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A kind of indirect ELISA antibody detection kit and detection method

A technology for enzyme-linked immunosorbent assay and antibody detection, which is applied in measurement devices, instruments, scientific instruments, etc., can solve the problems of low sensitivity of detection methods, high detection background, and long detection time, so as to shorten the detection time and improve the signal-to-noise ratio. , the effect of reducing the negative background

Active Publication Date: 2021-06-04
GUANGDONG HAID ANIMAL HUSBANDRY & VETERINARY RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the existing detection methods, there are problems such as high detection background, that is, the sensitivity of the established detection method is too low, and the detection time is too long.

Method used

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  • A kind of indirect ELISA antibody detection kit and detection method
  • A kind of indirect ELISA antibody detection kit and detection method
  • A kind of indirect ELISA antibody detection kit and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Preparation method of antibody diluent: accurately weigh 1.0g bovine serum albumin (BSA), 0.10g thimerosal sodium, 1.0g ethylenediaminetetraacetic acid disodium (EDTA-2Na) in a 1000mL clean and sterilized beaker, add 700mL sterilized Dissolve bacteria in distilled water, then use a disposable 50mL syringe filled with absorbent cotton, slowly filter 20mL camel serum that has been measured into a beaker, and then use a 100mL sterilized graduated cylinder to measure 0.1M PBS 100mL and add it, stir and mix well Finally, adjust the pH to 7.2 with 1M NaOH solution, then continue to add distilled water to make up to 1000mL, and finally add 1mL of penicillin and streptomycin (1000IU) double antibody.

Embodiment 2

[0049] Preparation of goat anti-pig HRP-labeled secondary antibody dilution: Accurately measure 100ml of the basic antibody dilution into a clean and sterilized 200ml cell culture medium glass bottle, add goat anti-pig HRP-labeled IgG (KPL), fully stir and mix, respectively Prepare 1:500, 1:1000, 1:2000, 1:5000, 1:8000, 1:10000 goat anti-pig HRP-labeled secondary antibody dilutions.

Embodiment 3

[0051] Detect porcine reproductive and respiratory syndrome virus (PRRSV) antibody (indirect ELISA): use the goat anti-pig HRP labeled secondary antibody dilution solution of embodiment 2 to dilute PRRSV standard negative and positive serum by 1:40 times, then respectively divide two kinds of Add 100 μL / well of the diluted serum to the ELISA plate pre-coated with PRRSV-specific peptides, incubate at room temperature for 30 minutes and then wash, wash the microplate three times with 1× washing solution, 300 μL / well, and shoot Dry; then add 100 μL / well of the goat anti-pig HRP-labeled secondary antibody dilution solution of Example 2 1:8000, incubate at room temperature for 30 minutes, repeat the above washing step once; add 100 μL substrate to each well, and develop color in the dark for 10 minutes, Finally, 50 μL of stop solution was added to each well, and read with a microplate reader at a wavelength of 450 nm.

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Abstract

The invention discloses an indirect ELISA antibody detection kit, which comprises an antibody diluent and an enzyme-labeled secondary antibody. The antibody diluent contains the following components in mass fraction: 75-95% of 1× PBS buffer, 0.1-8% bovine serum albumin, 0.01-0.5% thimerosal sodium, 0.05-5% anticoagulant, 0.1-15% non-conventional feeding animal serum and 0.05-2% green chain Mycin double antibody; the dilution of the enzyme-labeled secondary antibody is 1: (500‑10000). The invention also discloses an indirect ELISA antibody detection method. The invention can reduce the negative background of the common enzyme-linked immunoassay method, and improve the signal-to-noise ratio and the sensitivity of the detection method.

Description

technical field [0001] The invention relates to the technical field of biological detection reagents, in particular to an indirect ELISA antibody detection kit and detection method. Background technique [0002] Enzyme-linked immunosorbent assay (ELISA) method is widely used in the determination of antibodies and antigens, the principle of which utilizes the high binding affinity of proteins or polypeptides to solid surfaces such as ELISA plates. In the indirect ELISA system for the determination of serological antibodies, due to the inherently high binding affinity of serum immunoglobulins to solid surfaces, this affinity is much higher than that of any currently used blocking agents, moreover, the overall The concentration of immunoglobulin is high, mg / ml, while the concentration of antigen-specific antibody immunoglobulin is very low, ng or μg / ml, and non-antibody immunoglobulin can cause significant non-specific interactions with antigen molecules. These factors all lea...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/535
CPCG01N33/535
Inventor 杨洁李其昌陈善真陈克宏刘博奇王贵平
Owner GUANGDONG HAID ANIMAL HUSBANDRY & VETERINARY RES INST
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