JP3692055B2 - Determination method of antigen-antibody reaction - Google Patents

Description

【００２１】
７）結果の判定
結果の判定は以下の基準に従った。
（１）通常測定
・測定値が８ＩＵ／ｍＬ未満・・・陰性
・測定値が８ＩＵ／ｍＬ以上・・・陽性
（２）中和試験
・中和率が５０％未満・・・陰性
・中和率が５０％以上・・・陽性
【００２２】
（実施例２） ＨＢｓ抗原測定試薬への適用−２
実施例１の通常測定用第１試薬及び中和用第１試薬の調製を以下のように行った以外は、実施例１と同様に行った。結果を表１に示した。
１）第１試薬（緩衝液）の調製
（１）通常測定用第１試薬の調製
ＢＳＡを１重量％含有する０．０５ＭＰＢＳに、ＰＥＧを１．０重量％の濃度になるように溶解した。上記溶液１Ｌに対し、更に、０．０３６ＭＰＢＳを８．０μＬ添加し、通常測定用第１試薬を調製した。
（２）中和用第１試薬の調製
ＢＳＡを１重量％含有する０．０５ＭＰＢＳに、ＰＥＧを１．０重量％の濃度になるように溶解した。上記溶液１Ｌに対し、更に、抗ＨＢｓウサギ抗体（精製品、緩衝液：０．０３６ＭＰＢＳ）（蛋白濃度：１．０ｍｇ／ｍＬ、特異抗体力価：５３００ＩＵ／ｍＬ（メディエースＨＢｓ抗体（積水化学工業社製）にて測定）を８．０μＬ添加し、中和用第１試薬を調製した。
【００２３】
【表１】

【００２４】
（比較例１）
通常測定用第１試薬の調製を以下のように行い、また、実施例１の中和用第１試薬に代えて下記１）−（２）の確認試験用第１試薬を用いて試験を実施した以外は、実施例１と同様に行った。結果を表２に示した。
【００２５】
１）第１試薬（緩衝液）の調製
（１）通常測定用第１試薬の調製
ＢＳＡを１重量％含有する０．０５ＭＰＢＳに、ＰＥＧを１．０重量％の濃度になるように溶解し、通常測定用第１試薬を調製した。
（２）確認試験用第１試薬の調製
ＢＳＡを１重量％含有するホウ酸Ｎａ−ＨＣｌ緩衝液（ｐＨ９．１５）に、ＰＥＧを１．０重量％の濃度になるように溶解し、確認試験用第１試薬を調製した。
【００２６】
（比較例２）
通常測定用第１試薬の調製を以下のように行い、また、実施例１の中和用第１試薬に代えて下記１）−（２）の確認試験用第１試薬を用いて試験を実施した以外は、実施例１と同様に行った。結果を表２に示した。
【００２７】
１）第１試薬（緩衝液）の調製
（１）通常測定用第１試薬の調製
ＢＳＡを１重量％含有する０．０５ＭＰＢＳに、ＰＥＧを１．０重量％の濃度になるように溶解し、通常測定用第１試薬を調製した。
（２）確認試験用第１試薬の調製
ＢＳＡを１重量％含有するホウ酸Ｎａ−ＨＣｌ緩衝液（ｐＨ９．１５）に、ＮａＣｌを０．５Ｍの濃度になるように、またＰＥＧを１．０重量％の濃度になるように溶解し、確認試験用第１試薬を調製した。
【００２８】
【表２】

