JP6705245B2 - Method for producing an immunoreaction reagent for measuring anti-thyroglobulin antibody - Google Patents

Description

The present invention is a method for producing an anti-thyroglobulin antibody measuring immunoreaction reagent used for in vitro diagnostics and the like, which improves measurement reproducibility and measurement sensitivity by purifying thyroglobulin immobilized on a solid phase. is there.

Thyroglobulin (hereinafter, Tg) is a glycoprotein having a molecular weight of about 660,000 and functions as a site for thyroid hormone synthesis. It is conventionally known that this autoantibody against Tg, that is, an anti-thyroglobulin antibody (hereinafter, TgAb) is related to autoimmune thyroid diseases such as Graves' disease and Hashimoto's disease, and the measurement of TgAb together with anti-microsomal antibody It is widely used as an index for diagnosis and treatment of diseases. There is also a report that TgAb is positive for autoimmune diseases other than thyroid disease. A semi-quantitative method using an agglutination method as a measurement principle has been used for the measurement of TgAb, but it was not sufficient in terms of sensitivity and specificity. Therefore, quantitative methods such as radioimmunoassay (RIA method), enzyme immunoassay (EIA method), chemiluminescent enzyme immunoassay method (CLEIA method), and chemiluminescent immunoassay method (CLIA method) were reported, and the usefulness of TgAb quantification was clarified. Has become.

The measuring principle of this immunoassay is a solid phase method, which is a two-step sandwich method using a human Tg antigen and an anti-human IgG antibody. The first immune reaction is initiated with the Tg immobilized on the solid phase and the sample. After incubating for a fixed time at a constant temperature, unreacted sample components are removed with washing water (B/F separation). After B/F separation, a second immune reaction is started by adding an enzyme-labeled anti-human IgG antibody. After incubating for a certain time at a certain temperature, unreacted enzyme-labeled antibody is removed with washing water (B/F separation). After that, a substrate is added to measure the enzyme activity bound to the solid phase, and the production rate of the fluorescent substance or the luminescent substance or the fluorescence intensity or the emission intensity is measured, whereby the TgAb concentration in the sample can be known. ..

As the Tg antigen used for this solid-phase antigen, a human-derived protein is used instead of a gene recombinant (recombinant) protein.

When the human Tg antigen donor has an autoantibody (TgAb) against Tg, it means that TgAb is bound to Tg before it is prepared as an immunoreaction reagent. When this Tg bound to TgAb is used as a raw material for the solid phase antigen, a phenomenon occurs in which the measured value is higher than the original measured value, or the background cannot be measured with high sensitivity.

Furthermore, when the amount of TgAb possessed differs depending on the antigen provider and the timing of provision, the quality of Tg as a raw material for solid phase antigen is not constant. As a result, phenomena such as lot difference and deterioration of measurement reproducibility occur.

From the above, it has been a problem that human-derived Tg is used as a raw material for a solid-phase antigen, which hinders highly accurate TgAb measurement.

In the field of clinical examination, in order to measure TgAb with high accuracy, suppression of background rise during measurement, suppression of difference between lots, and reproducibility of measurement are required at the same time.

Therefore, an object of the present invention is to effectively suppress background rise, to suppress difference between lots, and to improve the reproducibility of measurement. It is to provide a method for producing a reagent.

As a result of intensive studies to solve the above-mentioned problems, the present inventors purified Tg by removing the antibody bound to human-derived Tg, thereby suppressing the background increase and reducing lot-to-lot differences. The inventors have found that both suppression and measurement reproducibility are good, and have completed the present invention.

That is, the present invention is as follows.
(1) A method for producing an immune reaction reagent, which comprises subjecting human-derived Tg to affinity purification, and using the obtained Tg to produce a TgAb measurement immune reaction reagent.
(2) The production method according to (1), wherein the affinity purification is performed with one kind or a combination of two or more kinds selected from Protein A, Protein G and Protein L.
(3) The production method according to (1) or (2), which has a step of immobilizing Tg obtained by affinity purification on a solid phase.
(4) The production method according to (1) or (2), wherein human-derived Tg is immobilized on a solid phase and then affinity-purified.

Hereinafter, the present invention will be described in detail.

The human-derived Tg used in the present invention is provided by a human. Therefore, TgAb, which is an autoantibody against Tg, may be bound.

In the present invention, the removal of the antibody bound to human-derived Tg is performed by affinity purification using a resin to which Protein A, Protein G, Protein L, etc. are bound or magnetic beads. One type may be selected from Protein A, Protein G, Protein L and the like, or two or more types may be used in combination. At this time, when human-derived Tg is purified first and then the obtained Tg is immobilized on a solid phase, both resin and magnetic beads can be used for affinity purification. When human-derived Tg is immobilized on beads or fine particles containing a magnetic substance and then purified, a resin containing no magnetic substance can be used for affinity purification. When the human-derived Tg is immobilized on beads or fine particles that do not contain a magnetic substance and purification is performed, magnetic beads can be used for affinity purification.

