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Establishment of novel ultrasensitiveness ELISA method

A test method, enzyme-linked immunosorbent adsorption technology, applied in measuring devices, instruments, fluorescence/phosphorescence, etc., can solve the problems of limiting the sensitivity of ELISA methods and failing to reach the sensitivity of chromogenic substrates, so as to improve sensitivity and increase Effect of Fluorescence Yield

Active Publication Date: 2014-01-15
CHANGCHUN BCHT BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, the molecular size of the labeled anti-antibody (MW: 75kd) and HRP enzyme (MW: 40kd) is similar, so the number of enzyme molecules coupled to each anti-antibody molecule is small. Rarely, the enzyme concentration with which the anti-antibody binds to the solid phase does not reach the sensitivity of the chromogenic substrate, thus limiting the sensitivity of the ELISA method

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  • Establishment of novel ultrasensitiveness ELISA method
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  • Establishment of novel ultrasensitiveness ELISA method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Using the novel ELISA method of the present invention to detect anti-HIV-1gp120 monoclonal antibody

[0033] Experimental steps:

[0034] 1. Coating of ELISA plates.

[0035] 1. Dilute HIV-1gp120 with coating solution (carbonate buffer, pH 9.6) to 2 μg / mL, and add to the wells of the ELISA plate, 100 μL / well. Placed at 4°C for 18h.

[0036] 2. At room temperature, shake off the solution in the well and pat dry. Add PBS containing 2‰ (volume) Tween-20, 250 μL / well, shake on a horizontal shaker for 5 minutes, shake off the washing solution in the well, and pat dry. Repeat the wash 5 times.

[0037] 3. Add 200 μL / well of PBS containing 5% (mass volume ratio) skimmed milk powder, and place at 37°C for 4 hours.

[0038] 4. At room temperature, shake off the solution in the well and pat dry. Add PBS containing 2‰ (volume) Tween-20, 250 μL / well, shake on a horizontal shaker for 5 minutes, shake off the washing solution in the well, and pat dry. Repeat the wa...

Embodiment 2

[0051] Example 2 Detection of anti-HIV-1gp120 monoclonal antibody using classic ELISA method

[0052] Under the same experimental conditions as in Example 1, biotinylated HRP (use concentration 2.5×10 -5 mg / ml) instead of biotinylated TurboNuclease, use TMB chromogenic substrate (use concentration 0.05g / L) instead of fluorescent substrate, use streptavidin without gold nanoparticles (use concentration 5×10 -4 mg / ml) instead of 40nm gold nanoparticles-streptavidin, the sensitivity detection of the classic ELISA method was performed according to the steps described in Example 1.

[0053] The test results are shown in Figure 2(B). The average value of the negative control is 0.082±0.001, so when the OD value > 0.17, the dilution is positive, and when the OD value is ≤ 0.17, the dilution is negative. The results of this experiment Medium 10 4 The OD value of double dilution was 0.884, which was positive; 10 5 The OD value of double dilution was 0.138, which was negative. There...

Embodiment 3

[0054] Example 3 Detection of anti-HIV-1 gp120 monoclonal antibody using a novel ELISA method without gold nanoparticles

[0055] Under the same experimental conditions as in Example 1, streptavidin without gold nanoparticles (using a concentration of 5×10 -4 mg / ml) instead of 40nm gold nanoparticles-streptavidin, the sensitivity detection of the new ELISA method without using gold nanoparticles was carried out according to the steps described in Example 1.

[0056] The test results are shown in Figure 2(C). The average value of the negative control is 0.103±0.001, so when the fluorescence value > 0.22, the dilution is positive, and when the fluorescence value is ≤ 0.22, the dilution is negative. The results of this experiment Medium 10 5 The fluorescence value of double dilution is 0.625, which is positive; 10 6 The fluorescence value of double dilution is 0.20, which is negative, so the sensitivity of the novel ELISA method using the TurboNuclease enzyme of the present inv...

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Abstract

The invention provides a novel ELISA (Enzyme Linked Immunosorbent Assay) method with high detection sensitivity. The detection sensitivity of the novel ELISA method can reach 100 times of that of an existing ELISA method. In the novel ELISA method, a more sensitive novel fluorogenic substrate is used for replacing a chemical chromogenic substrate in the conventional ELISA method; moreover, a high-efficiency stable enzyme capable of catalyzing the fluorogenic substrate to generate a luminescent effect is used as a marker enzyme so as to realize that the luminescent effect of the substrate reaches the minimum enzyme quantity required for the detectable level; the novel ELISA method also uses a gold nanoparticle coupling streptavidin system with excellent biocompatibility for realizing an amplification effect of an enzymatic reaction; integration of improvement measures on the three aspects finally implements great improvement on the detection sensitivity of the ELISA method.

Description

technical field [0001] The invention relates to an immunological detection method, in particular to a novel enzyme-linked immunosorbent assay (ELISA) method. Background technique [0002] Enzyme-linked immunosorbent assay (ELISA) is an immunological detection method based on the specific reaction between antigens and antibodies for the detection of antigens or antibodies in samples. This method is simple, fast, sensitive, etc. features. Since Engvall and Perlmann in 1971 [1] Since the invention, the method has been developed rapidly and has been widely used in the detection of various antigens and antibodies. The basic principle is to covalently couple the enzyme molecule to the antibody or anti-antibody molecule. This coupling does not change the immunological characteristics of the antibody and the catalytic activity of the enzyme. Antibody specific binding, after adding the enzyme substrate solution, the substrate produces a color reaction under the catalysis of the la...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/543G01N21/64
CPCG01N33/543
Inventor 高峰姜春来范培虎孔维
Owner CHANGCHUN BCHT BIOTECH
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