JPS6036966A - Latex reagent for measuring rheumatic factor - Google Patents

Abstract

PURPOSE:To obtain high specificity and sensitivity by binding chemically or adsorbing physically an antigen to latex particles having a specific grain size and removing the excess amt. of the agtigen then bringing the corresponding antiserum or refined antibody into reaction therewith. CONSTITUTION:Latex is synthetic polymer latex particles of polystyrene, polybutadiene or polystyrene, etc. having a functional group on the surface and the surface thereof may be treated with a nonionic surfactant, etc. The grain size of the latex particles is 0.01-10mu, more preferably 0.1-1.0mu and has preferably a narrow width of the grain size distribution to improve the reproducibility of the result of measurement. The conceivable antigen to be bound chemically or adsorbed physically to the latex particles is the antigen originating in animals such as guinea pig, rabbit, goat, etc. and the antigen stable to heat, acids and alkalis is more preferable as far as said material is recognizable as the antigen. The antibody to be made correspondent to the antigen adsorbed to the latex particles is obtd. from animals such as guinea pig, rabbit, goat, etc. which can produce the antibody.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides rheumatoid factor (RF)
This relates to latex reagents for measurement (abbreviated as ).

More specifically, latex particles with an average particle size of 0.01 to 10P are loaded with an antigen by chemical bonding or physical adsorption, and then an antibody corresponding to the antigen is sensitized by an immunological reaction. The present invention relates to a latex reagent for RF measurement characterized by the following.

RF is a substance that appears in the serum of patients with rheumatoid arthritis (RA). This antibody is thought to be one of the autoantibodies since it has a specific affinity for self and homologous or foreign γ-globulin.

There are 11 major classifications of serological testing methods used for RF detection.
2) Latex agglutination reaction, in which human γ-globulin is adsorbed to hematologically inert latex.

Generally, the sensitivity of 1) sensitized hemagglutination reaction is inferior to that of 2) sensitized latex agglutination reaction, but it is superior in one ship equipment. The latex agglutination reaction is easy to operate and has high sensitivity, so it is widely used as a screening test, but it is also used for many diseases other than RA-=, such as tuberculosis for liver disease, leprosy, syphilis A virus disease, etc. Since the test results are positive for acute bacterial endocarditis, sarcoid-disease, collagen disease, etc., the differential diagnosis for these diseases must be based on the sensitized hemagglutination reaction.

For the sensitization hemagglutination reaction, a Rose reaction using sheep red blood cells and a corresponding anti-sheep red blood cell rabbit antibody and He' 11 were used.
There is also a reaction in which purified rabbit denatured γ-globulin is adsorbed to formalin/tannic acid-treated sheep red blood cells. In order to eliminate the influence of normal xenoagglutinin and heterophilic antibodies on sheep red blood cells in the test serum, 11) A method of measuring unsensitized sheep red blood cells as a control (RoSe method') N2) 6. Method of measuring after absorption by red blood cells (Heller method)) 3)
As a substitute for sheep red blood cells, there is a method of absorbing with a buffer solution containing membrane-dissolving components of sheep, horse and cow red blood cells.

In daily clinical tests, method 3) is frequently used because the operations in 1) and 2) are complicated.

However, in either method, absorption of RF by the test serum may be incomplete, and non-specific absorption of RF may be observed.

The present inventor conducted extensive research in order to obtain an RF Ill11 standard method that does not have the drawbacks of the above-mentioned conventional methods, and as a result found a new RF measurement reagent that has high specificity and sensitivity and is easy to operate. i.e.) serologically inactive average particle size 0.01-10P
LaTesoku/J, chemically bind or physically adsorb the antigen to each particle 7, remove the remaining excess antigen by centrifugation, etc. 1 react with the corresponding antiserum or purified antibody to form an RF reaction factor. RF characterized by combining
The object of the present invention is to invent a latex reagent for measurement.) The Rose method and H
Similar to the e1ler method, it is characterized by absorbing rabbit immunized antibodies by applying an antigen-antibody reaction, and does not require the absorption procedure when using sheep red blood cells as a carrier when measuring NRF, resulting in high specificity and sensitivity. An object of the present invention is to provide a latex reagent for RF measurement that exhibits the following properties.

Next, the present invention will be explained in detail.

The latex used in the present invention may be synthetic polymer latex particles such as polystyrene (9-polybutadiene) or boristhinone having a functional group on the surface, the surface of which has been treated with a nonionic surfactant or the like. The particle size of the latex particles is 0.01 to 107x, preferably 0.01x.
1-1. In order to improve the reproducibility of measurement results, it is desirable that the width of the particle size distribution is narrow. The specific gravity of the latex particles used is between 0.9 and 1.4.

Antigens that can be chemically bonded or physically adsorbed to latex particles include those derived from animals such as guinea pigs, rabbits, and goats.1If the antigen can be recognized, it is best to use one that is stable against heat, acids, and alkalis. desirable.

Antibodies corresponding to antigens adsorbed to latex particles can be obtained from animals capable of producing antibodies, such as guinea pigs, rabbits, and goats. Although a rabbit immune antibody is used in the Rose method and the Heler method, the RF reaction factor is not limited thereto.

Antibodies obtained by trench immunization can be left as antiserum, but A.
Furthermore, an r-globulin fraction purified by ammonium sulfate fractionation method - DEAE ion exchange chromatography method 4 - Method A gel filtration method, ethanol fractionation method 1 affinity chromatography method, etc. may also be used.

