ABSTRACT
To produce specific antibodies against malachite green ( MG) , one special hapten was synthesized and characterized, and conjugated to carrier protein as immunogen. The immunogen showed excellent reactogenicity and immunogenicity. One specific monoclonal antibody (mAb, named MG-DA4-C7) with high sensitivity and specificity for MG in indirect competitive enzyme-linked immunoassay ( icELISA ) was screened. The isotype was IgG1 and the light chain was κ type. After optimization of ELISA conditions, the proposed icELISA showed a 50% inhibition value ( IC50 ) of 0. 96 μg/L, a linear range ( IC20-IC80 ) of 0. 1-8. 1 μg/L and a limit of detection ( LOD, IC10 ) of 0. 05 μg/L for determination of MG. The assay showed cross-reactivity of 18. 1%, 26. 5% with crystal violet and brilliant green, respectively, and negligible cross-reactivity with other metabolites of MG (<0 . 1%) . The average recoveries of MG from spiked fish samples were from 87. 3% to 107. 3%. Good correlation (R2=0. 999) was obtained between the results of icELISA and those of liquid chromatography-tandem mass spectrometry analysis. The proposed icELISA is suitable for the determination of MG in fish samples in a simple and sensitive manner.
ABSTRACT
The prevalence studies on specific IgE to toluene diisocyanate (TDI)-human serum albumin (HSA) conjugate in TDI-induced asthma have shown variable results. In this study, we attempted to compare specific IgE bindings to TDI-HSA conjugate and its specificity using 3 different conjugates. Sera were collected from 20 TDI-induced asthma and 10 controls. Specific IgE were measured by ELISA using three TDI-HSA conjugates; two from Carnegie Mellon (CM; 98 and 99 CM conjugates) and one from Ajou University. To evaluate specificity and cross-reactivity, ELISA inhibition tests were applied. Positive and negative predictive values between Ajou conjugate and 98 CM conjugate were 75% and 100%. Those between Ajou and 99 CM were 100% and 93.8%. One patient showed an isolated positive response to the Ajou with negative responses to the other two conjugates. ELISA inhibition test using this patient's serum revealed the significant inhibitions by the Ajou and minimal inhibitions by the others. On the other hand, another patient showed an isolated positive response to 99 CM with negative responses to the others, and ELISA inhibition test showed significant inhibition by 99 CM with minimal inhibitions by the others. These results suggest that specific IgE bindings to a new antigenic determinant of TDI-HSA conjugate can be heterogeneous and differ from one individual to another.
Subject(s)
Humans , Asthma/immunology , Asthma/chemically induced , Enzyme-Linked Immunosorbent Assay , Immunoglobulin E/biosynthesis , Serum Albumin/immunology , Toluene 2,4-Diisocyanate/immunology , Toluene 2,4-Diisocyanate/adverse effectsABSTRACT
The antigenic determinants of Trichosanthin (TCS) were studied using cyanogen bromide cleavage of TCS and immunoblotting technique. 21 McAbs prepared against TCS could be divided into 5 groups according to the patterns of their reactivities with CNBr-fragments of TCS (CBTf1-4). (1) 7 antibodies bind to CBTf 1;(2) 2 antibodies bind to CBTf2;(3)2 antibodies bind to CBTf 4;(4)7 antibodies are cross-reactive with these fragments; (5) 3 antidodies bind to none of 4 fragments. Using two fragments CBTf1 and CBTf2 as competitors in a ELISA competition test, quantitative determination of antigenicity of TCS fragments by 4 McAbs to native TCS has shown that the maximal amount of bound antibody for CBTf1 with 2 McAbs reached 50% and for CBTf2 with another McAb nearly 50%. From these data, we concluded that TCS fragments have kept antigenic determinants as native TCS and it is very essential for us to study the structure-function relationship of TCS.
ABSTRACT
In this paper, the antigens of both Vibrio cholerae on 55 strains of El Tor and on 28 strains obtained with genetical method were studied by ELISA method using 12 strains of McAb against Vibrio cholerae (VCOMcAb). The results indicated that the 55 strains of Vibrio cholerae El Tor were expressed 11(A-K) of the recognized antigenic determinants, and fell into XⅧ(Ⅰ-XⅧ) distinct McAb reactivity types or groups, and the 28 varients of Vibrio cholerae from genetical method were expressed 9 (A-Ⅰ) of the recognized antigenic determinants, and fell into Ⅹ (Ⅰ-Ⅹ) distinct McAb reactivity types or groups. From those VCOMcAbs, the “F3” could recognize all 55 strains of Vibrio cholerae El Tor, indicating it has specificity; other 11 strains of VCOMcAb have a variety of reactivity to antigens on the 55 strains of Vibrio cholerae El Tor in both Inaba and Ogawa and their percentages of reactivity are different. This shows that these strains may have subtypes or subgroups.
ABSTRACT
Using 22 monoclonal antibodies (McAbs) as probe to analyse the antigenic determinants of crystallized Tian Hua Fen (THF) or Trichosanthin protein in ELISA additivity test was reported. The 22 McAbs were come from five fusions, each from different fusion well. The isotype of 22 McAbs were identified by immunodiffusioo test, 20 McAbs belong to IgG_1 subclass, 1 IgG_(2a) subclass and 1 IgM class. Based on the results obtained from ELISA additivity test, 22 McAbs could be clustered into six distinct groups. It indicates that there are six different antigenic determinants in crystallized THF, each can be recognized by some McAbs. The results coincide with the calculation of B cell recognized antigenic determinants obtained by prediction from amino acid sequences of THF protein, The work has laid a foundation for further exploring the structure-function relationship of THF protein on molecular level .
ABSTRACT
Monoclonal antibodies, CAb-15 and CAb-16, were produced by using of spleen cells from Balb/c mice immunized with cell suspension of fresh hepatoma tissues with mouse myeloma Sp 2/0 cells. Western blotting showed that the antigen determinant of CAb-15 was situated on the ?_1 [Ⅲ] polypeptide chains and that of CAb-16 on the triple helical domain. The monoclonal antibodies were also used for immunohistochemical localization by ABC method in formalin-fixed and paraffin-embedded sections of normal tissues from liver, kidney, aorta, skin, cartilage, placenta, stomach, breast and granulation tissues. The results showed that these antibodies are useful in immunohistochemical study for collagen.