JP2589088B2 - Determination of anti-streptolysin O - Google Patents

Description

The present invention relates to a method for quantifying anti-streptolysin O (hereinafter abbreviated as "ASO"), and more particularly, to an automated immunoturbidimetric assay which is an improved immunoturbidimetric assay. To a method for quantifying ASO suitable for

[Conventional technology]

Streptolysin O (hereinafter abbreviated as "SLO") is a hemolytic toxin produced by human-derived streptococci and has antigenicity. When streptococcus produces SLO in the body, ASO, an antibody against it, is produced. . Therefore, for example, by measuring the amount of ASO in serum, it is possible to diagnose streptococcal infections, particularly rheumatic fever and acute glomerulonephritis, which are secondary diseases thereof.

Conventionally, methods for measuring ASO include the Landzlandal method and the microtiter method, which measure ASO by utilizing the fact that SLO, which is an antigen, reacts with ASO, which is an antibody in sample serum, and neutralizes toxin activity. Method: Lattex turbidimetric method, in which SLO, which is an antigen, is sensitized to carrier latex, and antigen-antibody reaction is detected as latex agglutination by an automatic analyzer to measure ASO; ASO and SLO are reacted on a slide glass by antigen-antibody A latex slide agglutination method of observing the amount of agglutination with the naked eye and measuring ASO has been employed.

[Problems to be solved by the invention]

However, the Lanzlandal method and the microtiter method necessarily require rabbit, sheep or human type O erythrocytes to be carried out, and have the disadvantage that if the erythrocytes are not fresh, the measured values will vary. . In addition, the measurement method itself is complicated, and the reaction time is relatively long, that is, 1 hour and 30 minutes. Therefore, there is a problem that automation of the measurement is difficult.

In addition, the latex turbidimetric method has problems in that the measured values vary due to non-specific aggregation of the latex, and that the automatic analyzer is clogged due to drying of the reagent.

Furthermore, since the latex slide agglutination method is also a visual method, there is a problem that it is difficult to automate the measurement.

Therefore, there has been a demand for the development of a method for quantifying ASO which has high accuracy and can be suitably applied to a general-purpose biochemical automatic analyzer at present.

[Means for solving the problem]

The present inventors have conducted intensive studies to solve the above-mentioned problems, and found that a polyoxyethylene ether-based nonionic surfactant having a phenyl group (hereinafter, simply referred to as “nonionic surfactant”). Is used as an aggregation promoter,
Completed the present invention by finding that ASO can be measured easily and with high accuracy by measuring the change in absorbance due to the occurrence of aggregation after the start of the antigen-antibody reaction, and that this method can be advantageously applied to an automatic analyzer. did.

That is, the present invention is a method for quantifying ASO, which comprises adding SLO and a nonionic surfactant to a sample, and measuring the amount of change in absorbance due to the resulting aggregation.

In practicing the present invention, first, preferably, an excess of SLO and a nonionic surfactant are added to a sample containing ASO.

Various body fluids are used as the specimen, and serum is generally preferred. Further, the preferred temperature range of the agglutination reaction system between SLO and ASO is from room temperature to about 40 ° C., and the pH range is from 4.5 to 8.5. Therefore, it is necessary to adapt the reaction system to these conditions in order to practice the present invention. . At the time of adding the SLO and the nonionic surfactant and thereafter, it is necessary to perform stirring so that the system becomes uniform. Further, the nonionic surfactant used in the present invention is, for example, its HL
Those having a B value of 13 to 20 or more are particularly preferred. More specifically, for example, polyoxyethylene octyl phenyl ether (EO = 30 to 100), polyoxyethylene nonyl phenyl ether (EO = 30 to 100), and the like, and these can be used alone or They can be used in combination.

The addition of the SLO and the nonionic surfactant to the sample may be performed simultaneously, or the SLO may be added to the sample to which the nonionic surfactant has been added in advance. In general, the amount of SLO
200 to 1,000 units or more per nonionic surfactant
About 20% by weight.

Next, the amount of change in absorbance due to aggregation caused by the antigen-antibody reaction is measured.

The measurement wavelength used in the method of the present invention is generally 300 to 400 nm. Also, since it is important to measure the change in absorbance at the time of significant aggregation formation immediately after the start of the reaction,
Generally, it is desirable to terminate the absorbance measurement within 5 to 7 minutes after the start of the reaction, preferably within 3 minutes after the start of the reaction. The measuring instrument is not particularly limited as long as it is a general-purpose automatic analyzer having a photometer. In order to further improve the accuracy in the method of the present invention, the amount of change in absorbance may be obtained from the measured value of change in absorbance per unit time by the least square method.

Finally, the ASO amount is measured from the absorbance change amount by using the calibration curve.

The calibration curve is easily prepared by using a sample solution containing no ASO, for example, a standard serum containing no ASO, and ASO having a known concentration.

In order to easily carry out the method of the present invention, components necessary for carrying out the method, that is, SLO which is an essential component
It is advantageous to use a kit for an analytical reagent containing a buffer, a preservative, an inorganic salt and the like for keeping the pH of the specimen within a certain range and preventing turbidity, in addition to the nonionic surfactant and the nonionic surfactant.

An example of such an analysis reagent kit is as follows.

