UDK 613.2/.3:615.98:582.28:543.544.5.068.7 DESIGN OF ANTIGEN SYNTHESIS AND PREPARATION AND CHARACTERIZATION OF SPECIFIC AND EURYTOPIC ANTIBODIES AGAINST B-GROUP AFLATOXINS

The aim of this study was to prepare B-group aflatoxins(BGAFs) antibody with strong specificity and good eurytopicity. According to the molecular structure and active site of aflatoxin B1 (AFB1), the BGAFs artificial antigen AFB1-BSA was prepared by 6 methods such as oxime active ester(OAE)，methylation of ammonia(MOA)，mixed anhydride(MA)，semi acetal(SA)，epoxide(EP) and enol ether derivative(EED) and identified by UV and SDS-PAGE. Polyclonal antibodies against AFB1(AFB1 pAb) were prepared by immunizing New Zealand rabbits with AFB1-BSA, and the titers of AFB1 pAb was detected by indirect ELISA, the sensitivity of AFB1 pAb was analyzed by indirect competitive ELISA(icELISA) and the specificity and eurytopicity of AFB1 pAb was analyzed by cross-reactivity(CR) test. The results showed that AFB1-BSA was synthesized successfully and the best one was OAE method among 6 synthesis methods of BGAFs artificial antigen and its conjugation ratio of AFB1 to BSA was about 8.46∶1. The immune efficacy of OAE method was the best, its AFB1 pAb had high titers of 1∶（1.28×104） by indirect ELISA, a good sensitivity with the 50% inhibition concentration(IC50) of 10.32 μg/L to AFB1 by icELISA and a high CR to AFB2 of 75.21%, AFG1 of 44.13%, AFG2 of 14.72%, AFM1 of 16.36% and AFM2 of 1.44%, respectively. In this study, AFB1 pAbs with high titer, sensitivity, specificity and eurytopicity were prepared, which laid a matter and technical foundation for the establishment of BGAFs immunoassay.

immunoassay. In particular, immunoassay has become a technology because of its strong specificity, high sensitivity, simple operation, large-scale screening and on-site detection. Indispensable technical means, the key to establishing a BGAFs immunoassay method is to obtain excellent antibodies, and hapten design and antigen synthesis are the prerequisites for preparing excellent antibodies (Gefen T.et. al., 2015). There have been related reports on the research of BGAFs antigen synthesis methods at home and abroad (Mongkon, W. et. al., 2017W. et. al., , Xiao L.W, et. al., 2017, but there are no reports on the design of different hapten molecules, antigen synthesis and comparative analysis of antibody characteristics. In this study, AFB1 was used as the starting material for the reaction. Polyclonal antibodies (pAbs) were prepared through different AFB1 hapten molecular design and antigen synthesis methods, and their characteristics were analyzed to screen out the best hapten and antigen synthesis methods. It lays the foundation for the preparation of high-quality monoclonal antibodies of BGAFs with high sensitivity, broad recognition spectrum and strong specificity (Zhou, Y. et. al., 2007).
Aim The aim of this study was to prepare B-group aflatoxins (BGAFs) antibody with strong specificity and good eurytopicity.

Materials and Methods
Main reagents, solutions and experimental animals AFB1, AFB2, AFG1, AFG2 standard products, Singapore Pribolab product; Cationized bovine serum albumin (cBSA), goat anti-rabbit enzyme-labeled secondary antibody (GaRIgG-HRP), American Sigma product. The diluent used in the enzyme-linked immunosorbent assay (ELISA) is 0.01 mol·L -1 pH7.4 phosphate buffer solution (PBS); the washing solution is PBS containing 0.5 g·L -1 Tween -20 (PBST); the blocking solution is PBST containing 50 g·L -1 porcine serum; the coating solution is 0.1 mol·L -1 carbonate buffer solution (CBS) with pH 9.6. The experimental animals were 18 male New Zealand white rabbits at the age of 2 months and weighing 1±0.2 kg. They were provided by the Experimental Animal Center of Xinxiang Medical College. They were divided into 6 groups, each with 3 rabbits.
BGAFs artificial antigen synthesis design According to the active sites on the molecular structure of AFB1 (Figure 1), the following six methods are proposed to prepare artificial antigen AFB1-BSA (Table 1).
3 Aldehyde Schiff Aldehyde SA The condensation reaction of AFB1 under the action of H2SO4 produces AFB2a with active sites of aldehyde groups, whose aldehyde groups can form unstable Schiff bases with the amino groups of BSA. Through the reduction of NaBH4, the antigen AFB2a-BSA is synthesized [13,14] .

