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Plate-type immunofluorescence kit for detecting group reactive antibodies and preparation method of plate-type immunofluorescence kit

An immunofluorescence and antibody detection technology, which is applied in biological testing, measuring devices, material inspection products, etc., can solve the problems of high laboratory requirements, time-consuming, and small volume of Terasaki microplates, and achieve rapid screening and specificity And the effect of good repeatability and high sensitivity

Active Publication Date: 2021-04-27
JIANGSU WEIHE BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The Terasaki microplate dedicated to the first method is small in size and is not suitable for general-purpose microplate readers. It requires the laboratory to use a Biotek microplate reader to interpret the results, and the reaction time is close to 2 hours, which takes a long time
Although the second method shortens the reaction by half and improves the specificity and sensitivity, it has higher requirements for the laboratory and the supporting equipment is expensive

Method used

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  • Plate-type immunofluorescence kit for detecting group reactive antibodies and preparation method of plate-type immunofluorescence kit
  • Plate-type immunofluorescence kit for detecting group reactive antibodies and preparation method of plate-type immunofluorescence kit
  • Plate-type immunofluorescence kit for detecting group reactive antibodies and preparation method of plate-type immunofluorescence kit

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Screening for characteristic dominant sequences of genes encoding HLA-I and HLA-II mixed antigens

[0032] Genes encoding HLA-I and HLA-II class antigens were retrieved from IMGT / HLA.

[0033] The antigenic determinant reactive epitopes of each antigenic protein were predicted by DNAstar-protean, and the gene fragments without reactive epitopes were excluded. Then use NetOGlyc and NetNGlyc to predict the N-glycosylation sites and O-glycosylation sites of the coding genes of each antigen protein, and exclude the reactive epitopes containing N-glycosylation sites and O-glycosylation sites fragments. Due to the high homology between the antigens of HLA-I and II, the fragments with higher homology of antigenic determinant reactive epitopes in the coding genes of the remaining antigenic proteins were selected as HLA-I hybrids. The characteristic dominant sequences of antigens and HLA-II mixed antigens are shown in Table 7:

[0034] Table 7 Characteristic sequenc...

Embodiment 2

[0038] The specific process of protein expression of the characteristic dominant sequence is as follows:

[0039] The designed characteristic dominant sequence was designed and synthesized by General Biosystems Co., Ltd. primers, and the restriction sites of BamH I and Xho I (Takara company) were added to the primer setting, and PCR amplification reaction was carried out to amplify Products were identified using 1.5% agarose gel electrophoresis. The purified PCR product and pcDNA3.1 were digested with BamH I and Xho I, and the separated target gene and plasmid pcDNA3.1 (Biobowell Biotechnology Co., Ltd.) were recovered by gel electrophoresis and treated with T4 DNA ligase (Takara Company) at 16°C. Overnight ligation reaction, take a small amount of linker to transform the competent cell DH5α and culture overnight, and the transformed bacteria are coated with LB solid medium with 100 μg / ml ampicillin (10g / L tryptone, 5g / L yeast extract, 10g / L chloride Sodium, pH=7.4) plates, i...

Embodiment 3

[0042] The protein samples expressed and purified by SEQ ID No.1-17 were diluted to 4 μg / mL and coated on the Elisa plate respectively, and each protein sample was coated with two reaction wells and control wells, 100 μL / well, 4 Coat at ℃ for 18h, wash twice with 200μL / well washing solution, block with 120μL / well blocking solution at 37℃ for 1h, pour off the blocking solution, and pat dry. Add 100 μL of HLA antibody-positive samples of the corresponding proteins in turn to the reaction wells (SEQ ID No.1: A1 positive sample, SEQ ID No.2: A25 positive sample, SEQ ID No.3: A68 positive sample, SEQ ID No.4: B7 Positive sample, SEQ ID No.5: B18 positive sample, SEQ ID No.6: C1 positive sample, SEQ ID No.7: C4 positive sample, SEQ ID No.8: C5 positive sample, SEQ ID No.9: C7 Positive sample, SEQ ID No.10: C17 positive sample, SEQ ID No.11: C18 positive sample, SEQ ID No.12: DR1 positive sample, SEQ ID No.13: DR4 positive sample, SEQ ID No.14: DQ2 Positive sample, SEQ ID No.15: DQ4...

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Abstract

The invention discloses a technical platform for establishing plate-type immunofluorescence reaction-PCR instrument detection, and provides a plate-type immunofluorescence kit for detecting group reactive antibodies and a preparation method of the plate-type immunofluorescence kit. The kit comprises a 96-well plate coated with an antigen and an antibody, and a fluorescein labeled antibody; and a fluorescent quantitative PCR instrument is adopted as a detection instrument. According to the method and the kit for detecting the group reactive antibodies by the plate-type immunofluorescence reaction-PCR instrument, purified HLA-I and HLA-II mixed antigens are used for quickly screening samples; the reaction of the HLA-I and HLA-II antibodies can be distinguished and detected more accurately through fluorescence numerical values, and the sensitivity, specificity and repeatability are high.

Description

technical field [0001] The invention relates to the technical field of in vitro detection, in particular to a technology platform based on plate immunofluorescence reaction-PCR instrument detection, and provides a plate immunofluorescence kit for detecting population reactive antibodies and a preparation method thereof. Background technique [0002] After more than 50 years of development, allogeneic organ transplantation has become a radical method for the treatment of various organ tissue failures, and the subsequent rejection of organ tissues is directly related to the success of clinical transplantation. When the recipient's immune system contacts the alloantigen on the allograft, it will generate an immune response against the donor, and the human leukocyte antigen (HLA) peptide in the donor will be presented to T cells and B cells, through After a series of responses, stimulate the production of anti-donor HLA antibodies that mediate acute and chronic rejection reactio...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/58G01N33/543
CPCG01N33/6854G01N33/582G01N33/543
Inventor 陈炤源王浩章婷婷李天程
Owner JIANGSU WEIHE BIOTECH
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