The Tg thus purified is used to produce a TgAb measurement immunoreaction reagent. This reagent has, for example, Tg immobilized on a solid phase and an enzyme-labeled anti-human IgG antibody. The measuring principle of this immunoassay reagent is a solid phase method, which is a two-step sandwich method using a human Tg antigen and an anti-human IgG antibody. The first immune reaction is initiated with the Tg immobilized on the solid phase and the sample. After incubating for a fixed time at a constant temperature, unreacted sample components are removed with washing water (B/F separation). After B/F separation, a second immune reaction is started by adding an enzyme-labeled anti-human IgG antibody. After incubating for a certain time at a certain temperature, unreacted enzyme-labeled antibody is removed with washing water (B/F separation). After that, a substrate is added to measure the enzyme activity bound to the solid phase, and the production rate of the fluorescent substance or the luminescent substance or the fluorescence intensity or the emission intensity is measured, whereby the TgAb concentration in the sample can be known. ..

In the present invention, Tg immobilized on a solid phase refers to Tg immobilized on a solid phase carrier such as beads or fine particles, which is insoluble in a solution in which an antigen-antibody reaction is carried out. If not, it also includes soluble Tg bound with biotin for binding an avidin-enzyme complex.

As the solid phase carrier, beads or fine particles can be used. Fine particles are particularly preferable, and they may be inorganic substances such as glass, metal and ceramics, or organic substances such as high molecular weight polymers. Further, those fine particles may contain a magnetic substance. The particle size of the fine particles is preferably 0.1 to 50 μm, more preferably 1 to 10 μm.

The enzyme-labeled anti-human IgG antibody refers to an enzyme-conjugated anti-human IgG antibody that is soluble in a solution in which an antigen-antibody reaction is carried out. However, even if the enzyme is not directly bound to the antibody, an avidin-enzyme complex is generated. Also included are soluble biotin-conjugated antibodies for conjugation.

The anti-human IgG antibody in the present invention may be a polyclonal antibody or a monoclonal antibody, and is derived from virtually any animal species producing the antibody, for example, rabbit, goat, sheep, pig, horse, mouse or rat. Antibodies can be used. The form of the antibody may be a complete antibody or an antibody fragment such as F(ab′) ₂ or Fab′ obtained by cleaving it with an enzyme treatment or a chemical treatment.

The enzyme is not particularly limited, but examples thereof include alkaline phosphatase, peroxidase and the like.

In the present invention, a protein may be allowed to coexist during the production of the immunoreaction reagent, and they are not particularly limited, and for example, bovine serum albumin, collagen peptide, casein, casein sodium, skim milk and the like can be used. The concentration range of 0.1 to 20% (weight/volume) is preferable, and the concentration range of 1 to 10% (weight/volume) is particularly preferable. In addition, sugar, a buffer solution and salts may be coexistent, and they are not particularly limited, but sucrose, mannitol, trehalose, inositol and the like can be used as long as they are sugar. As the buffer solution, for example, Tris, MOPSO, MOPS, MES or the like can be used, and as the salt, for example, sodium chloride, potassium chloride, magnesium chloride, zinc chloride or the like can be used. In addition to these, other reagent components and the like may coexist as necessary.

In this way, by purifying human-derived Tg, autoantibodies (TgAb) derived from Tg donors are removed, and then purified Tg is immobilized on a solid phase, which is used as a solid-phase antigen. TgAb measurement immunoreaction Reagents can be manufactured. Further, by immobilizing human-derived Tg on a solid phase and then purifying it, an autoantibody (TgAb) derived from a Tg donor can be removed, and a TgAb measuring immunoreaction reagent using this as a solid-phase antigen can be produced. it can.

ADVANTAGE OF THE INVENTION According to this invention, the TgAb measurement immunoreaction reagent used for the in-vitro diagnostic agent etc. which can suppress background rise effectively, can suppress the difference between lots, and has favorable measurement reproducibility. Can be obtained. For example, since the TgAb measurement immunoreaction reagent obtained in the present invention can be accurately measured, the present invention can also realize highly accurate TgAb measurement.

Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is not limited to the examples. Each measurement was carried out by a two-step sandwich method using a fully automatic enzyme immunoassay device (AIA-CL2400, manufactured by Tosoh Corporation) as an immunoassay device and an immunoreaction reagent for the device as an immunoassay reagent. In addition, each immunoreaction reagent was prepared as described below.

