Nanobody mimicking citrinin antigen and its application
A nano-antibody and citrinin technology, applied in the biological field, can solve the problems of inspectors' health and environmental threats, and achieve the effect of low cost and high application value
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Embodiment 1
[0022] Example 1. Preparation of anti-citinillin monoclonal antibody
[0023] (1) Preparation of immune antigen
[0024] Citrinin is a small molecular hapten which is not immunogenic, but it can be immunogenic by coupling with a large molecular carrier protein. According to the structural analysis of citrinin, it has three groups of hydroxyl, carboxyl and carbonyl, all of which can be coupled with proteins by appropriate chemical methods. However, the carboxyl group and hydroxyl group are important groups in its antigenic determinant. If they are used as linking groups to connect with proteins, the antigenicity of citrinin will change, and the body cannot be induced to produce antibodies against citrinin molecules. Therefore, the formaldehyde addition method is adopted to pass through the C on the citrinin molecule. 1 The active hydrogen at the position was used to prepare the artificial antigen of citrinin. The specific steps are as follows: dissolve 0.5 mg citrinin in 150...
Embodiment 2
[0029] Example 2. Construction and identification of natural single domain heavy chain antibody phage display library
[0030] (1) Construction of natural single domain heavy chain antibody phage display library
[0031] Total RNA was extracted from two healthy non-immunized alpaca leukocytes. The cDNA was obtained by reverse transcription with Oligo dT, and the gene encoding the variable region of the heavy chain antibody was obtained by semi-nested PCR. The phagemid vector pHEN1 and the variable region encoding gene amplified by PCR were respectively used Sfi I. not After I double enzyme digestion, T4 DNA ligase was used to ligate overnight, and the ligated product was electrotransformed into Escherichia coli TG1, and 10 μL of the transformed bacterial solution was taken to be diluted to determine the library capacity. The rest of the bacterial solution was spread on a 2×YT culture plate containing 100 μg / mL. All colonies were scraped off with 2×YT medium containing ...
Embodiment 3
[0034] Example 3. Affinity panning and identification of mimetic citrinin antigen nanobodies
[0035] (1) Affinity panning of nanobodies
[0036] Anti-CIT monoclonal antibody 4G6 was diluted with 10 mM PBS (pH 7.4), coated on a microtiter plate at a final concentration of 100 μg / mL, and incubated overnight at 4°C. The next day, after washing 10 times with PBST (PBS containing 0.1% Tween-20 (v / v) ), add 300 μl of blocking solution (3% BSA-PBS) and incubate at 4°C for 1 hour. After 1 hour, discard the blocking solution, wash 5 times with PBST, and add 100 μl of the prepared phage display antibody library (dilute the phage with PBS to about 1.0×10 11 c.f.u), incubate the reaction at 37°C for 1 hour. Unbound phages were discarded, washed 10 times with PBST, bound phages were eluted with 0.2 M Glycine-HCl (pH 2.2), and immediately neutralized with 15 μl 1 M Tris-HCl (pH 9.1). Take 10 μl of the eluted phage to determine the titer, and the rest is used to infect 20 mL of E. col...
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