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An ELISA kit for detecting liver fluke IgG4 antibody and its preparation method

A technology for detecting antibodies and kits is applied in the field of ELISA kits for detecting liver fluke IgG4 antibodies and the field of preparation thereof, which can solve the problems of cumbersome operations, unfavorable batch operations, and difficult storage and transportation of samples.

Active Publication Date: 2017-03-22
深圳华康生物医学工程有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these methods are time-consuming and labor-intensive, and the samples are not easy to store and transport. The test results of the samples collected each time are quite different, and the operation is cumbersome, which is not conducive to the batch operation of the disease control department and medical institutions. High, and the early diagnosis of the disease cannot be made, which is not conducive to the promotion and application of clinical units
Enzyme-linked immunosorbent assay (ELISA) and other immunological detection methods have solved the shortcomings of time-consuming, labor-intensive, and high missed detection rates of etiological methods to a certain extent, but at present, natural insect egg antigen extracts are used as kit coatings. Antigens, natural antigens have defects such as large batch-to-batch differences, more impurities after purification, and few effective antigen components, which may easily cause quality problems such as poor specificity, low sensitivity, and large batch-to-batch differences.

Method used

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  • An ELISA kit for detecting liver fluke IgG4 antibody and its preparation method
  • An ELISA kit for detecting liver fluke IgG4 antibody and its preparation method
  • An ELISA kit for detecting liver fluke IgG4 antibody and its preparation method

Examples

Experimental program
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Effect test

Embodiment 1

[0067] Embodiment 1: Preparation of CsSP46 protein

[0068] Cloning of liver fluke CsSP46 gene, construction and identification of expression vector, such as figure 1 Shown:

[0069] (1) Design primers according to the gene sequence of liver fluke:

[0070] Upstream primer: 5'-ATA CATATG ATGGGACGTCCCCATGCAGATG-3' (SEQ ID NO.3), wherein the underlined part is the Xho I restriction site;

[0071] Downstream primer: 5'-GGG CTCGAG TTACTTGGGAGTCTTTGATGATTG-3' (SEQ ID NO.4), wherein the underlined part is the Nde I restriction site.

[0072] The genome of liver fluke was extracted by conventional small amount bacterial DNA extraction method, ie alkaline lysis method.

[0073] PCR amplification: PCR amplification is performed using the extracted plasmid as a template, and the reaction system is as follows:

[0074]

[0075] Set up a set of negative controls with water as the sample.

[0076] The reaction conditions are as follows:

[0077]

[0078] The obtained PCR ...

Embodiment 2

[0095] Example 2: Selection of horseradish peroxidase-labeled secondary antibodies

[0096] Take 10mg horseradish peroxidase and dissolve in 2mL 0.4mol / L NaHCO of pH8.0 3 Add 0.2mL of 1% DNFB absolute ethanol solution, stir at room temperature for 1h; add 2mL of 0.06mol / L NaIO 4 Add 2mL of 0.16mol / L ethylene glycol, stir at room temperature for 1h, put it into a dialysis bag, dialyze against 2000mL of 0.01mol / L carbonate buffer solution of pH9.5 at 4°C overnight, change the solution for 3 Add 2 mL of carbonate buffer solution containing 10 mg of anti-human IgG4 to 6 mL of formylated horseradish peroxidase solution, stir at room temperature for 3 h in the dark; add 10 mg of NaHB 4 , put it at 4°C overnight; put it into a dialysis bag, dialyze against 0.01mol / L, pH7.2 PBS, 4°C for 24h, change the medium three times; centrifuge at 3000r / min for 30min to remove the precipitate. The supernatant was chromatographed on a sephadexG-200 gel column, eluted with PBS, aliquoted in small...

Embodiment 3

[0099] Embodiment 3: the assembly of kit

[0100] (1) Coating the microtiter plate: the purified recombinant antigen CsSP46 was coated on a 96-well microtiter plate, and coated overnight at 4°C. After taking it out the next day, wash the plate 3 times with PBST, 3 minutes each time. 1% BSA prepared in PBST solution was used as blocking solution, 100ul of blocking solution was added to each well, and blocked at 37°C for 1h. After sealing, the plate was washed 3 times with PBST, 3 min each time. Put it into a special packaging bag for 96-well microplate plate, seal it with a sealing machine and store it at 4°C.

[0101] (2) Horseradish peroxidase-labeled anti-human IgG4: Horseradish peroxidase-labeled anti-human IgG4 can be obtained through technical service outsourcing or commercially purchased, prepared by conventional methods in the field, and prepared in 0.01mol / L of 2% BSA Store in phosphate buffer, pH 7.4.

[0102] Preparation of other solutions in the kit:

[0103] (...

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Abstract

The invention relates to an ELISA kit for detection of liver fluke IgG4 antibody and a preparation method thereof. The kit comprises an enzyme label plate and an enzyme label detection antibody, wherein the enzyme label plate is coated with specific antigen CsSP46 of liver fluke; and the enzyme label detection antibody is an enzyme-labeled anti-human IgG second antibody or anti-human IgG4 second antibody. Amino acid sequence of the specific antigen CsSP46 comprises a fragment as shown in the SEQ ID NO.1. When used in ELISA detection of hepatic clonorchiasis, a recombinant antigen prepared from the kit has higher sensitivity than eggs detection and has higher sensitivity and specificity than existing crude antigens and other recombinant antigens. The kit of the invention can be used in clinical examination of patients with hepatic clonorchiasis and treatment effect evaluation after treatment and control efficiency assessment and evaluation of population in endemic areas by the Center for Disease Control and Prevention, and has a wide market prospect and huge economic and social benefits.

Description

technical field [0001] The invention relates to the technical field of biological immune detection, in particular to a kit and a preparation method thereof, in particular to an ELISA kit for detecting liver fluke IgG4 antibody and a preparation method thereof. Background technique [0002] Liver fluke, also known as Clonorchis sinensis, is commonly called liver fluke because its adults mainly parasitize in the hepatic and bile ducts of the final host. Adults mainly parasitize in the liver and bile ducts of humans, dogs, cats, pigs, etc. Adult worm extracts and metabolites contain antigenic components, which can induce a variety of immunopathological reactions after infecting the human body. In the serum of liver fluke patients, IgG is the easiest to detect After the host is infected with liver fluke, it can induce a strong humoral immune response. The main immunoglobulin involved in humoral immunity is IgG, and its level is positively correlated with the degree of infection....

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68
CPCG01N33/6893
Inventor 曾周祥傅剑华蔡华蔡栋李明磊庄兴华操美芳
Owner 深圳华康生物医学工程有限公司
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