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Plate-type immunofluorescence kit for platelet antibody detection and preparation method thereof

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, and achieve the effects of high sensitivity, improved detection sensitivity, and strong result specificity

Pending 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

This method requires high laboratory requirements, and special equipment is required.

Method used

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  • Plate-type immunofluorescence kit for platelet antibody detection and preparation method thereof
  • Plate-type immunofluorescence kit for platelet antibody detection and preparation method thereof
  • Plate-type immunofluorescence kit for platelet antibody detection and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Selection of Characteristic Predominant Sequences of Coding Genes of HLA-Class I Mixed Antigens

[0033] The coding genes of HLA class I antigens were retrieved from IMGT / HLA.

[0034] 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 among the various antigens of the HLA-I class, the fragments with higher homology of the epitope of the antigenic determinant reaction epitope were selected as the HLA-I class mixed antigen in the coding genes of the remaining antigenic proteins. The characteristic dominant sequence is shown in Table 11:

[0035] Table 11 Characteristic sequences ...

Embodiment 2

[0039] The specific process of protein expression of the characteristic dominant sequence is as follows:

[0040] 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

[0043] The protein samples after expression and purification of SEQ ID No.1-11 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, sample diluent diluted three times), add 100 μL sample diluent to the control well, room temperature (22-28°C), ...

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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 platelet antibody detection and a preparation method thereof. The kit comprises a 96-well plate coated with an antigen and an antibody and a fluorescein labeled antibody, and the detection instrument is a fluorescent quantitative PCR instrument. The method and the kit for detecting the platelet antibody by the plate-type immunofluorescence reaction-PCR instrument are high in sensitivity, good in specificity and repeatability and capable of covering various factors causing the platelet antibody, results among holes can be compared more visually and clearly through fluorescence numerical values, misreading and misreading are avoided, and missed diagnosis and misdiagnosis are reduced.

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 platelet antibody detection and a preparation method thereof. Background technique [0002] With the continuous deepening of research on blood transfusion medicine and the wide application of component blood transfusion in clinical practice, platelet transfusion, as an important clinical blood transfusion treatment, is of great value in the treatment of bleeding tendency and platelet dysfunction caused by platelet deficiency. However, poor or even ineffective platelet transfusion is a common problem at present, and the destruction of foreign platelets by a large number of platelet antibodies produced in patients may be the main reason. Among them, patients with long-term multiple blood transfusions are more likely to produce platele...

Claims

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Application Information

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Patent Type & Authority Applications(China)
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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