JPH0416764A - Immunoassay - Google Patents

Abstract

PURPOSE:To obtain immunoassay using high purity gold colloid by using an antigen or antibody sensitized gold colloid particles having a specific particle size or less. CONSTITUTION:Gold colloid particles are prepared by adjusting the concn. of sodium citrate to be used and a stirring method when auric chloride is reduced and the particle size thereof is set to 7 nm or less. By this method, even in a hue changing method accompanied by agglutination reaction and sandwich immunoassay, excellent sensitivity, stability and reactivity are obtained. When a particle size is set to 7 nm or less and 85% or more of colloid particles is adjusted to a uniform particle size, a further effective result is obtained.

Description

[Detailed description of the invention] (Industrial application field) The present invention relates to the field of immunoassays using colloidal gold.

(Problems to be solved by conventional technology and invention) Hor
J, Hi by Isberger and Rossert
stochem.

Cytochew, 25.295=305 (1977
) describe an agglutination assay for mannan using colloidal gold as a label. In addition, JP-A-63-25553, JP-A-64-32169, etc. disclose the use of a dispersion of colloidal gold particles in sandwich immunoassay.

The present inventors basically aimed to develop a highly sensitive immunoassay method using colloidal gold while following these already known methods.

(Means to solve the problem) When performing immunoassay using highly sensitive gold colloid, by adjusting the particle size of the gold floid particles to 7 nm or less, and by adjusting the particle size to 7n+w or less and achieving 85% By using colloidal gold particles with a uniform particle size, the sensitivity is 4 to 10 times higher than conventional methods, and the stability and reactivity are much better in both the color change method associated with agglutination reaction and the sandwich immunoassay method. found this and completed the present invention.

The basic principle of the color tone changing method using colloidal gold is as described in J.H.
istochew, Cytoche+m, 25.295
~305 (1977), as disclosed in Figl. The sandwich immunoassay method using colloidal gold was published in 1983.
-32169 and many other applications have been filed. However, the particle size is constant, and the particle size is constant and uniform (85
% or more) is not known to increase sensitivity.

According to the objects of the invention, analytes (such as lf, hCG,
Hormones such as LH, hemoglobin, and IgG.

serum proteins such as IgM, viruses such as HB, bacteria, etc.)
An immunoassay method is provided for determining the presence of.

As a method for preparing gold colloid with a constant particle size, when reducing chloroauric acid, the concentration of quench and sodium phosphate used and the stirring method are adjusted according to the method described in JP-A-63-25553, I.
mmunochemtry 8:1081.1971
It is prepared by referring to the literature such as et al., but it is preferable to fractionate by centrifugation or the like in order to obtain particles with a constant diameter.

The content of the invention will be specifically illustrated below with reference to Examples.

4H! 0) Add 1rn12 while stirring, and after 1 minute add 2%
After adding 5 mff of sodium citrate solution and stirring for a further 20 minutes, it is cooled to 30°C. After cooling, adjust the pH to 7.0 with 0.1M potassium carbonate solution, and add 1% as a stabilizer.
After adding 1 mff of PEG20,000 and stirring for 10 minutes, the mixture was filtered with a 0.22 μm Millipore filter.

Example 2 (1) Preparation of colloidal gold solution sensitized with anti-hCG polyclonal antibody Anti-hCC (DAKOPATTS, code No. A23)
1) 10mM HEPES.

It was diluted with pH 7.1 to give a concentration of 200 μg/mQ. This liquid is 3m7! and gold colloid liquid 30 prepared in Example 1
Transfer mQ to a centrifuge tube and stir thoroughly.

Tweite AI buffer (IOmM HEPES, 0.3
M D-Mannitol, 0.05% PEG 20000
% 0.1% BSA pH 7, l) was added to 1
After stirring for an hour, it was stored at 4°C overnight.

