Doses of Immunogen Contribute to Specificity Spectrums of Antibodies against Aflatoxin

Li, Peiwu; Wu, Jing; Zhang, Li; Fan, Zhiyong; Yu, Tingting; Jiang, Feng; Tang, Xiaoqian; Zhang, Zhaowei; Zhang, Wen; Zhang, Qi

doi:10.3390/toxins9050172

Cited by 9 publications

(7 citation statements)

References 23 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Taking aflatoxin B1 as the research object, the effect of immunogen dose on antibody specificity and class broad specificity was discussed. e results showed that low-dose immunogen can improve the specificity of the antibody, and high-dose immunogen can increase the broad specificity of the antibody, and the research results provided reference for the development of new antibodies [54]. However, whether the results of this study are applicable to the preparation of antibodies to ZEN and other small molecule compounds remains to be further explored in the future.…”

Section: Synthesis Methods Of Zen Immunogen and Characteristicsmentioning

confidence: 78%

Synthesis of Zearalenone Immunogen and Comparative Analysis of Antibody Characteristics

Wang

Zhang

et al. 2021

International Journal of Analytical Chemistry

View full text Add to dashboard Cite

Background. This study aimed to explore the zearalenone (ZEN) immunogen synthesis method, immunogenicity, and antibody characteristics and to lay a foundation for the establishment of immunoassay methods for ZEN single residue and ZEN and its analogs total residue. Methods. Based on the molecular structure and active sites of ZEN, oxime active ester (OAE), condensation mixed anhydride (CMA), formaldehyde (FA), and 1,4-butanediol diglycidyl ether method (BDE) were designed and used for immunogen (ZEN-BSA) synthesis. The immunogens were identified by infrared (IR) and ultraviolet (UV) spectra and gel electrophoresis (SDS-PAGE) and were then used to immunize Balb/c mice to prepare ZEN polyclonal antibody (ZEN pAb). The titers and sensitivity of the ZEN pAb were determined by indirect noncompetitive ELISA (inELISA) and indirect competitive ELISA (icELISA), respectively, and its specificity was assessed by the cross-reaction test (CR). Results. ZEN-BSA was successfully synthesized, and the molecular binding ratios of ZEN to BSA were 17.2 : 1 (OAE), 14.6 : 1 (CMA), 9.7 : 1 (FA), and 8.3 : 1 (BDE), respectively. The highest inELISA titers of ZEN pAb of each group were 1 : (6.4 × 103) (OAE), 1 : (3.2 × 103) (CMA), 1 : (1.6 × 103) (FA), and 1 : (1.6 × 103) (BDE), respectively. The 50% inhibition concentrations (IC50) for ZEN by icELISA of each group were 11.67 μg/L (OAE), 16.29 μg/L (CMA), 20.92 μg/L (FA) and 24.36 μg/L (BDE), respectively. ZEN pAb from the mice immunized with ZEN-BSA (OAE) and ZEN-BSA (CMA) had class broad specificity to ZEN and its analogs. The CRs of ZEN pAb with α-ZAL, β-ZAL, α-ZOL, β-ZOL, and ZON were 36.53%, 16.98%, 64.33%, 20.16%, and 10.66%, respectively. ZEN pAb from the mice immunized with ZEN-BSA (FA) and ZEN-BSA (BDE) had high specificity for ZEN. The CRs of ZEN pAb with its analogs were all less than 1.0%. Conclusion. This study demonstrated that the preparation of the class broad-specificity antibodies of ZEN and its analogs can be achieved by immunizing animals with the immunogen ZEN-BSA prepared by the OAE method, while the preparation of highly specific antibodies can be achieved by immunizing animals with the immunogen ZEN-BSA prepared by the FA method. These findings lay the material and technical foundation for immunoassay of ZEN single residue and ZEN and its analogs total residue.

show abstract

Section: Synthesis Methods Of Zen Immunogen and Characteristicsmentioning

confidence: 78%

Synthesis of Zearalenone Immunogen and Comparative Analysis of Antibody Characteristics

Wang

Zhang

et al. 2021

International Journal of Analytical Chemistry

View full text Add to dashboard Cite

show abstract

“…Five six-week-old female Balb/c mice were immunized using a high ZEN-BSA conjugate dose at long intervals with multiple-site subcutaneous injections [22,43]. For the first immunization, each mouse was subcutaneously injected on four sites (on the back) with 200 µg of the ZEN-BSA conjugate dissolved in sterilized PBS and emulsified with an equal volume of FCA.…”

Section: Zen Mab Preparation 461 Animal Immunizationmentioning

confidence: 99%

“…The influencing factors of the immunization method include the injection route, dosage, interval, adjuvant usage, frequency, and individual animal differences, with the dosage and the interval being the key factors. Some scholars have shown that a low immunogen dose induces a narrowspectrum specificity of antibodies, while a high dose has a broad-spectrum of specificity of antibodies [43]. An immunogen elicits the generation of memory B cells, which require certain immunization intervals to clone and proliferate, producing several specific antibodies.…”

