JP3212128B2 - Immunological assay using colloidal gold - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for immunological analysis of biological components.

[0002]

2. Description of the Related Art Radioimmunoassay (RIA), enzyme immunoassay (EIA), immunoturbidimetry, nephelometry, etc.
Various immunological measurement methods, such as a method and a latex agglutination method, are known. However, the RIA uses radioactive isotopes and requires special equipment, and the EIA has many problems such as many reaction steps, complicated operation, and long reaction time.

[0003] The immunoturbidimetric method and the nephelometric method are methods for optically measuring a sedimentable antigen-antibody complex formed in a solution.
This is an excellent method. Although the two methods differ only in the method of measuring turbidity, there is no essential difference, but both require a dedicated measuring device. In general,
In an agglutination method such as immunoturbidimetry, when the amount of antigen is large relative to the amount of antibody, the production of the antigen-antibody complex increases depending on the amount of antigen, but the production of the complex reaches a maximum at a certain amount of antigen. On the other hand, in an antigen-excess region where the amount of antigen is further increased, the formation of a complex is reduced, and a so-called prozone phenomenon occurs. For this reason, it is necessary to determine whether the measured value is before or after the maximum value, and it is indispensable to incorporate a check mechanism for the prozone phenomenon into the measuring device, which is complicated and expensive. It became.

In the latex agglutination method, an antibody bound on polymer carrier particles such as polystyrene latex is reacted with an antigen. Qualitatively, agglutination of the particles is performed using a slide plate or a microtiter plate. The presence or absence of the antigen is determined by visually observing the aggregated image. Also, quantitatively, the turbidity is optically measured as in the immunoturbidimetry.
However, also in this case, as in the case of the immunoturbidimetric method, a prozon phenomenon occurs in the antigen excess area, and thus the risk of a negative measurement value is inevitable. As described above, in the measurement utilizing the antigen-antibody reaction, there has been a strong demand for the emergence of a measurement method in which the reaction is not likely to be negative even when a high concentration of antigen is used.

[0005]

SUMMARY OF THE INVENTION The present invention aims to provide an improved method for measuring an antigen in an antigen excess region using a colloidal gold agglutination method. The colloidal gold agglutination method is a type of particle aggregation method using colloidal gold as the carrier particles to which the antibody is bound, but the color tone of the reaction solution changes greatly due to the aggregation, so the change in the color tone can be visually observed. By making the determination, the antigen can be qualitatively detected. The antigen concentration can be quantified by measuring the absorbance. The quantification is performed by calculating the concentration of the antigen in the sample from a standard curve of a known concentration of the antigen. Measurement of the absorbance is 520-580 nm, preferably 530-550 nm
It is carried out in the wavelength range of the above, from the value of the absorbance after reacting for a certain period of time, or from the difference in absorbance at two points after the start of the reaction, or from the amount of change in the absorbance decreasing at the beginning of the reaction per unit time, the antigen The concentration can be determined. further,
It can also be measured from the absorbance at one wavelength of 520-580 nm and the absorbance at one wavelength of 600-800 nm.

[0006] As described above, in the reaction between an antigen and an antibody or carrier particles, in the region from the excess antibody region to the optimum specific region, as the amount of antigen increases, the formation of a complex due to the antigen-antibody reaction increases. However, when the antigen concentration exceeds a certain level, the amount of production gradually decreases and finally stops reacting. Conventional measurement methods using an antigen-antibody reaction have been performed in a range from an antibody excess range to an optimum specific range. The present invention utilizes an antigen-excess region, that is, a prozone phenomenon, which is a reaction region not used in conventional measurement methods. The present invention
A sufficient amount of antigen is added to the reaction solution to cause
In addition, the antigen-antibody reaction is performed only in the antigen excess area,
This makes it possible to measure the amount of antigen in the medium.