Example 1 Preparation of gold colloid A 10% chloroauric acid solution (HA
uC4a ・(2) Preparation of gold colloid with a particle size of 1 nm or less The anti-hCG polyclonal antibody-sensitized gold colloid solution prepared in (1) of Example 2 was centrifuged at 10°C and 9000 PPM for 20 minutes, and the upper groove was separated. Collected in a centrifuge tube, 10
'C! , centrifuge for 30 minutes at 15,000 RPM. Remove this upper groove and add 30ml of AI buffer to the precipitate.
12 was added and suspended uniformly, and then further heated at 10°C and 150°C.
Centrifuge for 30 minutes at 00 PPM. The same operation was performed twice, and 10 ml of AI buffer was added to the resulting precipitate to prepare.

(3) Preparation of gold colloid with a particle size of 1 nm The solution prepared in (1) of Example 2 was heated at 10"C!, 900
Centrifuge at 0 RPM for 10 minutes, collect the upper groove into another centrifuge tube, and centrifuge at 10° C. and 15,000 RPM for 30 minutes. The supernatant was removed, and At buffer 30++Q was added to the precipitate to homogeneously suspend it, followed by further centrifugation at 10° C. and 15,000 RPM for 30 minutes. The same operation was carried out twice, and At buffer 10a+Q was added to the resulting precipitate.

(4) Preparation of gold colloid with a particle size of 4 to 5 nm (1
) was centrifuged at 10°C and 6000 PPM for 20 minutes, and the supernatant was collected into another centrifuge tube and centrifuged at 10°C.
, 9000 PPM for 30 minutes.

Remove this upper groove and add 30mf of At buffer to the precipitate.
After adding f and making it homogeneous, it was further heated at 10℃ and 9000℃.
Centrifuge in PPM for 30 minutes. Perform the same operation twice,
The resulting precipitate was prepared by adding 10 mQ of AI buffer.

(5) Preparation of gold colloid with a particle size of 7 nm The solution prepared in (1) of Example 2 was heated to 15,000 P at 10°C.
Centrifuge at PM for 20 minutes, collect the upper groove into another centrifuge tube, and centrifuge at 10° C. and 9000 PPM for 30 minutes.

Remove this upper groove and add 30 mQ of AI buffer to the precipitate.
was added and suspended evenly, and then further heated at 10°C, 9,000 yen.
Centrifuge in ORPM for 30 minutes. The same operation was performed twice, and A1 buffer 10n+Q was added to the resulting precipitate to prepare.

(6) Preparation of gold colloid with a particle size of 9 nm Prepared in (1) of Example 2 I;
Centrifuge at PPM for 10 minutes, collect the upper groove into another centrifuge tube, and centrifuge at 10° C. and 9000 RPM for 30 minutes.

Remove this upper groove and add 30v2 Al buffer to the precipitate.
was added and suspended uniformly, and then further heated at 10°C and 9000°C.
Centrifuge for 30 minutes at RPM. Perform the same operation twice,
The resulting precipitate was prepared by adding 10 ml of At buffer.

(7) Preparation of gold colloid with a particle size of 17.5 nin. The solution prepared in (1) of Example 2 was heated at 10'C at 15000 RPM for 30
Centrifuge for a minute and remove the upper groove. Next, 30 mα of Al buffer is added to the precipitate to homogeneously suspend it, and then centrifuged at 10° C. and 5000 RPM for 30 minutes. The same operation was performed twice, and 10 ml (l) of sample buffer was added to the resulting precipitate to prepare.

(8) Preparation of gold colloid with non-uniform particle size The solution prepared in (1) of Example 2 was heated at 10°C and 15,000 yen.
Centrifuge at RPM for 30 minutes and remove the upper groove.

Next, 30-mL of A1 buffer is added to the precipitate to homogeneously suspend it, followed by further centrifugation at 10° C. and 15.00 ORPM for 30 minutes. The same operation was performed twice, and 10 m+2 of At buffer was added to the resulting precipitate to prepare.