(Example 1)
Human Tg antigen was affinity-purified with Protein G resin to obtain purified Tg. The obtained Tg was immobilized on magnetic fine particles (2.5 μm) to obtain Tg-immobilized magnetic fine particles. The Tg-immobilized magnetic fine particles were added to a Tris buffer solution containing bovine serum, collagen peptide, sucrose and sodium chloride, and freeze-dried. On the other hand, alkaline phosphatase-labeled anti-human IgG antibody was added to a Tris buffer solution containing collagen peptide, trehalose, magnesium chloride and zinc chloride, and freeze-dried.

A zero-concentration sample, plasma, and serum were measured for the reagent with the above-mentioned automatic immunoassay device, and the luminescence intensity of a chemiluminescent substrate which is a substrate of alkaline phosphatase was measured. Each sample was measured 5 times, and the average value was used as the measured value. The low-concentration detection limit (MDC) was calculated based on the measured value of the 0-concentration sample and 2SD. For plasma and serum samples, concentration and measurement reproducibility were calculated based on the measured values. The results are shown in Table 1.

(Example 2)
Tg was purified and reagents were prepared in the same manner as in Example 1 except that human Tg antigens in different lots were used. Sample measurement was performed on the reagent by the same method as in Example 1 using the automatic immunoassay device. The results are shown in Table 1.

(Comparative Example 1)
Reagents were prepared in the same manner as in Example 1, using the same lot of Tg as in Example 1 except that affinity purification of Tg was not performed with Protein G resin. Sample measurement was performed on the reagent by the same method as in Example 1 using the automatic immunoassay device. The results are shown in Table 1.

(Comparative example 2)
Reagents were prepared in the same manner as in Example 1 except that the same lot of Tg as in Example 2 was used and Tg was not affinity-purified with Protein G resin. Sample measurement was performed on the reagent by the same method as in Example 1 using the automatic immunoassay device. The results are shown in Table 1.

０濃度サンプル品の発光強度（Ｃｏｕｎｔ／ｓｅｃ．）は、比較例１の９２２に対して実施例１は１３６と、約８００低減させる結果があり、低濃度検出限界（ＭＤＣ）も０．３６８（ＩＵ／ｍＬ）から０．０３３（ＩＵ／ｍＬ）と１０倍以上良好となる結果であった。また、比較例２の５６７に対して実施例２は１６４と、約４００低減させる結果があり、低濃度検出限界（ＭＤＣ）も０．２４３（ＩＵ／ｍＬ）から０．１０８（ＩＵ／ｍＬ）と２倍以上良好となる結果であった。さらに、比較例１と比較例２にておいては差が４００以上ありロット間差が確認されたが、実施例１と実施例２にておいては差が３０弱であり、ロット間差を抑制していることがわかる。

The emission intensity (Count/sec.) of the 0-concentration sample product is 136 in Example 1 compared to 922 in Comparative Example 1, which is about 800, and the low-concentration detection limit (MDC) is 0.368 ( From IU/mL) to 0.033 (IU/mL), the result was 10 times or more favorable. In addition, Example 2 has a result of reducing by about 400 to 567 of Comparative Example 2 by 164, and the low concentration detection limit (MDC) is from 0.243 (IU/mL) to 0.108 (IU/mL). And the result was more than twice as good. Further, the difference between the lots was confirmed to be 400 or more between Comparative Example 1 and Comparative Example 2, but the difference between the lots was slightly less than 30 between Example 1 and Example 2, and the difference between lots was small. It can be seen that this is suppressed.

Comparison of the measured concentrations of plasma and serum samples between Example 1 and Example 2 showed that plasma samples were 38.8 (IU/mL) and 37.0 (IU/mL) and serum sample A was 511.9 (IU/mL). mL) and 489.7 (IU/mL), and serum sample B is 1,421.3 (IU/mL) and 1,443.5 (IU/mL), and the difference between the measured values is within 5% in all cases. It can be seen that the difference between lots is suppressed.

The reproducibility of the plasma and serum samples was 6.7%, 4.8% and 3.1% in Comparative Example 1, respectively, but in Example 1, 4.6%, 1.3% and 3%, respectively. The result was an improvement of 0.0% in all the samples. Further, in Comparative Example 2, they are 6.2%, 2.5% and 4.6%, respectively, but in Example 2, they are 3.5%, 2.3% and 4.1%, respectively, which are improved in all the samples. It was the result.

From the above results, it is shown that the performance of the immune reaction reagent was improved by purifying human-derived Tg and removing the autoantibody (TgAb) derived from the Tg provider.

Claims (1)

A method for producing a human-derived thyroglobulin by affinity purification, and using the obtained thyroglobulin to produce an anti-thyroglobulin antibody assay immunoreaction reagent , wherein affinity purification is one or two selected from Protein A, Protein G and Protein L. It is carried out in a combination of more than one kind, has a step of immobilizing thyroglobulin obtained by affinity purification on a solid phase, and the anti-thyroglobulin antibody measurement immunoreaction reagent is a thyroglobulin immobilized on a solid phase And an enzyme-labeled anti-human IgG antibody .