3、 4
Bifuran ring Oxidation Hydroxyl EP Using dichloromethane as solvent, the double bond of AFB1 bifuran ring is oxidized to form AFB1 epoxide, which reacts with the primary amine of BSA to form secondary amine, introduces a hydroxyl group on the epoxide, and couples with BSA in the form of monoamide Into AFB1-BSA [15,16] .
3、 4 Bifuran ring Glycolic acid carboxyl EED The molecular structure of AFB1 contains an active site bifuran ring, which can react with glycolic acid to generate AFB1 enol ether derivative (AFB1-GA) with active carboxyl group, which is used to couple the carboxyl group with BSA to synthesize AFB1-BSA [17] .
BGAFs artificial antigen identification UV Scan Dissolve AFB1 with methanol, prepare 1 mg·mL -1 AFB1 solution; use volume ratio (v/v) 4:6 methanol PBS solution to dissolve BSA and AFB1-BSA, prepare 1 mg·mL -1 BSA and AFB1-BSA solution; UV scan at a wavelength of 200 ~ 500 nm, through the calculation formula A = εCL (where A is the absorbance value, read by the instrument; ε is the molar extinction coefficient, which is a constant value; C is the solute concentration in the solution; L is Optical path, determined by the instru-ment), calculate the molecular binding ratio of AFB1 and BSA (Wang Y.N. et. al., 2014).

SDS-PAGE identification
The concentration of the concentrated gel and the separating gel are selected to be 5% and 12%, the voltage is 90 v and 60 v, the sample volume is 10 μL per well, and the protein content is 10 μg per well. The UV analyzer system software calculates AFB1 and BSA the molecular binding ratio.
Preparation of AFB1 pAb The artificial antigens synthesized by 6 different methods were used to immunize New Zealand white rabbits. Each antigen was used to immunize 1 group, a total of 6 groups, 3 rabbits in each group. The immunization dose is calculated according to the amount of protein BSA in AFB1-BSA, each is 100 μg, the volume is 1 mL, the back is injected subcutaneously at 4 to 6 points, a total of 5 immunizations, each interval is 3 to 4 weeks, after the fifth immunization for 2 weeks, blood was collected from the ear vein, the polyantiserum was separated by centrifugation, and the polyantiserum was purified by the saturated ammonium sulfate salting-out method to prepare AFB1 pAb (Ju RH et. al., 2015).
Specific identification With AFB1, AFB2, AFG1, and AFG2 as inhibitors, the IC50 of each inhibitor was determined by icELISA, and the percentage of the IC50 of AFB1 pAb to AFB1 and the IC50 of other inhibitors was used as the cross-reaction rate (CR%) , the calculation method is CR% = IC50 of AFB1 pAb to AFB1/ IC50×100 of AFB1 pAb to other inhibitors.

Results
GAFs artificial antigen identification results UV identification The results are shown in Figure 2. In the range of UV200～ 500 nm, the characteristic peak of BSA is at 278 nm, and the characteristic peak of AFB1 is at 363 nm. The artificial antigen AFB1-BSA is synthesized by 6 methods including OAE, MOA, MA, SA, EP, EED. Both contain the characteristic peaks of BSA and AFB1, indicating that the above 6 methods can synthesize artificial antigen AFB1-BSA. The calculated results of the molecular binding ratio of BSA to AFB1 (Liu, H.X. et. al., 2014) are shown in Table 2.  Note: Compared to the molecular weight of BSA and AFB1, BSA is 66.446, AFB1 is 312, BSA is much larger than AFB1, so the utilization rate of BSA is 100% when the utilization ratio is calculated.

SDS-PAGE identification
The results are shown in Figure 3. It can be seen that the bands of the 6 artificial antigens AFB1-BSA lag behind the bands of BSA, indicating that the molecular weight of AFB1-BSA is greater than that of BSA, and it can be determined that the synthesis of AFB1-BSA is successful.

Sensitivity analysis
The results are shown in Figure 5. It can be seen that the icELISA inhibition curve of 6 immunized rabbits has a good linear relationship. The OAE group has the best sensitivity, with an IC50 of 10.32 μg·kg -1 . The sensitivity of the other groups is inferior to that of the OAE group.  Specificity and broad-spectrum analysis The results are shown in Table 4. It can be seen that the antibodies prepared by the six methods can recognize AFB1 100%, and the OAE method has the best specificity and broadspectrum, with an IC50 of 10.32 μg·kg -1 and a CR with AFB2 of 86.46%; The CR with AFG1 and AFG2 were 44.13% and 14.72%, respectively. Antibodies prepared by other methods have good specificity and can recognize AFB1 100%, but their sensitivity and broad-spectrum are not as good as those prepared by OAE method. The results show that the best antigen synthesis method for preparing antibodies against BGAFs with high sensitivity, strong specificity and good broad spectrum is the OAE method.

