JPS5992353A - Measuring method of flocculating reaction using insoluble carrier particle - Google Patents

Abstract

PURPOSE:To magnify a measuring range to avoid the revelation frequency of antigen surplus phenomena and to minimize a diluting operation and to measure accurately and simply a high-concentration antigen by adding a free antibody to a reaction system together with the antibody sensitized to insoluble carrier particles. CONSTITUTION:An antibody sensitized to insoluble carrier particles, a free monospecific antibody capable of reacting with an antigen in a serum to be inspected, a normal serum of antibody producing animals and the serum to be inspected are allowed to react successively by shaking and mixing for several minutes in a liquid medium and said reaction product is diluted to a fixed volume by a diluent. Thereafter, light is irradiated to the flocculated liquid basing on the antigen-antibody reaction and the flocculating reaction is measured quantitatively by the variation of absorbance at this time. Fine particles of an organic polymer, individually dispersed coccus type bacteria such as staphylococcus, streptococcus etc., or inorganic oxides, mineral powder, metals are used for the insoluble carrier particles. Further, a buffer solution such as a physiological saline solution, phosphoric acid or tris-hydrochloric acid is used for the diluent.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for measuring an agglutination reaction using insoluble carrier particles.

In recent years, in the field of clinical testing, serological diagnosis of certain diseases has become important.

For this diagnosis, it is extremely important to quantify antigens and antibodies accurately, simply, and quickly. The present invention aims to solve this problem.

Generally, an antigen-antibody reaction occurs within a certain concentration range to form a complex. If this concentration ratio is extremely biased, it becomes difficult to form an antigen-antibody complex. When the amount of antigen is increased in the presence of a certain amount of antibody, the amount of precipitate due to complex formation increases in proportion to the antigen concentration up to a certain amount of antigen, but once the antigen concentration exceeds a certain point, the opposite occurs. The number of sediment deaths decreases. This phenomenon is well known as the solubilization of complexes due to antigen excess.

This can also occur in antigen measurement reactions using serological latex reagents. Serological latex reagents are usually made by sensitizing latex particles with a particle size of 0.005 to 1 μm with antibodies or antigens and dispersing them in a weakly alkaline buffer. However, since there is a limit to the amount of antibody or antigen that can be sensitized to latex particles at a given location, there is also a limit to the amount of antigen or antibody that can be measured. Therefore, when measuring high concentration antigens, due to the above-mentioned antigen excess phenomenon,
If left as is, a low value may be erroneously measured. In this case, attempts are made to avoid this error by diluting the specimen (sample), but this is complicated. For example, in the measurement of immunoglobulin IJ G (IgG), the sample serum is
It is customary to dilute 0 to IO,000 times, which is a major drawback when considering the need to simplify the measurement operation of clinical test drugs.

Conventionally, a method has been proposed in which an antigen-antibody agglutination reaction is performed using a reagent in which insoluble carrier particles such as latex are sensitized with antibodies or antigens, and the turbidity of the complex formed as a result of the reaction is optically measured. [Unexamined Japanese Patent Publication No. 54-108693]
, JP-A-55-159157, JP-A-57-1970, and Sawai and Ishikawa, Inspection and Technology; 10 (6), 555 (19
82) etc.]. However, even with these methods, when the sample concentration is high, extensive dilution operations are required without exception, large errors due to dilution, and incorrect measurement results due to antigen excess phenomenon due to the narrow measurement range. There is also a risk of releasing.

After considering the above, the present inventor found that when measuring high concentration antigens such as immunoglobulins present in sample serum, it is necessary to add free antibodies to the reaction system together with antibodies sensitized to insoluble carrier particles. As a result, we have discovered an accurate and simple method for measuring high-concentration antigens by expanding the measurement range to avoid the occurrence of antigen excess phenomenon, thereby minimizing the need for dilution of sample serum, and have completed the present invention. reached.

That is, the present invention provides a method in which insoluble carrier particles are sensitized with an antibody, the antibody is reacted with a sample, and the aggregation of this reaction mixture is optically measured. This is an agglutination reaction measurement method characterized by adding a free antibody to a reaction system to perform an antigen-antibody reaction.

According to the method of the present invention, the agglutination reaction is carried out using: ■ an antibody sensitized to insoluble carrier particles; ■ a free monospecific antibody capable of reacting with the antigen in the sample serum; ■ a normal surface culture of the antibody-producing animal; and ■ the sample serum. Sequentially, the reaction is caused by shaking and mixing in a liquid medium for several minutes, and after dilution in the dark with a diluent, the agglutination based on the antigen-antibody reaction is irradiated with light, and quantitative measurements can be made from the change in absorbance at that time. . Of course, the order of the pipetting operations described above is arbitrary, and there is no need to adhere to the above numerical order, but it is preferable to react only the sample serum last.

