Monoclonal antibody for resisting human NT-proBNP polypeptide
A monoclonal antibody, nt-probnp technology, applied in the direction of anti-animal/human immunoglobulin, anti-hormonal immunoglobulin, etc., can solve problems such as poor repeatability, poor clinical relevance of characterization, and complicated sample pretreatment methods, and achieve Simple preparation process, high antibody affinity, good protein surface activity and antigenic effect
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Embodiment 1
[0023] 1). Select NT-proBNP epitope polypeptide fragments aa1 and aa2:
[0024] DNAstar (Genetool) software was used to analyze the protein structure and antigenic epitope of the full-length amino acid sequence of human NT-proBNP in the early stage. According to the optimal conditions, two polypeptide fragments aa1 and aa2 were selected. The aa1 is SEQ ID NO.1. The sequence shown, the aa2 is the sequence shown in SEQ ID NO.2. The sequence SEQ ID NO.1 and the sequence SEQ ID NO.2 are as follows:
[0025] Sequence SEQ ID NO.1: ETSGLQEQRNHL
[0026] Sequence SEQ ID NO.2: IRGHRKMVLYTLRAPR
[0027] 2). Preparation of NT-proBNP epitope polypeptide fragments aa1 and aa2:
[0028] Weigh 100 mg of HMP resin and place it in the reaction chamber of an automatic peptide synthesizer, and the synthesizer automatically couples specific amino acids in different sequences, and the coupling rate is greater than 99%. The reaction steps are as follows:
[0029] ①Amino acid activation (HOBt / D...
Embodiment 2
[0058] 1). Select NT-proBNP epitope polypeptide fragments aa1 and aa2, the method is the same as in Example 1;
[0059] 2). Preparation of NT-proBNP epitope polypeptide fragments aa1 and aa2:
[0060] Weigh 100 mg of HMP resin and place it in the reaction chamber of an automatic peptide synthesizer, and the synthesizer automatically couples specific amino acids in different sequences, and the coupling rate is greater than 99%. The reaction steps are as follows:
[0061] ①Amino acid activation (HOBt / DCC method): take 0.15mol Fmoc-Ile and 0.15mol HOBt, dissolve with appropriate amount of DMF; take another 0.15molDCC, add slowly under stirring, stir and react at room temperature for 50 minutes, and obtain The activated amino acid solution is set aside;
[0062] ② Linking amino acids to the resin: under the action of a coupling reagent, the activated amino acid solution and the resin carrier (Wang resin) are subjected to an esterification reaction, and the reaction time is 250 m...
Embodiment 3
[0087] 1). Select NT-proBNP epitope polypeptide fragments aa1 and aa2, the method is the same as in Example 1;
[0088] 2). Prepare NT-proBNP epitope polypeptide fragments aa1 and aa2;
[0089] Weigh 100 mg of HMP resin and place it in the reaction chamber of an automatic peptide synthesizer, and the synthesizer automatically couples specific amino acids in different sequences, and the coupling rate is greater than 99%. The reaction steps are as follows:
[0090] ①Activation of amino acid (HOBt / DCC method): take 0.15mol Fmoc-Gln and 0.15mol HOBt, dissolve with appropriate amount of DMF; take another 0.15molDCC, add slowly under stirring, stir and react at room temperature for 40 minutes, and obtain after filtration The activated amino acid solution is set aside;
[0091] ② Linking amino acids to the resin: under the action of a coupling reagent, the activated amino acid solution and the resin carrier (Wang resin) are subjected to an esterification reaction, and the reaction ...
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