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Monoclonal antibody for detecting estrogen receptor alpha as well as preparation method and application thereof

A monoclonal antibody and estrogen receptor technology, applied in the field of immunodetection, can solve the problems of insufficient specificity and affinity of antibody reagents, difference in measurement results, false positives, etc., and achieve high accuracy, simple method and strong specificity. Effect

Active Publication Date: 2022-08-09
图凌(杭州)生物医药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, due to the lack of specificity and affinity of antibody reagents, there are differences in the results of ER-α and PR tests between different clinical testing institutions and different testing reagents, and even a certain proportion of false positives and false negatives in the test results result

Method used

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  • Monoclonal antibody for detecting estrogen receptor alpha as well as preparation method and application thereof
  • Monoclonal antibody for detecting estrogen receptor alpha as well as preparation method and application thereof
  • Monoclonal antibody for detecting estrogen receptor alpha as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1 Preparation of anti-ER-α monoclonal antibody and anti-PR monoclonal antibody

[0063] This example utilizes the following steps to prepare anti-ER-α and anti-PR rabbit monoclonal antibodies:

[0064] (1) Synthesize or obtain a polypeptide comprising the ER-α amino acid sequences aa560 to aa595, as shown below (SEQ ID NO: 1):

[0065] VEETDQSHLATAGSTSSHSLQKYYITGEAEGFPATV

[0066] Synthesize or obtain recombinant protein fragments comprising PR amino acid sequences aa750 to aa933, the amino acid sequence sequences are shown below (SEQ ID NO: 2):

[0067] LIQYSWMSLMVFGLGWRSYKHVSGQMLYFAPDLILNEQRMKESSFYSLCLTMWQIPQEFVKLQVSQEEFLCMKVLLLLNTIPLEGLRSQTQEMRSSYIRELIKAIGLRQKGVVSSSQRFYQLTKLLDNLHDLVKQLHLYCLNTFIQSRALSVEFPEMMSEVIAAQLPKILAGMVKPLLFHKK

[0068] (2) The above-mentioned ER-α polypeptide was combined with KLH (Thermo Scientific TM , Cat. No. 77649) was cross-linked and diluted to 1 mg / mL with PBS buffer. The recombinant protein fragment of the above PR was direct...

Embodiment 2

[0077] Example 2 Detection of anti-ER-α monoclonal antibody and anti-PR monoclonal antibody titers

[0078] The titer of the anti-ER-α rabbit monoclonal antibody and the anti-PR rabbit monoclonal antibody prepared in Example 1 was detected using an indirect enzyme-linked immunosorbent assay (iELISA) method, and the half effective concentration of each antibody was calculated according to the standard curve ( EC50).

[0079]96-well microtiter plates were coated with ER-α immunogenic polypeptide and PR recombinant protein fragment respectively, and the coating concentration was 1 μg / ml. The initial dilution concentration of the 4 primary antibody samples is 1000ng / mL, and then on the dilution plate, 5% nonfat milk powder is used as the diluent to make a 4-fold vertical dilution, a total of 7 gradients, and the 8th well is added with the diluent as a negative control. hole. The test results are shown in Table 1. figure 1 and figure 2 shown.

[0080] Table 1 iELISA detection...

Embodiment 3

[0106] Example 3 Detection of Affinity of Anti-ER-α Monoclonal Antibody and Anti-PR Monoclonal Antibody

[0107] Use the competitive ELISA method to detect the antibody affinity of anti-ER-α monoclonal antibody B and anti-PR monoclonal antibody B, and calculate the molar concentration corresponding to the half effective inhibitory concentration (50% of Effective Concentration, IC50) of the antibody according to the affinity standard curve, is the affinity constant.

[0108] Antigen-antibody binding is a reversible reaction: A+B AB (A represents antibody, B represents antigen, and AB represents antigen-antibody conjugate). When the reaction reaches equilibrium, the dissociation constant Kd=[A][B] / [AB], [AB] represents the concentration of the antigen-antibody conjugate in the reaction equilibrium, [A] represents the concentration of free antibody in the reaction equilibrium, [B] ] represents the concentration of free antigen at reaction equilibrium. When the amount of antibod...

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Abstract

The invention discloses a monoclonal antibody for detecting an estrogen receptor alpha as well as a preparation method and application of the monoclonal antibody, and belongs to the technical field of immunodetection. The monoclonal antibody for detecting the estrogen receptor alpha is obtained by immunizing an experimental rabbit with immunogen polypeptide as shown in SEQ ID NO: 1, and CDR-H1, CDR-H2 and CDR-H3 sequences of a heavy chain variable region and CDR-L1, CDR-L2 and CDR-L3 sequences of a light chain variable region of the monoclonal antibody are respectively as shown in SEQ ID NO: 5-10. When the monoclonal antibody for detecting the estrogen receptor alpha is used for detecting the estrogen receptor alpha, the specificity is high, the accuracy is high, and the monoclonal antibody has great clinical application value for clinical prediction, diagnosis, medication guidance and prognosis of breast cancer.

Description

technical field [0001] The invention belongs to the technical field of immunodetection, and in particular, relates to a monoclonal antibody for detecting estrogen receptor alpha and a preparation method and application thereof. Background technique [0002] Breast cancer accounts for 23% of malignant tumors, which is a common malignant tumor. The diagnosis, treatment and prognosis of breast cancer are closely related to the histopathological staging of breast cancer and the expression and degree of estrogen receptor-α (ER-α) and progesterone receptor (PR). It is known that PR can be induced by ER-α and play an important role in the regulation of ER-α protein, therefore, PR is often used as an indicator of ER-α function. The expression of ER-α and PR is related to the effect of endocrine therapy in patients. At present, the detection of ER-α and PR is widely used in the prognosis judgment of breast cancer patients and the guidance of medication. Therefore, the accurate dete...

Claims

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Application Information

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IPC IPC(8): C07K16/28G01N33/577G01N33/574
CPCC07K16/2869G01N33/577G01N33/57415G01N33/57484C07K2317/565C07K2317/56C07K2317/92C07K2317/76G01N2333/723
Inventor 胡旻章月凯陶娜娜
Owner 图凌(杭州)生物医药有限公司
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