The insoluble carrier particles used in the present invention are fine particles of organic polymeric substances, such as polystyrene, carboxy-modified,
Latexes obtained by emulsion polymerization such as sulfonic acid modified, styrene acrylonitrile copolymers, individually dispersed cocci-type bacteria such as staphylococci and streptococci, or inorganic oxidizers such as silica, solicar alumina, and alumina. materials, other mineral powders, metals, etc. are used. There are many known methods for sensitizing antibodies to such insoluble carrier particles, and any of these methods may be used. In addition, a buffer solution such as physiological saline, phosphoric acid, or Tris-hydrochloric acid is used as the diluting solution.

Objects that can be measured by the method of the present invention include immunoglobulins such as de-IgG, IgA, IgM, etc. in serum, argumino, ceruloplasmin, transferrin, antitrypin, etc., and in particular immunoglobulin in sample serum. This method is effective for measuring substances that exist in high concentrations.

In the method of the present invention, when a free antibody is added to an antibody sensitized to an insoluble carrier, non-specific agglutination may occur depending on the lot of antibody. In such cases, the normal serum of the animal used for antibody production is added to the reaction system to avoid this non-specific agglutination.

According to the present invention, the amount of antigen that can be measured can be increased and accuracy can also be expected. Accordingly, the dilution factor of the sample serum can be significantly reduced, and the measurement range can be significantly expanded.

EXAMPLES Hereinafter, the present invention will be further explained with reference to Examples, but the present invention is not limited thereto.

Example 1 Preparation of (+) anti-human IgG antibody A human IgG monosparific antibody produced from a domestic rabbit is used. (Antigen) Purify by affinity chromatography by passing the solution through a column of Sepharose 4B on which IgG & chains are immobilized. ) (2) Preparation of anti-Human) Ig (J antibody sensitized latex reagent) Latex (manufactured by Nippon Gosei Rubber Co., Ltd., Calpokin modified type, particle size 0.305μ) was suspended (0.5%) in a glycine buffer solution of p118. , this floating liquid 1
and 1 volume of anti-Human IgG antibody solution prepared at 5007 g/- with phosphate buffer and kept at room temperature for 30 minutes. Centrifuge at oooxG for 20 minutes to remove unadsorbed rabbit r-globulin molecules to the latex particles;
Next, the precipitated latex particles are again dispersed in a glycine buffer containing 1% f3sA to a density of 0.5 to prepare a sensitized latex.

(3) Measurement of latex agglutination reaction The above anti-Igc) latex reagent 20 pi, anti-1gG
Add 20μt of antibody solution, 20μ of normal rabbit serum diluted 3 times with phosphate buffer, and 20pi of sample serum or standard product into a small test tube, mix for 10 minutes, then add 4d of diluent and incubate for 340 hours. Hitachi 200-10 absorbance
The aggregation activity is determined by the change in absorbance (-ΔA340) with respect to the blank. (The concentration is calculated using one of the standard products, △A340, as a calibration.) Comparative Example 1 (1) In the measurement of latex agglutination reaction in (3) of Example 1, the effect of free antibody on preventing non-specific aggregation was evaluated by a normal patient. Instead of measuring without rabbit serum, a phosphate buffer (100+nM, pL (7,5) is used for measurement. After performing an agglutination reaction according to a prescribed method, the presence or absence of agglutination was determined with the naked eye. The results are shown in Table 1.

Table 1 (11) The effect of human serum components on preventing non-specific aggregation is measured using a phosphate buffer (10 omM, pH 7, s) instead of excluding normal rabbit serum. Aggregation was determined according to (1). The results are shown in Table 2. The latex reagent used was one sensitized with anti-α-fetoprotein antibody (rabbit). The preparation method is Example 1
(2).

Table 2 1: Items that do not show aggregation with the naked eye +: Aggregation is clearly observed with the naked eye.

[Brief explanation of the drawing]

FIG. 1 is a diagram comparing the absorption spectrum obtained when an antigen is added to a latex reagent and aggregated with that of a control (no antigen added). 3 6122448951903E! 07501500
IgG Concentration (, *7/J Procedural Amendment (Method) March 7, 1980 Kazuo Wakasugi, Commissioner of the Patent Office 1, Indication of the Case 1982 Patent Application No. 202166 Z Name of the Invention Aggregation using insoluble carrier particles Reaction measurement method 3, relationship with the amendment person case Patent applicant address 2-29-14 Ohnodai, Sagamihara City, Kanagawa Prefecture, Showa 5
February 22, 2008 (shipment date) 5. Column 6 for detailed explanation of the invention in the specification subject to amendment, table frame in the description of amendment (as attached) Attachment Table 1 Table 2 - With the naked eye No aggregation +, aggregation clearly visible to the naked eye

Claims (1)

[Claims] In this method, in which insoluble carrier particles are sensitized with an antibody, this is reacted with a sample, and the aggregation of this reaction mixture is optically measured. A method for measuring an agglutination reaction using insoluble carrier particles, characterized in that an antigen-antibody reaction is performed in addition to the system.