To make antigens compatible with latex particles, chemically bond them to carboxylated polystyrene using a coupling reagent such as carbodiimide, or chemically bond them to 1-aminated polystyrene using a coupling reagent such as glyclic aldehyde.Polystyrene latex to 30°C at 4-40°C in a buffer of pH 5-10, preferably pH 6,5-8.
There is a method of physically adsorbing the substance for minutes to 24 hours. As the buffer solution, for example, phosphate buffered saline and glycine buffered saline are used. After the reaction is complete, wash several times with a neutral buffer solution by centrifugation or A-filtration. Next is anti-no”;
(To carry antibodies on the bound latex particles, apply an antiserum or purified antibody corresponding to the antigen in a neutral buffer solution at 4 to 40°C for 30 minutes to 241141 using an antigen-antibody reaction. After the reaction is complete, wash several times by centrifugation or filtration with a nearby buffer solution, and finally add stabilizers such as bovine albumin (BSA) or zasocalose and sodium azide. The sensitized latex particles are suspended in a neutral buffer containing a preservative.

These sensitized latexes are stable for more than one year. Using the sensitized latex thus obtained, RF in serum can be easily measured by the microcuicou method and the slide agglutination method. In the microtyke method, a certain amount of a neutral diluent is dispensed into a microtray, then a certain amount of serum is added to the first container, and the solution is sequentially diluted with a diluter. On the other hand) Create a dilution system in the same way using the reference positive serum. Add a certain amount of sensitizing latex to the mixture to make it uniform, and after a certain amount of sensitization release (1'1), check the end point of 1 agglutination and calculate the agglutination value ('l + m). .

In addition, in the slide agglutination method, a certain amount of an arbitrarily diluted test solution is dropped onto a slide glass, a certain amount of a reference positive serum is also added separately, and then a certain amount of sensitizing latex is added dropwise to each slide and stirred gently.

By determining the resulting agglutination image, the RF agglutination titer can be determined within a few minutes.

The RF filling latex reagent of the present invention is a method for binding an RF reaction factor to latex particles.1 It is not a product in which purified denatured γ-globulin is chemically or physically adsorbed. 4'! Next, the antibody corresponding to the antigen, that is, the RF' reaction factor, is bound in an immunologically meaningful manner by an antigen-antibody reaction. j' C9, and is similar in that it binds immune antibodies to the Rose method and Heler method, but since it uses λ serologically inactive Kotobuki latex particles as a carrier), it can be used for red normal xenoagglutinins and heterophilic antibodies. It is advantageous in that there is no need to absorb or remove them with unsensitized blood cells, etc., and no non-specific reactions occur after the reaction with RF. Furthermore, by applying antigen-antibody reaction as a latex particle carrier)
There is no precedent for a latex reagent for RF measurement that sensitizes the F' reaction factor, and it is completely new and has excellent features such as ease of operation, sensitivity, and specificity.

Next, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited to the following Examples, which do not give rise to the gist of the present invention. Preparation of antibiotic albumin (BSA) antibodies
After diluting an equal amount of con-emulsion into a physiological saline solution, inject it subcutaneously into the foot pad of each limb using a rabbit. do. Two months later, 950 ml of blood was collected from the ear artery of one rabbit and centrifuged in a conventional manner to obtain about 20 ml of λ antiserum.

The obtained antiserum was confirmed to be a single antibody A because it formed a single sedimentation line with the antigen by one octellony method, one immunoelectrophoresis method, and one lambda alternating immunoelectrophoresis method.

Example 2. Preparation of bovine albumin sensitized latex Latex (manufactured by Takeda Pharmaceutical Co., Ltd.: 5DL59, specific gravity 1.
14. Particle size: 0.8P) in 0.1M ammonium chloride balanced saline solution, pH 8, so that the particle concentration becomes 0.5%.
To the solution diluted with 2, add Latex A and an equal volume of 0.IM ammonium chloride buffer salt solution containing 9100 Pg of crystallized bovine albumin (at 37°C). After reacting for 1 hour, centrifuge once to remove unadsorbed After washing and removing crystallized bovine albumin, A Latin 22 particles were suspended in a 0.1M ammonium chloride buffered salt solution to obtain a bovine albumin sensitized latex.

Example 3 Sensitization of antibiotic albumin antibodies to bovine albumin-sensitized latex Antibiotic albumin rabbit serum was added to bovine albumin-sensitized latex at 25 Pi, mixed, and reacted for 11 hours at 37°C. After the reaction was completed, centrifuged) 'After washing with 1M ammonium chloride buffered saline, add bovine albumin to a final concentration of 0.596 and saccharose to a final concentration of 1% as a stabilizer) to a latex concentration of 0.25%. A rabbit antibody-sensitized latex (suspended in ammonium buffered saline), that is, a latex reagent for RF measurement is obtained.

Example 4. Measurement of RF Add dilution solution to each hole of U-shaped microplate at 0°025m.
Dispense 1 portion at a time, add 0.025 ml of serum to the first portion, and dilute sequentially with a Gailutor.

Then apply one Example 3 to each hole. Dispense 0.025+nl of the latex reagent for RF measurement obtained in step 1), mix thoroughly, and leave at room temperature for 12 hours or more to check the end point of aggregation. If a reaction is occurring, the latex will be dispersed all over the bottom of the tube.If a reaction is not occurring, the latex will be concentrated in the center of hole A, so the end point of aggregation can be easily determined. can.

Typical chronic rheumatism patient serum 1 according to the above method
Table 1 shows the results of measurements for 0 cases and 10 cases of normal human blood. Also shown is a commercially available RAHA test (Mykuro Tita, manufactured by A) using the same serum.

Margin below

Claims (1)

[Claims] It is characterized in that latex particles with an average particle size of 0.01 to 10P are loaded with a foreign antigen by chemical bonding or physical adsorption, and then immunologically sensitized with the corresponding antibody. Latex reagent for equine factor measurement