Glutaraldehyde (GA)-Hapten Adducts, but without a Carrier Protein, for Use in a Specificity Study on an Antibody against a GA-Conjugated Hapten Compound: Histamine Monoclonal Antibody (AHA-2) as a Model

Abstract

In our recent study on monoclonal antibodies (mAbs AHA-1-5) against glutaraldehyde (GA)-conjugated histamine (HA), we identified one mAb (AHA-2) which can detect neuronal HA in the rat brain with an immunocytochemistry method (ICC) [Fujiwara et al. (1999) J. Biochem. 126, 503-509]. In the present study the specificity of AHA-2 mAb for use for ICC has been examined by means of competitive experiments involving HA and analogs, all of which had been allowed to react with GA followed by sodium borohydride, but not allowed to couple with the carrier protein. It was demonstrated that the antibody distinguished alterations in the chemical structure of the molecule, showing decreased immunoreactivity with all the GA-adducts of (R)-(-)-alpha-methylhistamine, 1- and 3-methylhistamine, L-histidine, and 1- and 3-methyl-L-histidine. On the other hand, AHA-1 mAb only reacted with GA-adducts of 3-MeHA (3-MeHA-GA) and HA (HA-GA), to almost the same degree, in relatively high concentration ranges. AHA-3, 4, and 5 mAbs reacted about 10-times more strongly with 1-MeHA-GA than with HA-GA, but reacted very little or not at all with the other analogs. These results may suggest that AHA-2 mAb recognized both the non-substituted imidazole and alpha-methine groups of a HA molecule in addition to the conjugation site of GA including the part(s) reduced with NaBH(4), and especially the imidazole group more strictly than the other mAbs. This may partly explain why AHA-2, among the five AHA mAbs, can detect neuronal HA with an ICC method. The present ELISA method for GA-hapten adducts should be applicable to other antibodies against GA-conjugated biologically active amines or amino acids, thus allowing the study of antibody specificity for ICC more easily and accurately than was previously possible with hapten-protein conjugates as antigens.

Immunocytochemistry for Amoxicillin and Its Use for Studying Uptake of the Drug in the Intestine, Liver, and Kidney of Rats

“…The conjugate of AMPC-GA was prepared in a manner similar to that for the conjugate of AMPC-GA-BSA, but the carrier protein (BSA) was omitted (14). The reaction mixture was directly used as a conjugate without any further purification for the inhibition enzyme-linked immunosorbent assays (ELISAs) described below (14).…”

“…The reaction mixture was directly used as a conjugate without any further purification for the inhibition enzyme-linked immunosorbent assays (ELISAs) described below (14).…”

Histamine quantification in human plasma using high resolution accurate mass LC–MS technology

“…However, RIA is highly time-consuming, and protective measures against radiations and exposure monitoring of the technicians due to use of radioactivity are necessary. In addition, cross reactivity with 3-methylhistamine, the main metabolite of HA in vivo, is still an issue for RIA and EIA methods [17].…”

Development of an LC-ESI–MS/MS method for the determination of histamine: Application to the quantitative measurement of histamine degranulation by KU812 cells

“…During the course of our characterizations of the bioactive constituents of Chinese natural medicines, the water and water/methanol extracts from T. yunnanensis wood were found to have remarkable inhibitory effects on the induction of histamine release from the human basophilic cell line, KU812 [10].Because of its low concentration in biological samples, quantification of the histamine requires a sensitive analysis method. Enzyme immunoassays and radioimmunoassays have been used for this purpose, but their specificity remains a problem as the antibodies used often have high crossreactivity with 3-methylhistamine [11], the main metabolite of histamine in vivo. High-performance liquid chromatography (HPLC) [12][13][14], capillary electrophoresis (CE) [15][16][17], and gas chromatography (GC) [18][19][20] are the techniques most commonly used for the determination of histamine and other biogenic amines.…”