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Method for detecting human leukocyte antigen HLA-G

A technology for HLA-G and leukocyte antigen, applied in the field of detection of serum soluble human leukocyte antigen G, can solve the problems of unsatisfactory sensitivity and accuracy, achieve good repeatability and accuracy, high detection sensitivity, and good specificity Effect

Active Publication Date: 2016-06-15
叶尚勉
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, its sensitivity and accuracy are not ideal

Method used

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  • Method for detecting human leukocyte antigen HLA-G
  • Method for detecting human leukocyte antigen HLA-G
  • Method for detecting human leukocyte antigen HLA-G

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Implementation 1. Recombinant HLA-G protein fragments

[0040] Materials and methods

[0041] 1. Cloning of target gene and construction of plasmid

[0042] ●Get the HLA-G gene sequence from the gene bank, select the regions with low homology with HLA-A, -B and -C, and design primers with Primer5.0 software. The upstream primer is 5'-gaagaggagacacggaacac-3'(255---274bp) and the downstream primer is 5'-ctccaggtaggctctccttt-3'(538---557bp). And the designed primers were handed over to Sangon Biological Engineering (Shanghai) Co., Ltd. for synthesis.

[0043] ●Because the human placental tissue has a high expression of HLA-G, the placental tissue was homogenized, and the total RNA was extracted by TRNzol method.

[0044] ●Two-step method for RT-PCR to amplify the target gene to be cloned. The reverse transcription (RT) reaction system and reaction conditions are shown in Table 1; the polymerase chain reaction (PCR) reaction system and reaction conditions are shown in T...

Embodiment 2

[0064] Implementation 2. Preparation of anti-HLA-G monoclonal antibody

[0065] Materials and methods

[0066] 1. Antigen Preparation

[0067] The HLA-G85aa-185aa fragment prepared in Example 1 was used as an antigen.

[0068] 2. Immunization of mice

[0069] ● After emulsifying 50 μg of HLA-G85aa-185aa fragments with an equal volume of Freund’s complete adjuvant until dripping water does not dissolve, intraperitoneally inject 6-8 week-old female Balb / c mice, the injection volume is 200 μl / mouse, 2 weeks later Perform a second immunization.

[0070] ● After emulsifying HLA-G85aa-185aa fragments with an equal volume of Freund's incomplete adjuvant until dripping does not dissolve, intraperitoneally inject mice with a volume of 200 μl / mouse, and perform the third immunization two weeks later.

[0071] ●After emulsifying the HLA-G85aa-185aa fragment with an equal volume of Freund's incomplete adjuvant until dripping water does not dissolve, inject into the mouse intraperitone...

Embodiment 3

[0088] Implementation 3. Preparation of anti-Beta-microglobulin monoclonal antibody

[0089] Materials and methods

[0090] 1. Antigen Preparation

[0091] Check the information and design the peptide sequence based on β2 microglobulin:

[0092] MSRSVALAVLALLSLSGLEAIQRTPKIQVYSRHPAENGKSNFLNCYVSGF (β2 microglobulin amino acid sequence: 1aa-50aa). The peptide was synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. and coupled to KLH.

[0093] 2. Immunization of mice

[0094] ●After emulsifying 50 μg of KLH-coupled β2 microglobulin polypeptide with an equal volume of Freund’s complete adjuvant until dripping does not dissolve, intraperitoneally inject 6-8 week-old female Balb / c mice, the injection volume is 200 μl / mouse, 2 A second immunization was performed a week later.

[0095] ●After emulsifying 50 μg of KLH-coupled β2 microglobulin polypeptide with an equal volume of Freund’s incomplete adjuvant until it does not dissolve in water, intraperitoneally inject mice wit...

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Abstract

The invention discloses a method for detecting a human leukocyte antigen HLA-G. A protein fragment of an amino acid sequence at the 85-185th position of a heavy chain of the HLA-G serves as an antigen to prepare an antibody 3C4, a protein fragment of an amino acid sequence at the 1-50th position of a light chain of the HLA-G is coupled with KLH to prepare an antibody 3H1 as an antigen, then 3C4 and 3H1 serve as an enzyme-linked immunosorbent assay reagent to detect the HLA-G, the amino acid sequence at the 85-185th position of the heavy chain of the HLA-G is as shown in SEQ ID NO:1, and the amino acid sequence at the 1-50th position of the light chain of the HLA-G is as shown in SEQ ID NO:2. The method is high in detection sensitivity and good in specificity and has good repeatability and accuracy, and therefore a new method is provided for detecting the HLA-G.

Description

technical field [0001] The invention belongs to the field of medical detection, in particular to a method for detecting serum soluble human leukocyte antigen G. Background technique [0002] Preeclampsia (PE) refers to symptoms such as headache, vertigo, nausea, vomiting, and epigastric discomfort on the basis of hypertension and proteinuria at about 24 weeks of pregnancy, also known as pregnancy-induced hypertension syndrome. It is a disease unique to pregnancy and one of the four major obstetric diseases. Its incidence rate is about 5-10%. Seriously endanger the safety of mother and child. Early screening for preeclampsia can help reduce harm to mothers and babies. [0003] The pathogenesis of preeclampsia is still not fully understood. However, it is now recognized by the medical community that abnormal placental implantation and abnormal development are the main causes of pre-eclampsia. Because after delivery, the placenta is expelled, and the symptoms of preeclamps...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/577
CPCG01N33/6854G01N2800/368
Inventor 叶尚勉
Owner 叶尚勉
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