[0007] In the colloidal gold agglutination method, from the antibody excess range to the optimum specific range, 520-
A decrease in absorbance at 580 nm is observed, and the antigen concentration can be quantified by measuring the absorbance or the rate of decrease in absorbance. On the other hand, in the antigen excess region, the decrease in absorbance decreases as the antigen concentration increases, and the antigen can be quantified. As the antibody used in the present invention, a polyclonal antibody is suitable, but one kind of monoclonal antibody is used for an antigen having an overlapping sequence and overlapping structure of amino acids or an antigen forming a multimer. it can. In addition, two or more monoclonal antibodies, or a mixture of a monoclonal antibody and a polyclonal antibody,
It is also possible to use. When mixed, two kinds of antibodies may be simultaneously bound to the colloidal gold, or may be separately bound and then mixed. As a carrier to which the antibody is bound, it is preferable to use colloidal gold having an average particle diameter of 20 to 100 nm.However, a binding method between the colloidal gold and the antibody is known, for example, the gold colloid is the method of Frans.
(Nature Phys. Sci., Vol. 241, p. 20-22 (1973)), and binding is performed by the method of Geoghegan & Ackerman (J.
Histochem.Cytochem., Vol.25, p.1187-1200 (1977))
And can be easily performed. The conditions such as the reaction temperature of the antibody-bound gold colloid with the antigen, the reaction pH, the type of buffer, the type of coexisting salt, and the salt concentration are not different from the conventional antigen-antibody reaction. To promote the reaction, polyethylene glycol, polyvinyl alcohol,
It is also effective to add a polymer such as dextran to the reaction system.

[0008]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, but it should not be construed that the present invention is limited thereto.

Example 1 (Preparation of Monoclonal Antibody-Bound Gold Colloid) Albumin was purified from human serum by column chromatography using Blue Sepharose, and this was used as an immunogen to immunize Balb / c mice. did. Cell fusion between spleen cells and myeloma cell line P3U1 was performed, and antibodies were screened by ELISA to obtain 10 kinds of anti-albumin monoclonal antibodies. Each antibody was purified from mouse ascites by protein A-sepharose and applied to colloidal gold having an average particle diameter of 50 nm prepared by the method of Frens.
5 μg was bound per ml colloid by the method of rman. The colloidal gold bound to the antibody is recovered by centrifugation and dissolved in phosphate-buffered saline (pH 7.2) containing 1% bovine albumin to give an absorbance of about 5.5 at 540 nm and 1 cm path length. The concentration was adjusted as described above. Using a 96-well microtiter plate, 100 μl of the antibody-bound colloidal gold and 50 μl of a 1 μg / ml human albumin solution were mixed and left for 30 minutes.As a result, antibody numbers HA0304 and HA08
The colloidal gold bound with 19 showed aggregation and the color changed from reddish purple to gray. HA0304 had stronger aggregation with the antigen.

Example 2 (Measurement Using Microtiter Plate) Human albumin solutions of various concentrations were prepared using a phosphate buffer (pH 7.2) containing 1% bovine albumin. 25μl
And 100 μl of the HA0304 antibody-bound gold colloid obtained in Example 1,
And 25 μl of 10% polyethylene glycol 6000 were poured into each well of a 96-well microtiter plate and gently shaken. 20 minutes later, Bio-Rad 3550 type press
The difference between the absorbance at 540 nm and 655 nm (measured value at two wavelengths) was measured using a trider. The result is shown in FIG. In the excess antibody region, the absorbance decreased as the antigen concentration increased in the albumin concentration of 0-2 μg / ml, but in the excess antigen region where the albumin concentration exceeded 2 μg / ml, the absorbance did not decrease as the antigen concentration increased. Was.

For the purpose of measuring albumin concentration in urine,
25 μl of urine instead of albumin solution was reacted with 100 μl of the gold colloid conjugated to HA0304 antibody, and the absorbance values shown in Table 1 were obtained. From the values of these absorbances alone, it is not possible to distinguish whether the albumin in the sample is in the antigen excess area or the antibody excess area. After 5 minutes, the absorbance was measured again to observe whether the agglutination reaction was promoted. As a result, it was determined that only sample No. 3 was in the antibody excess area and the others were in the antigen excess area. Based on the standard curve of FIG. The concentration was calculated.