【００２９】
実施例１及び２においては、ＨＢｓ抗原陽性血清について５検体とも通常試験では８ＩＵ／ｍＬ以上を示し、中和率も５０％を超えたため、陽性と判定された。一方、偽陽性血清については５検体とも通常試験では８ＩＵ／ｍＬを超えたが、中和率が５０％未満であるため、陰性と判定された。
比較例１及び２においては、ＨＢｓ抗原陽性血清について５検体とも通常試験で８ＩＵ／ｍＬ以上を示し、中和率も５０％を超えたため、陽性と判定された。しかし、偽陽性血清については５検体とも通常試験では８ＩＵ／ｍＬを超え、更に５検体中４検体は中和率も５０％以上になったため、陽性と判定されてしまった。
【００３０】
【発明の効果】
本発明は上述の構成よりなるので、検体を別容器に移しかえたりする手間なく、抗原及び／又は抗体が消費されたかどうかを調べることができ、非常に簡便に抗原抗体反応の中和試験を行うことができる。[0001]
BACKGROUND OF THE INVENTION
The present invention belongs to the field of immunological measurement, and particularly relates to measurement using an antigen-antibody reaction, and relates to a simple and highly accurate determination method of the measurement result.
[0002]
[Prior art]
Antigen-antibody reactions are routinely used for the detection of trace components. In an immunological measurement method, it is important to determine whether the measured result is due to an antigen-antibody reaction. For example, Japanese Patent Laid-Open No. 02-80956 discloses that an agglutination reaction reagent is mixed with an agglutination reagent on a substrate surface such as a slide, and an agglutination image resulting from an antigen-antibody reaction is observed and determined. Non-specific aggregation depending on whether or not aggregation is dissociated by adding a chemical substance containing high concentrations of chaotropic ions, changing the ion concentration to 0.5M or higher, and changing the ion concentration to 0.5M or higher. A method for checking whether or not is disclosed.
[0003]
Also, JP 11-511255 A forms a reagent containing a fixing material in which a binding member specific to an IgM antibody is fixed, and binds to a solid phase material to cause a deviation from the IgG antibody from the IgM antibody. A method for determining by making the pH sufficiently low is disclosed.
[0004]
The method using an immune reaction is a method with high measurement specificity because it uses a specific antigen-antibody reaction. However, non-specific agglutination reaction may occur depending on the state of the specimen, and an error may occur in the measurement result.
[0005]
In order to solve such a problem, as a means for confirming whether an antigen-antibody reaction or a non-specific agglutination reaction, in the above example, the separation condition (pH of the antigen-antibody reaction when the reaction that occurred is an antigen-antibody reaction) (pH , Changes in ionic strength, changes in chaotropic ions, etc.), but in such methods, there may be discrepancies even in non-specific reactions other than antigen-antibody reactions. It may not be possible to determine whether or not.
[0006]
[Problems to be solved by the invention]
In view of the above situation, the present invention provides a determination method capable of establishing an immunological measurement method capable of obtaining an accurate measurement result regardless of whether or not a non-specific aggregation reaction has occurred. Objective.
[0007]
[Means for Solving the Problems]
The present invention is an antigen-antibody reaction determination method for measuring an antibody or antigen corresponding to a supported antigen or antibody using an insoluble carrier carrying the antigen or antibody, and the reagent configuration for the measurement comprises: Two or more reagents consisting of a suspension of an insoluble carrier and a buffer solution are used, and these reagents are mixed at the time of measurement. The measurement is performed twice, and the reaction is performed at the first sample measurement. For the sample in which the reaction occurred, replace the buffer used for the measurement in the reagent configuration with a buffer containing the same antibody or antigen as the one supported on the insoluble carrier, and then perform the second measurement to obtain the first reaction. In contrast, the antigen-antibody reaction determination method determines whether the reaction is a specific reaction or a non-specific reaction by measuring the degree of decrease in the reaction with the buffer.
The present invention is described in detail below.
[0008]
In the present invention, a buffer solution of an ordinary measurement reagent comprising a suspension of an insoluble carrier, a buffer solution, and a buffer solution (hereinafter referred to as an antibody or antigen having the same specificity as the antibody or antigen previously supported on the insoluble carrier). In this case, the measurement is performed again while consuming the antigen or antibody in the sample. By taking such measures, it is possible to investigate whether antigens / antibodies have been consumed without having to transfer the specimen to another container. Confirmation can be made.
[0009]
As an antigen adsorbed or bound to an insoluble carrier and contained in a neutralization buffer, an immunoassay such as a protein antigen, lipid antigen, hapten, hapten bound to a carrier (for example, a protein such as BSA), etc. If it can be used for a law, the property will not ask | require. In the case of a purified protein or the like, animal species, tissues and the like need not be of the same origin as long as they have the same specificity as that adsorbed or bound to the insoluble carrier.
[0010]
As an antigen that is adsorbed or bound to an insoluble carrier and contained in a neutralization buffer, either a polyclonal antibody or a monoclonal antibody can be used, and an enzyme-digested fragment thereof can also be used. . Further, the antibody adsorbed and bound to the insoluble carrier is not necessarily an antibody derived from the same species, and one may be a monoclonal antibody and the other may be a polyclonal antibody.
[0011]
The number of liquids is not limited as long as the measurement system is composed of a latex suspension, a buffer solution, or the like. That is, a reagent configuration in which two or more types of buffer solutions and two or more types of latex suspensions are divided may be used. In addition, if there are at least two types of latex suspension and at least one buffer solution at the time of measurement, storage is divided into two or more buffer solutions and two or more latex suspensions at storage. Neither does the method interfere with the present invention.
[0012]
There is no limitation on the neutralization rate. In order to facilitate the determination, it is preferable that the neutralization rate can be set to a high value of 50% or more as a determination criterion. However, even when it is difficult to obtain high-titer antibodies, highly purified antigens, etc., neutralization is possible. If the presence or absence can be determined, the present invention will not be hindered.
[0013]
【Example】
Hereinafter, the present invention will be described in more detail by giving examples and comparative examples of the present invention. The present invention is not limited to the following examples.
(Example 1) Application to reagent for measuring HBs antigen-1
1) Preparation of first reagent (buffer solution) (1) Preparation of first reagent for normal measurement Phosphate-saline buffer solution (0.05 M phosphate) containing 1% by weight of bovine serum albumin (hereinafter also referred to as BSA) A buffer solution (pH 7.0), 0.1 M NaCl (hereinafter also referred to as 0.05 MPBS) and 1.0% by weight of polyethylene glycol having an average molecular weight of 500,000 (manufactured by Wako Pure Chemical Industries, Ltd., hereinafter also referred to as PEG) It dissolved so that it might become the density | concentration of. To 1 L of the above solution, 3.0 μL of normal rabbit serum was further added to prepare a first reagent for normal measurement.
(2) Preparation of first reagent for neutralization PEG was dissolved in 0.05M PBS containing 1% by weight of BSA to a concentration of 1.0% by weight. To 1 L of the above solution, 3.0 μL of anti-HBs antiserum (rabbit) (specific antibody titer 8000 IU / mL (measured with Mediace HBs antibody (manufactured by Sekisui Chemical Co., Ltd.)) was added. One reagent was prepared.
[0014]
2) Preparation of second reagent (anti-HBs antibody-sensitized latex solution) Phosphate-saline buffer solution (0.036M phosphate buffer solution (0.036M phosphate buffer solution) containing anti-HBs rabbit antibody (purified product) at a concentration of 1.0 mg / mL pH 6.6), 0.1 M NaCl (hereinafter also referred to as 0.036 MPBS) in 1.25 mL, polystyrene latex having an average particle size of 0.3 μm (solid content 10% (W / V), manufactured by Sekisui Chemical Co., Ltd.) 1 mL And 0.036 M PBS were added and stirred at 30 ° C. for 60 minutes. Next, 0.05 M PBS containing 1% by weight of BSA was added to this solution, stirred for 60 minutes at 30 ° C., and then washed by centrifugation at 18,000 rpm for 20 minutes at 4 ° C. The washing operation was performed 3 times. To the resulting precipitate, 100 mL of 0.05M PBS containing 1% by weight of BSA was added to suspend the latex, and then subjected to a dispersion treatment with an ultrasonic crusher to obtain a solid content of 0.1% (W / V). An anti-HBs antibody-sensitized latex solution was prepared. This was used as the second reagent.
[0015]
3) HBs antigen measurement reagent As the HBs antigen measurement reagent, the normal measurement first reagent and the second reagent were used in the normal measurement, that is, in the measurement of the HBs antigen. In the neutralization test, that is, the HBs antigen confirmation test, the first and second reagents for neutralization were used.
[0016]
4) Standard serum HBs antigen solution, human serum containing HBs antigen at concentrations of 0, 50, 100, 300, and 500 IU / mL was used as a standard sample for neutralization test.
In addition, HBs antigen positive sera: A to E and false positive sera F to J were used as specimens. Here, the false positive serum is a serum collected from a healthy person, but is determined to be positive in a normal test.
[0017]
5) Normal measurement method (1) Preparation of calibration curve 20 μL of the specimen and 150 μL of the first reagent for normal measurement were mixed and stored at 37 ° C., and then 150 μL of the second reagent was added and stirred. Thereafter, the absorbance at a wavelength of 750 mm after 1 minute and after 5 minutes was measured, and this difference was defined as the amount of change in absorbance (ΔAbs). Hitachi 7150 type automatic analyzer was used for the measurement.
A standard curve was prepared in advance by preparing a standard curve. In the following measurements, the amount of change in absorbance of the sample was substituted into the standard curve, and the amount of HBs antigen in the sample was calculated.
[0018]
(2) Measurement of specimen Using the HBs antigen positive serum and false positive serum as specimens, the amount of HBs antigen was measured according to the measurement method of 5)-(1) above. The results are shown in Table 1.
[0019]
6) Neutralization test method A neutralization test was carried out on the specimens determined to be positive in the normal measurement of 5)-(2) above.
Measurement was carried out in the same manner as in 5)-(1) above except that the first reagent for measurement was replaced with the first reagent for neutralization using HBs antigen positive serum and false positive serum as samples. The results are shown in Table 1. Moreover, the neutralization rate in a table | surface was computed according to the following formula.
[0020]
[Expression 1]

