can be reduced to the judgment suspension area. No. 9 was able to suppress non-specific agglutination. Similarly, at 0.05% or more, nonspecific agglutination by bovine serum albumin antibody was not observed.
[0042]
Example 3
(1) Preparation of pepsinogen II antibody-sensitized latex reagent (second reagent ) 3 mg of anti-pepsinogen II monoclonal antibody (A) was dissolved in 9.5 mL of sodium phosphate buffer, and 0.3 μm of polystyrene latex was dissolved. After adding 0.5 mL (solid content 10% by weight buffer) and incubating at 37 ° C. for 2 hours, 2 mL of 2% BSA-phosphate buffer was added thereto, and the mixture was incubated at 37 ° C. for 90 minutes to complete the post-coating. went.
Next, centrifugation (centrifugation at 15,000 rpm for 15 minutes) was performed, the supernatant was discarded, and the precipitate was washed with a bovine serum albumin solution (0.1 M glycine, 0.05 M sodium chloride, 0.05% sodium azide, 0.1% sodium azide). 2% BSA) to prepare a pepsinogen II antibody-sensitized latex (A). Similarly, a pepsinogen II antibody-sensitized latex (B) was prepared using the anti-pepsinogen II antibody (B). (A) and (B) were mixed to prepare a pepsinogen II antibody-sensitized latex reagent.
[0043]
(2) Preparation of buffer for pepsinogen II measurement (first reagent) A blocking reagent was added to a 1% bovine serum albumin-glycine buffer to prepare a 0.16% latex supported by bovine serum albumin. did. The control method (Comparative Example 4) had the same composition except that no blocking reagent was contained.
[0044]
(3) Measurement of pepsinogen II The measurement was performed in the same manner as in the measurement of the TP antibody in Example 1. Table 3 shows the results. In Table 3, the values in the column of RIA are values measured by a radioimmunoassay.
[0045]
[Table 3]

[0046]
Sample No. Numeral 10 is a specimen having bovine serum albumin antibody in the serum specimen. As a control method, a sample No. having a bovine serum albumin antibody was used. 10 is positively dissociated by a non-specific agglutination reaction. On the other hand, in the measurement with the reagent of the present invention, non-specific agglutination by the bovine serum albumin antibody was not observed.
[0047]
【The invention's effect】
According to the present invention, a non-specific reaction due to an antibody or a cross-antibody to a substance that is a component of an immunoreagent can be eliminated, so that false positives are reduced and specificity is improved, and a more reliable clinical test is achieved. Becomes possible.

Claims (6)

A human-derived specimen is reacted with an immunoreagent in an immunoassay comprising reacting a specific component in a human-derived specimen with an immunoreagent in which a specific antigen or antibody corresponding thereto is carried on an insoluble carrier. Prior to reacting with a blocking reagent which is present in said human-derived specimen and which comprises an antibody to a component of said immunoreagent or an antigen to cross-antibody carried on an insoluble carrier. . The method according to claim 1, wherein the component of the immunoreagent is a protein used for post-coating for stabilizing the immunoreagent. 3. The method according to claim 2, wherein the protein is bovine serum albumin or casein. (1) an immunoreagent in which a specific antigen or antibody against a specific component in a human-derived specimen is supported on an insoluble carrier; and (2) an antibody present in the human-derived specimen and directed to a component of the immunoreagent or An immunoassay kit comprising a combination with a blocking reagent in which an antigen for a cross antibody is carried on an insoluble carrier. The kit according to claim 4, wherein the component of the immunoreagent is a protein used for post-coating for stabilizing the immunoreagent. The kit according to claim 4, wherein the protein is bovine serum albumin or casein.