[0012]

[Table 1]

Example 3 (Measurement by Rate of Decrease in Absorbance) Phosphate buffer solution (pH 7.0) was placed in a 1 ml cuvette having an optical path length of 1 cm.
Add 680 μl of phosphate buffer containing 1% bovine albumin
20 μl of each concentration of human albumin solution prepared using (pH 7.0) was reacted with 300 μl of the HA0304 antibody-bound gold colloid used in Example 1 at 37 ° C., and the absorbance at a wavelength of 546 nm was measured for 6 seconds. Measurements were taken at intervals of 5 minutes. The measurement results are shown in FIG. 2, where the horizontal axis (x-axis) indicates the antigen concentration, and the vertical axis (y-axis) indicates the maximum rate of decrease in absorbance per minute. The time at which the rate of decrease in absorbance is maximized depends on the antigen concentration.The higher the antigen concentration, the faster the time, and the lower the time, the slower the time.The time at which the maximum speed is reached depends on whether the antigen is in the antigen excess area or the antibody excess area. Can be determined,
If it is less than about 80 seconds, it is determined that the antigen is in the excess area. An experiment was performed in which urine was used in place of the above albumin solution and the reaction was performed to measure the albumin concentration in urine, and the results in Table 2 were obtained.

[0014]

[Table 2]

Example 4 (Quantification of urinary albumin in an antigen excess area) Phosphate buffer containing 1 μg / ml human albumin (pH
The reaction was carried out under the same conditions as in Example 3 except that 7.0) was used as a buffer. The results of the standard curve are shown in FIG. 3, where the antigen concentration is shown on the horizontal axis (x-axis), and the difference between the absorbance immediately after the start of the reaction and 3 minutes later (two-point measured value) is shown on the vertical axis (y-axis). I have. Table 3 shows the results of measurement of albumin in urine.

[0016]

[Table 3]

[0017]

In the conventional immunological assay, the true value is determined depending on whether the measured value is in the antibody excess area or the antigen excess area due to the prozone phenomenon. Although the determination could not be made immediately, as a result of the present invention, it became possible to measure regardless of these.

[Brief description of the drawings]

FIG. 1 shows a standard curve in human albumin measurement. The horizontal axis (x-axis) shows the albumin concentration (μg / ml) and the vertical axis (y
(Axis) represents the difference in absorbance at 540 nm and 655 nm.

FIG. 2 shows the relationship between the concentration of antigen (albumin) in human albumin measurement and the maximum rate of decrease in absorbance per unit time. The horizontal axis (x-axis) is the albumin concentration (μg / ml)
And the vertical axis (y-axis) represents the maximum decrease in absorbance per minute at 546 nm.

FIG. 3 shows the relationship between the antigen (albumin) concentration in human albumin measurement and the decrease in absorbance at a certain time after the start of the reaction. The horizontal axis (x-axis) is the albumin concentration (μg / m
l) and the vertical axis (y-axis) represents the difference in absorbance at 546 nm between immediately after the start of the reaction and 3 minutes after.

──────────────────────────────────────────────────続 き Continuation of the front page (56) References JP-A-61-280568 (JP, A) Clinical Pathology VOL. 39 NO. 11 pp. 1184-1190 1991 Clinical Pathology VOL. 35 NO. 8 Pages 868-873 1987 (58) Fields investigated (Int. Cl. ⁷ , DB name) G01N 33/557 G01N 33/50 G01N 33/543 G01N 33/553

Claims (1)

(57) [Claims] (1) A solution of colloidal gold bound with an antibody is
Add the original sample and react to reduce the absorbance or
How to measure the antigen concentration in a sample based on the rate of decrease in absorbance
In the method, the antigen excess is added to the reaction system in advance to
The antigen concentration in the sample is determined by making the measurement area the antigen excess area.
How to measure degrees.