Immunochromatography test paper for detecting human PGI protein and preparation method thereof
A technology of fluorescent immunochromatography and test paper, which is applied in the field of peptide chemistry and immunology, and achieves the effects of high specificity, wide detection range and good sensitivity
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[0055] The preparation method of the PGI epitope peptide of the present invention can be a chemical synthesis method: the antigen epitope peptide is synthesized by a solid-phase method using an American ABI431A type polypeptide automatic synthesizer. The molecular weights of the antigenic epitope peptides (1) and (2) of the present invention are 1657.24 and 1616.93 respectively, which can be determined by mass spectrometry, and the synthesized antigenic epitope peptide sequences are identified by polypeptide sequence determination. The purity of the peptides was evaluated by thin-layer chromatography and high-performance liquid chromatography, and the concentration of the epitope peptide was determined.
[0056] 2. PGI antigen
[0057] The present invention also provides a PGI antigen prepared by coupling one of the human PGI epitope peptides (1) and (2) of the present invention with a carrier protein. Specifically, the present invention provides PGI antigens (1) and (2), whe...
Embodiment 1
[0108] Example 1: Preparation of PGI epitope peptides (1) and (2).
[0109] The preparation method uses chemical synthesis method: the PGI antigen epitope peptides (1) and (2) are synthesized respectively by solid-phase method using the American ABI431A automatic peptide synthesizer. The purity of the epitope peptide was assessed by high performance liquid chromatography, and the concentration of the peptide was determined. The molecular weights of the antigenic epitope peptides (1) and (2) of the present invention are 1657.24 and 1616.93 respectively, which are determined by mass spectrometry, and the synthesized polypeptide sequences are identified by polypeptide sequence determination.
[0110] 1. Synthesis of PGI epitope peptides (1) and (2)
[0111] The above peptides were synthesized by solid-phase method. The main idea of solid-phase peptide synthesis is: first connect the carboxyl group of the carboxyl-terminal amino acid of the peptide chain to be synthesized with...
Embodiment 2
[0198] Example 2: The PGI antigen epitope peptides (1) and (2) obtained in Example 1 are linked to carrier protein to prepare PGI antigens (1) and (2), respectively, and the obtained antigens (1) and (2) are used to Animals are immunized to prepare specific monoclonal and polyclonal antibodies using the antigen (1), and specific monoclonal and polyclonal antibodies are prepared using the antigen (2).
[0199]1. Antigen preparation: PGI peptides (1) and (2) were respectively linked with carrier protein KLH (keyhole limpet hemocyanin) by BDB (Bis-diazotizedbenzidine dichloride) method to prepare PGI antigens (1) and (2) .
[0200] Take 10.0mg of PGI peptide (1) or (2), dissolve it with 1ml 0.1M PBS buffer (pH7.4); dissolve KLH10mg with 20ml of 0.2M borate buffer (pH9.0); then mix the two Mix, cool to 0°C, take BDBCl 2 110 μL, reacted at room temperature for 1.5 h, dialyzed overnight, then aliquoted, and stored at -20 °C.
[0201] In the present embodiment, the formula of PBS ...
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