Anti-human SAA (Serum Amyloid A) monoclonal antibody and preparation method and application thereof

A monoclonal antibody, antigen epitope technology, applied in biochemical equipment and methods, chemical instruments and methods, microorganism-based methods, etc., can solve problems such as no other reports, and achieve the effect of high affinity and specificity

Active Publication Date: 2019-03-22
DIASYS DIAGNOSTIC SYST SHANGHAI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are no other reports on the detection metho...

Method used

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  • Anti-human SAA (Serum Amyloid A) monoclonal antibody and preparation method and application thereof
  • Anti-human SAA (Serum Amyloid A) monoclonal antibody and preparation method and application thereof
  • Anti-human SAA (Serum Amyloid A) monoclonal antibody and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Preparation and purification of SAA monoclonal antibody 03-7-3

[0034] 1. Material: His-SAA fusion protein, 8-week-old female BALB / c mice

[0035] 2. Methods and results

[0036] 2.1 Antigen preparation

[0037] 2.1.1 Construction of human SAA recombinant protein expression plasmid

[0038] The gene sequence of SAA (Gene ID: 6288) was searched in GenBank, and polymerase chain reaction (PCR) primers were designed according to the sequence. Human cDNA was used as a template to amplify the DNA fragment of the human SAA gene. The PCR product was detected by 1.0% agarose gel electrophoresis and the corresponding fragment was recovered with a gel recovery kit. The PCR amplified SAA gene fragment product was ligated into the expression vector pET28a( The His tag contained in the vector is used for purification), transformed into the TaKaRaDH5α host strain, and a single clone is picked for plasmid extraction and sequencing verification. The sequencing result is consistent w...

Embodiment 2

[0055] Example 2: Determination of the amino acid sequence of the variable region of SAA monoclonal antibody 03-7-3

[0056] 1. Material: Trizol (Invitrogen), the primers are synthesized by Shenggong Bioengineering Company, and the reverse transcription and PCR reagents are purchased from

[0057] TaKaRa company.

[0058] 2. Methods and results:

[0059] 2.1 Total RNA extraction and first strand synthesis of cDNA

[0060] The hybridoma cells in the logarithmic growth phase were collected, and total RNA was extracted according to the operating procedure of Trizol. Use spectrophotometer and agarose gel electrophoresis to qualitatively and quantitatively identify total RNA.

[0061] According to PrimeScript of TaKaRa TM II Description of 1st Strand cDNA Synthesis Kit Synthesize cDNA.

[0062] 2.2 SAA monoclonal antibody 03-7-3 heavy chain variable region (VH) and light chain variable region (VL) gene fragment amplification and sequencing

[0063] According to TaKaRa company Taq enzyme inst...

Embodiment 3

[0067] Example 3: SAA-ApoA1-HDL complex detection method established based on SAA monoclonal antibody 03-7-3 and its clinical application evaluation in the risk and diagnostic value of liver cirrhosis

[0068] 1. Material:

[0069] SAA monoclonal antibody 03-7-3; serum samples from healthy people on physical examination (normal liver function and two pairs and a half negative for hepatitis B) and patients with liver cirrhosis.

[0070] 2. Methods and results:

[0071] 2.1 Collection of clinical samples and related clinical information:

[0072] Collect 12 serum samples of healthy control group and 12 serum samples of liver cirrhosis group. The healthy control group is a healthy population with physical examination. The liver function indexes (alanine aminotransferase, aspartate aminotransferase, total bilirubin and direct indirect bilirubin, etc.) are all within the normal physiological value range, and hepatitis B is two and a half pairs It was negative, and there was no history of i...

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Abstract

The invention discloses an SAA monoclonal antibody 03-7-3, the heavy chain of the SAA monoclonal antibody 03-7-3 is IgG1, the light chain is k, and the amino acid sequences of the variable regions ofthe heavy chain and the light chain are respectively shown as SEQ ID NO. 3 and SEQ ID NO. 4. The SAA monoclonal antibody 03-7-3 disclosed by the invention can specifically identify disease-related SAA-ApoA1-HDL complex and SAA proteins, and on the basis of the SAA monoclonal antibody 03-7-3, an assay method for the SAA-ApoA1-HDL complex and the SAA proteins is established. The SAA-ApoA1-HDL complex is significantly associated with liver cirrhosis, and is a potential risk factor for the liver cirrhosis; and the concentration of the SAA proteins is significantly associated with the liver cirrhosis due to hepatitis B and liver cancer, and is a potential risk factor for the liver cirrhosis due to hepatitis B and the liver cancer. Therefore, the SAA monoclonal antibody 03-7-3 disclosed by the invention has important clinical value for the prediction of the potential risk of the liver cirrhosis and the liver cancer and the development of diagnostic markers for the diseases.

Description

Technical field [0001] The invention belongs to the field of biological detection, and specifically relates to an anti-human serum amyloid A (Serum Amyloid A, SAA) murine monoclonal antibody 03-7-3 and a hybridoma cell line secreting the monoclonal antibody, and uses the monoclonal antibody The antibody is based on the clinical detection of SAA-ApoA1-HDL complex (hereinafter referred to as SAA / ApoA1) and the detection method of SAA protein in human peripheral blood to predict the potential risk of liver cirrhosis and or liver cancer and evaluate its clinical diagnostic value . Background technique [0002] Cirrhosis (Cirrhosis) is a common clinical chronic progressive liver disease, diffuse liver damage formed by one or more causes of long-term or repeated action. Liver cirrhosis is a pathological process of abnormal proliferation of fibrous connective tissue in the liver when liver cells undergo necrosis and inflammation. Excessive fibrosis makes the liver atrophy and hardens, ...

Claims

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Application Information

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IPC IPC(8): C07K16/18G01N33/68G01N33/574G01N33/577C12N5/20C12R1/91
CPCC07K16/18C07K2317/515G01N33/57438G01N33/577G01N33/68
Inventor 张艳王荣芳晏鹤张杰钱震斌
Owner DIASYS DIAGNOSTIC SYST SHANGHAI
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