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Method for detecting human soluble asialoglycoprotein receptor (sASGPR)

A sialoglycoprotein and human body detection technology, applied in the field of immunology detection, can solve the problems of high preparation cost, inaccurate results, time-consuming and labor-intensive, etc., and achieve good repeatability, easy preservation, and cheap price

Active Publication Date: 2019-07-23
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The double-antibody sandwich method kit needs to use two different antibodies against different sites of ASGPR. The antibody preparation process is time-consuming and labor-intensive, and the preparation cost is relatively high, resulting in expensive kits and high detection costs for users; in addition, The detection limits of some existing ELISA kits are not suitable for the detection of sASGPR in human serum, which makes the detection of serum samples inconvenient and the results are not accurate enough

Method used

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  • Method for detecting human soluble asialoglycoprotein receptor (sASGPR)
  • Method for detecting human soluble asialoglycoprotein receptor (sASGPR)
  • Method for detecting human soluble asialoglycoprotein receptor (sASGPR)

Examples

Experimental program
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Effect test

Embodiment 1

[0039] Example 1 Preparation of ligand GSA

[0040] Add 20mg of human serum albumin to a clean reaction flask, then add 20mL 0.5M MES biological buffer (pH5.25) to fully dissolve human serum albumin, and then add 108.6mg galactosamine and 62mg EDC to fully dissolve it. After all the samples are completed, put the reaction flask in a 37°C oil bath and react for 16 hours. After the reaction is over, add 3.4mL1M CH to the reaction flask 3 COOH solution (pH 4.5) to terminate the reaction. After the reaction is terminated, the reaction solution is transferred to In the MLtra centrifugal filter, centrifuge at 5000×g for 20 minutes, discard the filtrate in the lower centrifuge tube, and add 10mM CH to the upper filter 3 The COOH solution (pH 7) was restored to the original volume of the sample, centrifuged again at 5000×g, and ultrafiltration was repeated several times to remove unreacted galactosamine and reduce the concentration of sodium acetate. The final GSA solution obtained aft...

Embodiment 2A

[0041] Example 2 Purification of ASGPR Standard

[0042] Take 150mL of the HepG2 cell supernatant collected in advance and centrifuge for 5min (3500×g) in a centrifuge. Take the centrifuged supernatant and add it to 50mL Concentrate in a MLtra centrifugal filter (3500×g), and finally concentrate to a final volume of 8 mL. Add 8 mL of lysis buffer to the concentrated HepG2 cell supernatant, mix well, and lyse at 4°C for 2-3 hours. After the lysis is completed, centrifuge at 20000×g for 30 minutes, transfer the supernatant to a new centrifuge tube, and add 800μL of 1M CaCl 2 , Incubate on ice for 30 min. Centrifuge again at 20000×g for 30 min, and discard the precipitate. The lactose sepharose bead column was pre-equilibrated with 50 mL washing buffer I, and the lactose sepharose beads were fully transferred to a centrifuge tube and combined with the centrifuged cell supernatant at 4°C overnight. The combined lactose sepharose beads were fully transferred to the column, and the ...

Embodiment 3

[0043] Example 3 Selection of primary antibody

[0044] Purchase two ASGPR1 mouse-derived primary antibodies, namely: ASGPR1 / 2(E-1) (Santa Cruz Biotechnology, sc-166633) and ASGPR1(A-5) (Santa Cruz Biotechnology, sc-393849), referred to as Mouse anti-human ASGPR-1 monoclonal antibody, mouse anti-human ASGPR-2 monoclonal antibody. Coat 2 96-well microtiter plates with 10μg / mL GSA, add 100μL GSA to each well, and coat at 4℃ for 24h. After coating, wash with PBST 5 times, 3min each time, and pat dry. Add 300 μL of 1% BSA to each well to seal the whole well, and seal it overnight at 4°C. After closing, wash with PBST 3 times, 3 min each time, and pat dry. Set ASGPR diluents as negative control wells, quality control wells, and 0.15μg / mL, 0.50μg / mL, 2.00μg / mL, 8.00μg / mL ASGPR standards as sample wells, and add the negative control and sample to the enzyme in turn In the standard plate, the sample is made into 4 replicate wells, and the sample volume per well is 100μL. After adding ...

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Abstract

The invention discloses a method for detecting a human soluble asialoglycoprotein receptor (sASGPR), and belongs to the field of immunological detection. The ELISA method for detecting the human sASGPR is economical, fast, accurate and high in practicability. According to the method, on the basis of a specific ligand of the ASGPR and specific identification of the ASGPR, a galactosylated serum albumin (GSA) is selected as the specific ligand of the ASGPR, the GSA is prepared from human serum albumin and has the advantages of being low in cost, easy to prepare, easy to preserve and the like, the detection limit of the method is applicable to detection of the sASGPR in a human serum sample, special and large instruments and equipment do not need to be used, and the method can be carried outin an ordinary laboratory, has the advantages of being high in specificity, good in stability, easy and convenient to operate, low in cost and the like, and provides certain reference value for clinical liver function assessment.

Description

Technical field [0001] The invention relates to a method for detecting human soluble asialoglycoprotein receptors, and belongs to the field of immunological detection. Background technique [0002] Liver disease has been an important and high-incidence disease worldwide for decades. Liver damage such as cirrhosis, hepatitis, and fatty liver is the only way for liver cancer. Liver damage is a serious threat to the health of people in my country and the world. And life. At present, the main detection methods for liver damage include histopathological diagnosis, serological diagnosis and imaging diagnosis. Among them, the main clinical serum markers for serological diagnosis include alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and serum bilirubin (BIL), but these indicators have low specificity The shortcomings of the liver injury will affect these indicators, other diseases can also cause abnormal changes in these indicators. Theref...

Claims

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Application Information

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IPC IPC(8): G01N33/68
CPCG01N33/68
Inventor 胡静尹健施奇敏
Owner JIANGNAN UNIV
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