[0021]
7) Judgment of results The judgment of the results was in accordance with the following criteria.
(1) Normal measurement / measurement value is less than 8 IU / mL ... Negative / measurement value is 8 IU / mL or more ... Positive (2) Neutralization test / neutralization rate is less than 50% ... Negative / neutralization Rate is more than 50% ... positive [0022]
(Example 2) Application to HBs antigen measurement reagent-2
Example 1 was performed in the same manner as Example 1 except that the first reagent for normal measurement and the first reagent for neutralization were prepared as follows. The results are shown in Table 1.
1) Preparation of first reagent (buffer) (1) Preparation of first reagent for normal measurement PEG was dissolved in 0.05M PBS containing 1% by weight of BSA to a concentration of 1.0% by weight. To 1 L of the above solution, 8.0 μL of 0.036 M PBS was further added to prepare a first reagent for normal measurement.
(2) Preparation of first reagent for neutralization PEG was dissolved in 0.05M PBS containing 1% by weight of BSA to a concentration of 1.0% by weight. Anti-HBs rabbit antibody (purified product, buffer solution: 0.036 MPBS) (protein concentration: 1.0 mg / mL, specific antibody titer: 5300 IU / mL (Mediace HBs antibody (Sekisui Chemical Co., Ltd.)) 8.0 μL) was added to prepare a neutralizing first reagent.
[0023]
[Table 1]

