WANG Yu-Ting, WANG Xiao-Hui, ZHAO Lin, LI Yue-Xin, WANG Di, GU Shao-Peng, HE Jin-Xin
Soybean glycinin (SG) is one of the major anti-nutritional factors in soybeans and also an important allergen triggering allergic reactions. The problem of soybean allergy has become increasingly prominent, posing an unignorable public health risk. In animal husbandry production, SG can induce intestinal allergic reactions in weaned piglets, leading to poor growth and thus impairing piglet production performance. Nanobodies (Nbs) are the single variable domain fragments (variable domain of heavy chain of heavy-chain antibody, VHH) derived from the enzymatic hydrolysis of heavy-chain antibodies naturally produced in Camelidae (e.g., camels and alpacas). They exhibit the characteristics of low cost, high yield, excellent stability, high specificity, low immunogenicity and ease of genetic modification. In this study, alpacas were immunized five times with SG as the immunogen. Total RNA was extracted from the lymphocytes of immunized alpacas and reverse-transcribed into cDNA, followed by two rounds of PCR to amplify the Nb gene sequences. The amplified products were digested with restriction enzymes and ligated to the phage display vector pComb3XSS. The ligation products were concentrated, recovered and electrotransformed into ER2738 competent cells, thus successfully constructing an initial Nb phage library against SG. The results showed that the library capacity reached 3.6×109 CFU/mL with a 100% Nb gene insertion rate, and the titer increased to 5.75×1012 PFU/mL after rescue with the helper phage M13KO7. Significant enrichment of the library was achieved via four consecutive rounds of gradient panning with reduced coating concentration and increased washing times. Phage-enzyme linked immunosorbent assay (Phage-ELISA) identified 20 positive clones, which were confirmed to be the same specific Nb by sequencing analysis. This Nb was successfully expressed in a soluble form in Escherichia coli BL21(DE3), and Western blotting (WB) and enzyme linked immunosorbent assay (ELISA) verified its strong specific recognition ability for SG. A double-antibody sandwich ELISA method for SG detection was established using rabbit immunoglobulin G (IgG) as the capture antibody and the prepared Nb as the detection antibody. After optimizing the reaction conditions, a standard curve was plotted using Origin software, and logistic regression analysis yielded a half effective concentration (EC50) of 62.21 ng/mL. This established method was applied to detect 10 soybean feed samples, and the results were highly consistent with those obtained by high performance liquid chromatography (HPLC), demonstrating the high accuracy and practicability of the developed double-antibody sandwich ELISA method. This study provides a crucial material basis for further investigating the residual hazards of SG.
