JPS605900B2 - Quantification method for antigenic substances - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for quantifying antigenic substances (excluding immunoglobulin and alpmin), and is particularly useful in clinical tests to determine the severity of congenital abnormalities, nephritis, anemia, etc. This invention is a quantitative method for quickly and accurately determining serum proteins necessary for diagnosis and differentiation from other diseases.

There are two commonly used methods for quantifying serum proteins: one-dimensional plate immunodiffusion method and in vitro simple diffusion method.

All of these methods apply the in-gel diffusion method. In the one-way plate immunodiffusion method, a certain amount of antigen (serum) is added to the small pores of an agar plate containing specific antiserum, and when the antigen is left for a certain period of time and at a certain temperature, the antigen diffuses while reacting with the antibodies in the gel. Creates a white sediment around the small hole.

This is a method to quantify the amount of antigen by measuring the diameter of the membrane. On the other hand, in the in vitro simple diffusion method, antiserum is gelled with agar, packed in a glass tube, and an antigen (serum) is placed on top of the gel to form a precipitate through an antigen-antibody reaction.

In this case, the precipitate does not move within the gel, but dissociates in the presence of excess antigen, and the dispersed antigen forms antibodies and precipitates.
In this way, the sedimentation line advances through the antibody gel layer while maintaining a clear front. Since a sedimentation zone is formed when the antigen and antibody reach an optimal ratio, this method measures the amount of antigen by measuring the diffusion distance. However, all of these methods require a long time, at least 1 to 2 days, to cause diffusion and complete precipitation reactions, and even 1 to 2 days if the amount of antigen exceeds the scale.

Moreover, the measurement also had the disadvantage that errors occurred because the diameter was read with the naked eye using a magnifying glass or the like. In addition, a method using a turbidity meter has been considered, but this utilizes the correlation between the amount of antigen and the amount of sediment, but in general, precipitation reactions due to antigen-antibody reactions, when the amount of antibody is constant, It is known that there is a relationship between the amount of antigen and the amount of precipitate as represented by a curve as shown in FIG.

Therefore, the amount of antigen calculated from the same amount of sediment A actually has two measurement points: antigen deficient area X and antigen excess area Y, and further confirmation of the obtained value is required. This cannot be used as a method that can accurately measure a large number of specimens. As a result of various studies to compensate for these shortcomings, the present inventors have determined in advance the amount of antigen required to maximize the amount of antigen-antibody reaction precipitate (hereinafter referred to as " By determining the optimal ratio (referred to as "optimum ratio") and adding this amount of antigen to the reaction system to cause an antigen-antibody reaction, it is possible to directly determine the amount of antigen in the sample from the amount of sediment read from the turbidity meter. They found that this is true, and after further study, they completed the present invention.

In other words, the present invention is based on "an antigen-antibody reaction reaction system consisting of a test antigen and its antibody, in which an amount of antigen slightly exceeding the optimum ratio is allowed to coexist, an antigen-antibody reaction is performed, and the amount of the generated antigen-antibody reaction precipitate is reduced. This is a method for quantifying antigenic substances (excluding immunoglobulins and albumin) by measuring by turbidimetry.In the present invention, "an amount slightly exceeding the optimum ratio" refers to the amount of precipitate formed in Figure 1. This refers to the amount of antigen at which the amount of antigen slightly exceeds the maximum amount (optimal ratio Z) and the amount of precipitate produced decreases.

The antigenic substance used in the present invention is a substance capable of producing antibodies, such as serum proteins such as complement components ``patoglobin, transferrin, and mono-antitrypsin.''

According to the present invention, as shown in FIG. 1, by using only the curve above the antigen excess area Z, there is no need to take concentration fluctuations for each sample, and moreover, the antigen deficiency area The detection sensitivity is much better than that of the previous method.

In carrying out the present invention, first, a stock solution of antibodies against various antigenic substances is diluted with a physiological saline containing a buffer such as phosphate buffer, Tris buffer, or veronal buffer according to the antibody titer. .