[0024]
(Comparative Example 1)
The first reagent for normal measurement was prepared as follows, and the test was conducted using the first reagent for confirmation test 1)-(2) below instead of the first reagent for neutralization in Example 1. The procedure was the same as in Example 1 except that. The results are shown in Table 2.
[0025]
1) Preparation of first reagent (buffer solution) (1) Preparation of first reagent for normal measurement PEG is dissolved in 0.05M PBS containing 1% by weight of BSA to a concentration of 1.0% by weight, A first reagent for normal measurement was prepared.
(2) Preparation of first reagent for confirmation test PEG was dissolved in Na-HCl borate buffer (pH 9.15) containing 1% by weight of BSA to a concentration of 1.0% by weight, and the confirmation test A first reagent was prepared.
[0026]
(Comparative Example 2)
The first reagent for normal measurement was prepared as follows, and the test was conducted using the first reagent for confirmation test 1)-(2) below instead of the first reagent for neutralization in Example 1. The procedure was the same as in Example 1 except that. The results are shown in Table 2.
[0027]
1) Preparation of first reagent (buffer solution) (1) Preparation of first reagent for normal measurement PEG is dissolved in 0.05M PBS containing 1% by weight of BSA to a concentration of 1.0% by weight, A first reagent for normal measurement was prepared.
(2) Preparation of first reagent for confirmation test In a borate Na-HCl buffer solution (pH 9.15) containing 1% by weight of BSA, NaCl is adjusted to a concentration of 0.5M, and PEG is added to 1.0%. It melt | dissolved so that it might become a density | concentration of a weight%, and the 1st reagent for confirmation tests was prepared.
[0028]
[Table 2]

[0029]
In Examples 1 and 2, all five samples of HBs antigen positive sera showed 8 IU / mL or more in the normal test, and the neutralization rate exceeded 50%. On the other hand, all of the five false-positive sera exceeded 8 IU / mL in the normal test, but were judged negative because the neutralization rate was less than 50%.
In Comparative Examples 1 and 2, since 5 samples of HBs antigen positive sera showed 8 IU / mL or more in the normal test and the neutralization rate exceeded 50%, it was determined to be positive. However, all of the five false-positive sera exceeded 8 IU / mL in the normal test, and four of the five specimens were determined to be positive because the neutralization rate was 50% or more.
[0030]
【The invention's effect】
Since the present invention has the above-described configuration, it is possible to examine whether antigens and / or antibodies have been consumed without having to transfer the specimen to a separate container. It can be carried out.

Claims (1)

A method for determining an antigen-antibody reaction, wherein an insoluble carrier carrying an antigen or an antibody is used to measure the antibody or antigen corresponding to the carried antigen or antibody,
The reagent configuration for the measurement uses two or more liquid reagents composed of a suspension of an insoluble carrier and a buffer solution, and these reagents are mixed at the time of measurement.
The measurement is performed twice,
For a sample that has undergone a reaction during the first sample measurement, the buffer solution used for the measurement in the reagent configuration is replaced with a buffer solution containing the same antibody or antigen as that carried on the insoluble carrier, and then the second time. Measure
A method for determining an antigen-antibody reaction, wherein a determination is made as to whether the reaction is a specific reaction or a non-specific reaction by measuring the degree of decrease in the reaction with the buffer relative to the initial reaction.