Discussion and Conclusion
About the design of BGAFs antigen synthesis method The molecular weights of AFB1 and AFB2 in BGAFs are 312.27 and 314.29, respectively. They belong to small molecule haptens and have no immunogenicity. According to the theory of hapten-carrier effect, only by combining with large-molecule protein carriers to form artificial antigens can they be specific for haptens. Therefore, the design of antigen synthesis methods is very important (Zeng, H. et. al., 2014). Since the selection of different active sites and the introduction of different linking arm lengths will have a greater impact on the properties and structure of small molecules, which in turn will affect the quality of antibodies produced (Shi HY et. al., 2006). According to the molecular structure characteristics of BGAFs, this study selected the 1-position carbonyl group, 2-position active hydrogen, 3position hydroxyl group and aldehyde group, and the difuran ring between 3-position and 4-position as the active groups. Through different chemical reaction methods, respectively introduce available carboxyl, hydroxyl, aminomethyl and other active groups to realize the coupling with carrier protein to synthesize

Вісник Сумського національного аграрного університету
Серія «Ветеринарна медицина», випуск 4 (51), 2020 artificial antigens. About the synthetic route of BGAFs artificial antigen At present, the research on BGAFs artificial antigen synthesis method is still at the empirical level, and trial and error methods are mostly used. Although a variety of artificial antigen identification methods have been established, the immunogenicity of the artificial antigens prepared is ultimately through the effect of animal immunity. It was confirmed (Guo N. F. et. al., 2014). Based on a large number of relevant research literature, this article uses AFB1 as the starting material for the reaction, and uses 6 methods such as OAE method, MOA method, MA method, SA method, EP method and EED method to synthesize artificial antigens, and through UV, SDS-PAGE for antigen identification and animal immunization for antibody characteristics analysis, the most ideal antigen synthesis method for the preparation of BGAFs antibody was selected by OAE method. Its advantages are that the reaction system is easy to construct, the reaction conditions are mild, the operation steps are simple, and the product yield is high. However, in terms of the advanced nature of the technical route adopted in this research, the research and application of molecular simulation technology, computer-aided technology, etc. Needs to be improved (Morita, I. 2017).
Analysis on the immune effect of BGAFs artificial antigen.
The purpose of this research is to screen out BGAFs artificial antigen synthesis methods, and lay the material and technical foundation for the preparation of high-quality BGAFs antibodies with high sensitivity, strong specificity and broad recognition spectrum. This requires that in the design of BGAFs antigen synthesis, on the one hand, it is necessary to consider the specificity and sensitivity of the antibody to AFB1 to meet the detection technology requirements under the AFB1 limit standard; On the other hand, it is necessary to consider the sensitivity and broad-spectrum of the antibody to AFB2 to meet the technical requirements for detection under the BGAFs limit standard (Xie Hui et al. 2017) used MA method to synthesize AFB1-BSA, and screened hybridoma cell 3B9 to obtain AFB1 mab. The antibody specifically recognizes AFB1 with a sensitivity of 1.04 μg·kg -1 , CR of AFB2, AFG1, AFG2, and AFM1 are 2.2%, 33.9%, 1.8%, and 5.12%, respectively, which have no CR with AMF2 and poor broad-spectrum. Xiao Zhi et al. used SA method to synthesize AFB1-BSA, and screened hybridoma cell 3A12 to obtain AFB1 mab. The antibody specifically recognizes AFB1 with a sensitivity of 6.1 μg·kg -1 , and is compatible with CR of AFB2, AFG1, AFG2, and AMF1. They are 7.8%, 20.2%, 0.6%, and 3.68%, respectively. It has no CR with AFM2, and it also has the problem of poor broad-spectrum.

Conclusion
The results of 6 different antigen synthesis methods and the characteristics of the antibodies produced showed that the OAE method was the best, the produced AFB1 pAb antibody titer was high, and the indirect ELISA titer reached 1: (1.28×10 4 ); the sensitivity to AFB1 was good, IC50 is 10.32 μg·kg -1 ; it has strong specificity and can recognize AFB1 100%. The CR with AFB2, AFG1, AFG2, AMF1, and AFM2 are 75.21%, 44.13%, 14.72%, 16.36% and 1.44%, respectively. The other five methods designed by this research have certain defects in varying degrees. Therefore, the author recommends that they should not be used except for research work.
In this study, based on the molecular structure characteristics of AFB1 and the existing active sites, six BGAFs antigen synthesis methods were designed, through UV, SDS-PAGE identification and analysis of the characteristics of AFB1 pAb produced by immunized animals, a high-titer, sensitive, specific, and broad-spectrum AFB1 pAb was obtained. It shows that antigen synthesis design is the prerequisite for the preparation of high-quality antibodies, and the OAE method is an effective way to realize the preparation of high-quality antibodies for BGAFs, laying a material and technical foundation for the establishment of BGAFs immunoassay methods.