Fluorescence immunochromatography test paper for detecting human Lp-PLA2 proteins and preparation method of fluorescence immunochromatography test paper
A fluorescence immunochromatography, lp-pla2 technology, applied in biological testing, measuring devices, analytical materials, etc., to achieve the effects of optimized preparation conditions, wide detection range, and improved fluorescence signal-to-background ratio
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Embodiment 1
[0111] Example 1: Preparation of Lp-PLA2 epitope peptides (1) and (2).
[0112]The preparation method uses chemical synthesis method: Lp-PLA2 antigenic 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 1690.09 and 1623.92 respectively, which are determined by mass spectrometry, and the synthesized polypeptide sequences are identified by polypeptide sequence determination.
[0113] 1. Synthesis of Lp-PLA2 epitope peptides (1) and (2)
[0114] 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 synthes...
Embodiment 2
[0202] Embodiment 2: the Lp-PLA2 antigen epitope peptide (1) and (2) obtained in Example 1 are linked with carrier protein to prepare Lp-PLA2 antigen (1) and (2), utilize gained antigen (1) and (2) Animals are immunized separately to prepare specific monoclonal antibodies and polyclonal antibodies using the antigen (1), and specific monoclonal antibodies and polyclonal antibodies are prepared using the antigen (2).
[0203] 1. Antigen preparation: Lp-PLA2 peptides (1) and (2) were respectively linked with carrier protein KLH (keyhole limpet hemocyanin) by BDB (Bis-diazotizedbenzidine dichloride) method to prepare Lp-PLA2 antigen (1) and (2).
[0204] Take 10.0mg of Lp-PLA2 peptide (1) or (2), dissolve it with 1ml 0.1M PBS buffer (pH7.4); dissolve 10mg of KLH with 20ml of 0.2M borate buffer (pH9.0); then Mix the two, 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.
[0205] In the present embod...
Embodiment 3
[0219] Example 3: Specific identification of human Lp-PLA2 monoclonal antibodies (1) and (2)
[0220] Detection was performed by ELISA. Human Lp-PLA2 protein, fibrinogen, and C-reactive protein (all purchased from Shanghai Lianshuo Company) were used as detection antigens to coat ELISA plates, and the prepared Lp-PLA2 monoclonal antibodies (1) and (2) For the specific reaction with the human Lp-PLA2 protein, normal BALB / c mouse serum was used as negative control, and PBS solution was used as blank control.
[0221] Result: Lp-PLA2 monoclonal antibody (1) and (2) only react positively (P / N>2.1) with Lp-PLA2 respectively, and are negative with fibrinogen, C-reactive protein reaction, illustrate that the present invention Lp-PLA2 monoclonal antibodies (1) and (2) have specificity respectively.
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