Section: Introductionmentioning

confidence: 99%

Preparation and Characterization of Monoclonal Antibodies with High Affinity and Broad Class Specificity against Zearalenone and Its Major Metabolites

Wang

Zhang

et al. 2021

Toxins

View full text Add to dashboard Cite

This study aimed to detect and monitor total Zearalenone (ZEN) and its five homologs (ZENs) in cereals and feed. The monoclonal antibodies (mAbs) with a high affinity and broad class specificity against ZENs were prepared, and the conditions of a heterologous indirect competitive ELISA (icELISA) were preliminarily optimized based on the ZEN mAbs. The immunogen ZEN-BSA was synthesized using the oxime active ester method (OAE) and identified using infrared (IR) and ultraviolet (UV). The coating antigen ZEN-OVA was obtained via the 1,4-butanediol diglycidyl ether method (BDE). Balb/c mice were immunized using a high ZEN-BSA dose with long intervals and at multiple sites. A heterologous indirect non-competitive ELISA (inELISA) and an icELISA were used to screen the suitable cell fusion mice and positive hybridoma cell lines. The ZEN mAbs were prepared by inducing ascites in vivo. The standard curve was established, and the sensitivity and specificity of the ZEN mAbs were determined under the optimized icELISA conditions. ZEN-BSA was successfully synthesized at a conjugation ratio of 17.2:1 (ZEN: BSA). Three hybridoma cell lines, 2D7, 3C2, and 4A10, were filtered, and their mAbs corresponded to an IgG1 isotype with a κ light chain. The mAbs titers were between (2.56 to 5.12) × 102 in supernatants and (1.28 to 5.12) × 105 in the ascites. Besides, the 50% inhibitive concentration (IC50) values were from 18.65 to 31.92 μg/L in the supernatants and 18.12 to 31.46 μg/L in the ascites. The affinity constant (Ka) of all of the mAbs was between 4.15 × 109 and 6.54 × 109 L/mol. The IC50 values of mAb 2D7 for ZEN, α-ZEL, β-ZEL, α-ZAL, β-ZAL and ZAN were 17.23, 16.71, 18.27, 16.39, 20.36 and 15.01 μg/L, and their cross-reactivities (CRs, %) were 100%, 103.11%, 94.31%, 105.13%, 84.63%, and 114.79%, respectively, under the optimized icELISA conditions. The limit of detection (LOD) for ZEN was 0.64 μg/L, and its linear working range was between 1.03 and 288.55 μg/L. The mAbs preparation and the optimization of icELISA conditions promote the potential development of a rapid test ELISA kit, providing an alternative method for detecting ZEN and its homologs in cereals and feed.

show abstract

“…The CR values of the 8H10 mAb for AFB1, AFB2, AFG1, and AFG2 were 100%, 60.47%, 65.97%, and 14.83%, respectively. Li et al [ 38 ] reported that the dose of immunogen had a great contribution to the specificity and broad spectrum of AFs mAb, and the low dose of immunogen helped to obtain a specific narrow-spectrum mAb. The high dose of immunogen helped to obtain a specific broad-spectrum mAb.…”

Section: Preparation Of a Single Universal Antibody For Taf Immunomentioning

confidence: 99%

Advances in Antibody Preparation Techniques for Immunoassays of Total Aflatoxin in Food

et al. 2020

View full text Add to dashboard Cite

Aflatoxin (AF) contamination is a major concern in the food and feed industry because of its prevalence and toxicity. Improved aflatoxin detection methods are still needed. Immunoassays are an important method for total aflatoxin (TAF) analysis in food due to its technical advantages such as high specificity, sensitivity, and simplicity, but require high-quality antibodies. Here, we first review the three ways to prepare high-quality antibodies for TAF immunoassay, second, compare the advantages and disadvantages of antigen synthesis methods for B-group and G-group aflatoxins, and third, describe the status of novel genetic engineering antibodies. This review can provide new methods and ideas for the development of TAF immunoassays.

show abstract

Doses of Immunogen Contribute to Specificity Spectrums of Antibodies against Aflatoxin

Cited by 9 publications

References 23 publications

Synthesis of Zearalenone Immunogen and Comparative Analysis of Antibody Characteristics

Synthesis of Zearalenone Immunogen and Comparative Analysis of Antibody Characteristics

Preparation and Characterization of Monoclonal Antibodies with High Affinity and Broad Class Specificity against Zearalenone and Its Major Metabolites

Advances in Antibody Preparation Techniques for Immunoassays of Total Aflatoxin in Food

Contact Info

Product

Resources

About