Effect of Epitoge Density on the Induction of the IgE Immune Response in Mice

Nakagawa, Takemasa; Blaser, Kurt; Weck, A.L. de

doi:10.1159/000232629

Cited by 11 publications

(5 citation statements)

References 12 publications

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“…Coupling the density of the chemical with the carrier protein was reported to affect antibody production. Briefly, immunogens with higher coupling densities usually produce monoclonal antibodies with higher substrate affinities (Klaus and Cross, 1974 ; Nakagawa et al , 1980) . In the present study, coupling density AFB1-CMO to BSA was around 6.0 and represented a similar to that of previous report (Zhou et al , 2007) .…”

Section: Resultsmentioning

confidence: 99%

Production of Group Specific Monoclonal Antibody to Aflatoxins and its Application to Enzyme-linked Immunosorbent Assay

Kim¹,

Cha²,

Bischoff³

et al. 2011

Toxicological Research

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Through the present study, we produced a monoclonal antibody against aflatoxin B1 (AFB1) using AFB1- carboxymethoxylamine BSA conjugates. One clone showing high binding ability was selected and it was applied to develop a direct competitive ELISA system. The epitope densities of AFB1-CMO against BSA and KLH were about 1 : 6 and 1 : 545, respectively. The monoclonal antibody (mAb) from cloned hybridoma cell was the IgG1 subclass with λ-type light chains. The IC50s of the monoclonal antibody developed for AFB1, AFB2, AFG1 and AFG2 were 4.36, 7.22, 6.61 and 29.41 ng/ml, respectively, based on the AFB1-KLH coated ELISA system and 15.28, 26.62, 32.75 and 56.67 ng/ml, respectively, based on the mAb coated ELISA. Cross-relativities of mAb to AFB1 for AFB2, AFG1 and AFG2 were 60.47, 65.97 and 14.83% in the AFB1-KLH coated ELISA, and 59.41, 46.66 and 26.97% in the mAb coated ELISA, respectively. Quantitative calculations for AFB1 from the AFB1-Ab ELISA and AFB1-Ag ELISA ranged from 0.25 to 25 ng/ml (R2 > 0.99) and from 1 to 100 ng/ml (R2 > 0.99), respectively. The intra- and inter-assay precision CVs were < 10% in both ELISA assay, representing good reproducibility of developed assay. Recoveries ranged from 79.18 to 91.27%, CVs ranged from 3.21 to 7.97% after spiking AFB1 at concentrations ranging from 5 to 50 ng/ml and following by extraction with 70% methanol solution in the Ab-coated ELISA. In conclusion, we produced a group specific mAb against aflatoxins and developed two direct competitive ELISAs for the detection of AFB1 in feeds based on a monoclonal antibody developed.

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Section: Resultsmentioning

confidence: 99%

Production of Group Specific Monoclonal Antibody to Aflatoxins and its Application to Enzyme-linked Immunosorbent Assay

Kim¹,

Cha²,

Bischoff³

et al. 2011

Toxicological Research

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show abstract

“…Coupling density of the chemical with the carrier protein was reported to affect antibody production. Immunogens with higher coupling densities usually produce mAb with higher substrate affinities [8,11]. The immunogen used in the present study had a relatively high coupling density compared to those used in previous reports [2].…”

Section: Discussionmentioning

confidence: 99%

Production of a highly group-specific monoclonal antibody against zearalenone and its application in an enzyme-linked immunosorbent assay

et al. 2012

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A monoclonal antibody (mAb) against zearalenone (ZEN) was produced using ZEN-carboxymethoxylamine and -BSA conjugates. Antibody produced by one clone showing a very high binding ability was selected and found to have a higher affinity for ZEN compared to a commerciall ZEN antibody. We developed two direct competitive ELISA systems using the selected antibody (ZEN-coated and anti-ZEN antibody-coated ELISA). Quantitative ranges for the anti-ZEN antibody-coated ELISA and ZEN-coated ELISA were from 25 to 750 ppb and from 12.5 to 100 ppb, respectively. The detection limit of both methods as measured with standard solutions was 10 ppb. The intra-plate and inter-well variation of both ELISAs were less than 10%. The IC 50 values for α-zearalenol, β-zearalenol, α-zearalanol, and β-zearalanol compared to ZEN were 108.1, 119.3, 114.1, and 130.3% for the ZEN-coated ELISA. These values were 100.7, 120.7, 121.6, and 151.6% for the anti-ZEN antibody-coated ELISA. According to the anti-ZEN antibody-coated ELISA, the average recovery rates of ZEN from spiked animal feed containing 150 to 600 ng/mL of ZEN ranged from 106.07 to 123.00% with 0.93 to 2.28% coefficients of variation. Our results demonstrate that the mAb developed in this study could be used to simultaneously screen for ZEN and its metabolites in feed.

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“…Benzylpenicilloyl (BPO) conjugation of bovine serum albumin (Fluka, Buchs, Switzerland 05470) and KLH (Calbiochem 374811) was performed according to the method described by Nakagawa et al [9]. Conjugation of protein with phosphorylcholine [10] and biotin was done as mentioned.…”

Section: Reagentsmentioning

confidence: 99%

Establishment of a Memory in vitro Murine IgE Response to Benzylpenicillin and its Resistance to Suppression by Anti-lL-4 Antibody

Heusser

Wagner

Brinkmann

et al. 1989

Int Arch Allergy Immunol

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Regulation of a memory IgE antibody response may be different from the induction of a primary response and may, therefore, be more relevant to the study of allergic diseases and the therapeutic manipulation of IgE antibody formation. In this paper a murine hapten-specific in vitro memory IgE antibody response to benzylpenicilloyl(BPO)-KLH is described. The response was analyzed by determining the number of antibody-producing cells (APC) in an ELISA spot assay. Of the total number of BPO-specific APC (10,000 APC/ 10⁶ cultured spleen cells), about 1 % were IgE-producing cells (100/10⁶ cultured cells), as detected on day 6 of culture. The level of the antibody response is antigen dose-dependent, and the detected APC are BPO specific. The memory IgE response is not inhibited by the addition of anti-IL-4 antibody (11B11), even at a high excess. In the presence of the mitogen lipopolysaccharide, it has been shown that switch of B cells to IgE is induced by IL-4, a process which can be inhibited by anti-IL-4 antibody. Because the antigen-induced IgE response cannot be inhibited by anti-IL-4 antibody, in vitro responding cells derived from BPO-KLH-preimmunized mice may, therefore, have already switched in vivo to IgE. On the other hand, B cells switching to IgE in a situation of cognate T-B cell interaction might receive IL-4 in a transsynaptical way from T cells which might not be accessible to inhibition by anti-IL-4 antibody. The identification of the two possibilities in situations of established allergic disorders will be decisive for determining whether pharmacological inhibition of IL-4 (or IL-4-induced switch) – e.g., by putative low molecular weight compounds – will ever be a meaningful approach to suppress allergic diseases.

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Effect of Epitoge Density on the Induction of the IgE Immune Response in Mice

Cited by 11 publications

References 12 publications

Production of Group Specific Monoclonal Antibody to Aflatoxins and its Application to Enzyme-linked Immunosorbent Assay

Production of Group Specific Monoclonal Antibody to Aflatoxins and its Application to Enzyme-linked Immunosorbent Assay

Production of a highly group-specific monoclonal antibody against zearalenone and its application in an enzyme-linked immunosorbent assay

Establishment of a Memory in vitro Murine IgE Response to Benzylpenicillin and its Resistance to Suppression by Anti-lL-4 Antibody

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