Antigenic Analyses of Sugi Basic Protein by Monoclonal Antibodies: II. Detection of Immunoreactive Fragments in Enzyme-cleaved <i>Cry j l</i>

Abstract

The 4 anti-Cry jI mAbs showing an epitope specificity different from each other, 046, 029, 026 and 027, were selected to analyze the structure of the antigenic determinant for each mAb on a Cry jI molecule. Immunoreactive fragments in enzyme-cleaved Cry j I were detected by means of the adsorption on the mAb column and of the binding to the mAbs on Elisa. The mAb 026 was found to be reactive to the fragments containing a Cry jI N-terminal region obtained by V8 protease or pepsin digestion, but not to those by lysylendopeptidase digestion. The mAb 027 was found to be capable of binding to the fragments containing a linear structure of Asn-Ala-Gly-Val-Leu-Thr-Cys-Ser-Leu-Ser-Lys, which were generated by V8 protease, lysylendopeptidase or pepsin digestion. Furthermore, the synthetic peptide Asn-Ala-Gly-Val-Leu-Thr-Cys-Ser-Leu-Ser-Lys-Arg could bind to 027, but not to 026, and could inhibit the binding of 027 to Cry jI or to its immunoreactive fragments. No fragments capable of reacting to the mAbs 046 and 029 could be found in this study, suggesting that 046 and 029 recognize a conformationally constituted epitope of Cry jI molecule which is destroyed by enzymatic cleavage. The epitope recognized by the mAbs 027 or 026 was found to be located in conformationally hidden parts of the molecule which was exposed to react to the mAbs only after the physicochemical or enzymatic treatment.

Using Monoclonal Antibodies to Characterize a Sequential Epitope on the Group I Allergen of Bermuda Grass Pollen

“…Using V8 protease, lysylcndopeptidase and pepsin to di gest the Cry j 1, its immunoreactive fragments had also been detected with MoAbs [18]. In addition, other reagents such as cynogen bromide and 2-nitro-5-thiocyanobcnzoic acid had also been applied for the studies of B cell epitopes with the use o f polyclonal antibodies [19,20], Peptide chains are cleaved by cyanogen bromide at the methionine, and cleaved by 2-nitro-5-thiocyanobenzoic acid at the cysteine.…”

Cupressaceae Pollinosis: Identification, Purification and Cloning of Relevant Allergens

“…Cry j 2, a protein of 37,000 D in molecular weight with an N-terminal amino acid sequence completely different from that of Cry j 1, did not show any allergenic cross-reactivity with Cry j 1, as demonstrated by IgE ELISA inhibition studies. Studies with a panel of 23 monoclonal antibodies (mAbs) raised against Cry j 1 and Cry j 2 [40, 41]led to the fine analysis of B cell epitopes displayed by the two allergens, confirming the absence of cross-reactive determinants on the two molecules. From a functional point of view, Cry j 2 has been classified as a polymethylgalacturonase, although existence of this activity has not been conclusively proven [42].…”

Comparison of Methods of Immobilization to Enzyme-Linked Immunosorbent Assay Plates for the Detection of Sugar Chains

“…Small synthetic peptides are commonly used to obtain information on the structure and location of antigenic epitopes, and several improved immobilization methods have been reported (1)(2)(3)(4). However, very few cases of improvement of immobilization of sugars have been reported.…”

“…However, very few cases of improvement of immobilization of sugars have been reported. The method using biotinylated oligosaccharides (5) is cumbersome because of the difficulty of separating biotin-labeled oligosaccharides from the mixture and is expensive because of the large amount of streptavidin necessary for the immobilization of bi- 1 To whom correspondence should be addressed. Fax: ϩ81-3-5978-5344.…”