Example 3 Confirmation of particle size and uniformity of sensitized gold colloid liquid In this example, a submicron particle size distribution analyzer (PACIFIC) was used as the measuring device.
5CIENTIFIC NIC0NP MODEL3
70) was used. Particle size 1 nm or less, Inm54~b1
The 7.5 nm sensitized gold colloid liquid exhibited a uniform particle size distribution of 85% or more. Heterogeneous colloid is 2.1nI11
is 64.1%, 33.4nm is 21.6%, 91.1a
m was 14.3%.

(See Table 1) Example 4 Degree test of colloidal gold liquid by color tone change method Particle size of sensitized colloidal gold liquid is 1 nm or less, 1nI1114 to 5nIl11
7nm, 9nm, 17.5nm and heterogeneous colloids were mixed with At to give an absorbance of 2.0 in 540nml.
Prepared with buffer and added 250 cells each to a 96-well plate.
Dispense μα.

hCG (S11:, MA, CG-5) was used as an antigen at 0.
100.400.1600.6400.25000 and
and 400,000 IU/L with 10aM HEPES, pH 7,
1, add 60pQ to each antibody, and add 60pQ to each antibody.
The color tone of the test solution was observed after a few minutes. As a result, the particle size was 1 nm.
and 7 nm sensitized gold colloid has a sensitivity of 1 for anti-X antibody reaction.
Commercial product A (particle size 7.8nII+, 9
7%, detection sensitivity 4001 U/L). It was confirmed that a non-uniform colloid with a large particle size of 17.5 nm has a detection limit of 640010/L, which is a low sensitivity, and that a colloid with a particle size of 7 nw or less is superior. (See Table 2) The evaluation was expressed as ++ (strongly reacted), + (reacted), and - (not reacted).

Example 5 Sensitivity test pile hcc of gold colloid liquid by Sanderch method
Polyclonal antibodies were incubated in loM HEPES, pH 7.
, 1 to 500 μg/m4, spotted 5 points of 10 μΩ each on a 0.45 μm Millipore filter, placed in a plastic Nyare, and kept warm at 37°C for 20 minutes. Add 5 m4 of blocking agent for pharmaceutical and immunological experiments) and block at 37°C for 30 minutes. After 30 minutes, the Millipore filter is taken out from the plastic petri dish and washed three times with 5 ml of At buffer using a filtration device. The antigen hCG (SIGMA
CG-5) to 0.10, 100.1000. and 10
．． 000 μg/m4 in 0.1% PBS/BSA, each 10 μg was spotted on the previously spotted anti-hCG polyclonal antibody, and reacted for 30 minutes at room temperature. After 30 minutes, wash 3 times with 5 m4 of AI buffer. A sensitized gold colloid solution with a particle size of inn was prepared with At buffer so that the absorbance at 540 nm was 5.0, 10 μg of it was spotted and reacted for 30 minutes, and then washed three times with 5 ml of At buffer. The degree of coloring was observed. Gold)Oide particle sizes of 1 nm or less, 4-5 nm, 7 nm, 9 nm%17.5 nm and non-uniform colloids sensitized by the same procedure were also tested. As a result, in a sensitivity test using the Sand-Deutsch method, the sensitized gold colloid with a particle size of 7 nm or less showed a sensitivity of 101 U, and a particle size of 9 nm.
It is superior to the sensitivity of non-uniform colloids of nm or more (1001 U/L), and commercially available product B (particle size 9.9n+a, 98%
, a detection sensitivity of 10011/L).

(See Table 3) The evaluation was expressed as ++ (strong reaction), 10 (reaction), or - (no reaction).

Claims (2)

[Claims] (1) In immunoassay using colloidal gold, the particle size was 7.
An immunoassay method characterized by using sub-nm antigen or antibody-sensitized gold colloid particles. (2) In immunoassay using colloidal gold, the particle size was 7.
An immunoassay method characterized by using antigen- or antibody-sensitized colloidal gold particles having a particle size of nm or less and a uniform particle size of 85% or more.