A kind of paraquat hapten ph-a, artificial antigen, antibody and its preparation method and application
A PH-A, artificial antigen technology, applied in the biological field, achieves the effects of simple preparation method, improved immunogenicity and strong specificity
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[0042] (1) Preparation of paraquat hapten PH-A
[0043] Since paraquat is a small molecule and has no immunogenicity, it cannot stimulate alpaca to produce an immune response, thereby producing antibodies. Therefore, it is necessary to couple the paraquat hapten to a carrier protein through protein linking technology to obtain immunogenicity. The active groups commonly used in protein coupling technology are amino, carboxyl, hydroxyl, sulfhydryl, etc. In view of the absence of these active groups in the molecular structural formula and it is not easy to connect arms; therefore, the use of 4'-(4-methylphenyl) )-benzaldehyde and carboxymethylhydroxylamine hemihydrochloride undergo oximation condensation derivatization reaction to obtain a hapten, which is denoted as hapten PH-A. The preparation method of paraquat hapten PH-A is as follows:
[0044] 100 mg of 4'-(4-methylphenyl)-benzaldehyde, 74 mg of carboxymethylhydroxylamine hemihydrochloride, in 5-10 mL of ethanol solution, ...
Embodiment 2
[0090] Example 2 Affinity panning and identification of Nanobodies
[0091] (1) Affinity panning of nanobodies
[0092] First, using PH-A-OVA as the coating source, the PH-A-OVA coating source was diluted with the coating solution to a final concentration of 10 μg / mL, and coated overnight at 37°C. The next day, after washing twice with PBST (0.01M PBS, 0.06% Tween-20 (v / v)), 1% isinglass was added to block for 2 h at 37°C. Drain the liquid in the well and pat dry, add 100 μL of phage library to each well (library titer is about 10 13 cfu / mL) and incubated at 37°C for 1 h. Unbound phages were discarded, washed 5 times with PBST (0.01M PBS, 0.05% Tween-20 (v / v)), 15 times with PBS (pH 7.0), and added Gly-HCl (0.2M, pH 2.2) Elute at 37°C for 10 min and immediately neutralize with 10 μL Tris-HCl (1 M, pH 9.0). 10 μL of the eluted phage was taken to determine the titer, and the rest was used to infect 4 mL of E.coil TG1 strain grown to log phase for amplification. On the third...
Embodiment 3
[0102] Example 3 Sequencing of the Encoding Gene of Nanobody Nb2-23 and Determination of Its Amino Acid Sequence
[0103] 1. Experimental method
[0104] The strain of Nanobody Nb2-23 obtained through indirect competitive ELISA identification was sent to a sequencing company for sequencing to obtain the nucleotide sequence of Nanobody Nb2-23; according to the DNA sequencing results and codon table, the nanobody Nb2-23 was obtained. amino acid sequence.
[0105] 2. Experimental results
[0106] The amino acid sequence of the VHH of the Nanobody Nb2-23 is shown in SEQ ID NO.1, and the nucleotide sequence of the gene encoding the Nanobody Nb2-23 is shown in SEQ ID NO.2.
[0107] The amino acid numbering and structural domain diagram of Nanobody Nb2-23 are as follows figure 2 As shown, it can be seen that the Nanobody Nb2-23 includes 4 framework regions (Framework region, FR) and 3 complementarity-determining regions (Complementarity-determining region, CDR); the framework reg...
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