Further, the antigenic substance is diluted with a buffer solution in an amount slightly exceeding the optimal ratio to the diluted antibody. moreover,
The sample to be measured is also diluted with a buffer solution so that the concentration falls within the measurement range. Next, the diluted specimen, antigenic substance and antibody at slightly higher than optimal ratios are mixed, and the turbidity is immediately measured.

(Measurement value at 0 hour. Depending on the dilution ratio of the sample, the turbidity may be ignored, and in that case, there is no need to measure at 0 hour.) After that,
After standing for a certain period of time at C, the turbidity is measured again, and the difference is taken as the turbidity due to the true antigen-antibody reaction. The values obtained in this way are measured in advance with a standard solution of each concentration of each antigenic substance on the same machine using a turbidity meter, and the amount of the antigenic substance in the sample is determined quickly and accurately by reading it from the calibration curve created. be able to.

In order to actually use the method of the present invention, the following reagent kit may be used.

That is, {a- Buffer solution for diluting the specimen 'b} Optimum amount of antigen 'c} Antibody solution at an appropriate concentration for antigen-antibody reaction 'd- Standard antigen solutions of various concentrations containing known amounts of antigen, respectively By preparing one set of antigenic substances,
Furthermore, by simultaneously incorporating the above-mentioned sets for measuring multiple types of antigenic substances, the user can select a desired kit and easily measure multiple antigenic substances on the same specimen at the same time.

Next, we will use the method of determining the "amount slightly exceeding the optimum ratio" in Examples 1 and 2, which will be described later, as a representative example.
How to determine the amount that slightly exceeds the optimal ratio will be explained using a "reference example."

[Reference example]

The antigen concentration is 5, 7, 9, 11, 12, 13, 14, respectively.
15, 17, and 19/Blood: Anti-transferrin antibody solution diluted with phosphate buffer to 6M in transferrin solution with varying concentrations as follows: 1.0
' was added to bring the total volume to 2.0 ships, and incubated at 3700 for about 60 minutes.

The amount of antigen-antibody reaction precipitate produced at each antigen concentration was measured using a turbidity meter, and the ratio (precipitate production rate) when the maximum value was set as 100 was expressed in a graph as shown in FIG. In this graph, the amount of antigen is 1 when the precipitation production rate is 100.
The optimal ratio is 3 m/t, and the "amount slightly exceeding the optimal ratio" means the amount of antigen when the sedimentation production rate begins to decrease.In other words, 14 m/t. The "amount slightly exceeding the optimal ratio" varies depending on the antibody, so it needs to be determined each time the antibody changes. Next, an example will be given and explained.

Example 1 0.001M phosphate buffer (pH 7.4) containing 14% transferrin The concentration of transferrin was changed to 0.95', an amount slightly exceeding the optimum ratio.
．． Add 0.05 I, 0.01M-phosphate buffer (bait 7
．． Antibody 1.0 diluted 6 times with physiological saline containing 4)
When the total amount of secretion was 2.0, and the turbidity was measured after 60 minutes at 37°C, a correlation between the added transferrin and the amount of sediment (turbidity) was determined as shown in FIG.

Example 2 Thirty human serum samples were diluted with 0.001M phosphate buffer (pH
7.4) diluted 20 times with physiological saline containing
Place 30 liters of each in a test tube, add 1.0' transferrin solution (equivalent to 14 liters) and mix.

Add 6 needle diluted antibody solutions of 1.0' to each 37
Let it react at q0. The amount of transferrin in human serum was determined from the graph obtained in Example 1 from the turbidity measured with a turbidity meter after 60 minutes, and the average value was 9/d' of 291.

This is in line with the average value for healthy people. In addition, one sample was arbitrarily taken from the human serum used here, a known amount of transferrin was added to it, and the measurement was performed in the same manner as above, and the recovery rate was investigated. As a result, a high recovery rate was obtained.

Example 3 0.001M phosphate buffer (pH 7.4) containing 15 habtoglobin 0.05 haptoglobin solution whose concentration was changed to 0.95' ``amount slightly exceeding the optimum ratio''
of 0.001M phosphate buffer (pH 7)
．． Antibody 1.0 diluted 20 times with physiological saline containing 4)
When the turbidity was measured after 60 minutes at 3700° C. as shown in Figure 3, the total amount was 2.0.