First reagent: Nonionic surfactant 2 to 20% by weight Preservative 0 to 0.2% by weight Inorganic salt (NaCl, sodium phosphate, etc.) 0.5 to 3% by weight Purified water balance (using a buffer solution to adjust the pH to 5.5 to Second reagent: SLO 1,000 to 10,000 units / ml Nonionic surfactant 2 to 20% by weight Preservatives 0 to 0.2% by weight Inorganic salts etc. 0.5 to 3% by weight Purified water balance (using a buffer solution) [Effect of the Invention] Since the quantification method of the present invention does not use a carrier such as a latex or an erythrocyte, an error depending on the type and freshness of the erythrocyte is generated, and a carrier such as a latex is used. Since the measured values do not vary due to non-specific aggregation of ASO and the change in absorbance at the beginning of the reaction is measured instead of the difference in absorbance after a certain period of time, ASO can be quantified with high accuracy. is there. Further, the addition of the coagulation accelerator shortens the coagulation time to about 5 to 10 minutes, so that the present invention can be applied to general-purpose biochemical automatic analyzers. Further, since a nonionic surfactant is used as the aggregation promoter, the labor for cleaning the reaction cell and the reagent dispensing mechanism is reduced, and the optical portion of the automatic analyzer and the reagent dispensing mechanism are protected.

〔Example〕

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

Example 143 ASO was determined for the serum of 143 samples using the following operation method and standard calibration curve. On the other hand, these sample sera were also determined by latex turbidimetry and microtiter method.
ASO was determined and the correlation between these methods and the method of the present invention was examined.

The correlation diagram between the method of the present invention and the latex turbidimetric method is shown in FIG.
FIG. 3 shows a correlation diagram between the method of the present invention and the microtiter method.
FIG.

(1) Equipment used Hitachi 7050 automatic analyzer (2) Reagent (i) First reagent HEPES buffer (pH 7.2) ... 0.05 M NaCl ... 0.15 M Sodium azide 0.1 wt% polyoxyethylene nonyl Phenyl ether 7.2% by weight (ii) Second reagent SLO solution Preparation of SLO Group A streptococci were cultured in Todo-Hewitt medium for 16 to 20 hours, and SLO was removed from the supernatant from which bacterial cells were removed by ammonium sulfate salting out. Obtained. This SLO is sufficiently dialyzed against a 0.005M phosphate buffer, adjusted to a hemolysis value of 1000 units / ml, and freeze-dried.

The SLO prepared in the preparation of the SLO solution was dissolved in the same component solution as the first reagent so as to have a hemolysis value of 1000 units / ml, and was used as a second reagent.

(3) Operation method FIG. 1 shows an outline of the measurement operation performed using the above-described equipment and reagents. First, the absorbance at 340 nm of a cell blank in which only water was put in the reaction cell was measured. Then, after water was discharged, 20 µ of the sample serum and 350 µ of the first reagent were added, and the first absorbance was measured. Thereafter, the measurement was performed at intervals of 20 seconds for a total of 32 times for 10 minutes. Here, the absorbance of the cell blank is automatically subtracted from each measured value by an arithmetic mechanism in the apparatus. Five minutes after the addition of the first reagent, 50 µ of the second reagent was added to start the reaction.

Of the above measured values, the 19th to 21st times (1 after addition of the second reagent)
(Min. To 1 min. 40 sec.), And the change in absorbance per unit time was calculated by the least squares method.

(4) Preparation of Standard Calibration Curve The following dilution series was prepared using ASO negative serum and 280 Todd unit serum.

Dilution ratio 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.
8 0.9 1.0 ASO (Todd unit) 0 28 56 84 112 140 168 19
6 224 252 280 Using 20 μ of each of these 11 types of sera, the amount of change in absorbance per unit time (ΔABS / min) was determined according to the above-described operation method, and the relationship with the ASO concentration was plotted to prepare a standard calibration curve. . This is shown in FIG.

(5) Results As is clear from FIGS. 3-A and 3-B, the method of the present invention has a good correlation with the conventional method.

[Brief description of the drawings]

FIG. 1 is a drawing showing an outline of a procedure for measuring absorbance by an automatic analyzer, and FIG. 2 is a drawing showing a standard calibration curve. FIG. 3-A is a drawing showing the correlation between the method of the present invention and the latex turbidimetry, and FIG. 3-B is a drawing showing the correlation between the method of the present invention and the microtiter method.

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Hideaki Shibata 2-1-1-1, Sugamo, Toshima-ku, Tokyo Nissui Pharmaceutical Co., Ltd. (56) References JP-A-53-44623 (JP, A) JP-A Sho 58-113758 (JP, A) JP-A-49-42822 (JP, A) JP-A-55-31960 (JP, A) JP-A-48-37184 (JP, A) JP-A-58-187862 (JP, A A)

Claims (3)

(57) [Claims] An anti-streptolysin O is characterized in that streptolysin O and a polyoxyethylene ether-based nonionic surfactant having a phenyl group are added to a sample, and the amount of change in absorbance caused by the resulting aggregation is measured. Assay method. 2. The method for quantifying anti-streptolysin O according to claim 1, wherein the change in absorbance is measured between 0 and 7 minutes after the addition of streptolysin O and the nonionic surfactant. 3. The method according to claim 1, wherein the measurement of the change in absorbance is obtained as a change in absorbance per unit time, and this value is processed and calculated by the least squares method. Assay method.