The correlation between toglobin and the amount of sediment (turbidity) was determined.
Furthermore, as a result of examining the recovery rate in the same manner as in Example 2, a high recovery rate was obtained. Example 4 A 0.001M phosphate buffer (pH 7.4) containing 318% of the third component of complement was mixed with a supplement whose concentration was varied to 0.95% (amount slightly exceeding the optimal ratio). Add 0.05' of third component C3 solution and add 1.0' of antibody diluted 2 parts with physiological saline containing 0.001 M phosphate buffer (pH 7.4). When the total amount was above 2.0 and the turbidity was measured after 60 minutes at 370, the correlation between the third component C3 of the added complement and the amount of sediment (turbidity) was determined as shown in FIG.

Furthermore, as a result of examining the recovery rate in the same manner as in Example 2, a high recovery rate was obtained.

Example 5 0.001M containing Q,1-antitrypsin 28 muta
- To 0.95' of Veronal buffer (pH 7.4) (an amount slightly exceeding the optimum ratio), 0.05 of Q,-antitrypsin solution of varying concentration was added, and 0.001M of Add 1.0% of the antibody diluted 20 times with physiological saline containing Ronal buffer (pH 7.4) to make a total volume of 2.0M.
When the turbidity was measured after 6 hours at 7q0, a correlation between the amount of added Q,-antitrypsin and the amount of sediment (turbidity) was determined as shown in FIG.

Furthermore, as a result of examining the recovery rate in the same manner as in Example 2, a high recovery rate was obtained.

Example 6 A reagent kit for quantifying haptoglobin was prepared as follows.

(a) Buffer test tube: A small test tube containing 0.9' of physiological saline containing 0.001M veronal buffer (pH 7.4).

'b} Antigen solution test tube: Antiserum containing 15 meta antigens 0.
A small test tube containing 98'.

[c} Antibody solution bottle: A bottle containing 1.0 diluted antibody solution prepared by diluting the antibody stock solution with physiological saline containing 0.001M-Veronal buffer (pH 7.4) to a concentration suitable for antigen-antibody reactions.

'dreptoglobin standard solution: A novel test tube containing haptoglobin standard serum solutions of various different concentrations.

The quantitative reagent kit constructed as described above is used in the following manner.

That is, a sample of 0.1' is collected into a buffer test tube [a} and mixed well. 0.02 of this mixed solution was further mixed with the antigen solution in a test tube 'c', and one volume of antibody solution was taken out from the antibody solution bottle 'c' and added to the mixed solution in test tube 'b'.
0 for 60 minutes. The turbidity is then measured using a turbidity meter. Separately, the same experiment as above is repeated using haptoglobin standard 0 solution 'd- of various concentrations instead of the specimen to obtain a standard curve, and the amount of haptoglobin in the specimen is determined from this standard curve.

[Brief explanation of drawings]

FIG. 1 is a curve showing the change in the amount of precipitate in the antigen-antibody reaction, FIG. 2 is a curve showing the correlation between the amount of transferrin and the amount of precipitate in Example 1, and FIG. 3 is a curve showing the correlation between the amount of transferrin and the amount of precipitate in Example 3.
FIG. 4 is a curve showing the correlation between the amount of haptoglobin and the amount of sediment in Example 4, and FIG. 3 is a curve showing the correlation between the amount of Q, antitrypsin and the amount of sediment in Example 5. FIG. 6 is a graph for determining the "amount slightly exceeding the optimum ratio" in the "reference example". Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6

Claims (1)

[Claims] 1. The antigen-antibody reaction system consisting of the test antigen and its antibody is made to coexist with an amount of antigen slightly exceeding the optimal ratio to perform the antigen-antibody reaction, and the amount of the generated antigen-antibody reaction precipitate is measured by turbidimetry. A method for quantifying antigenic substances (excluding immunoglobulin and albumin).