Glutaraldehyde (GA)-hapten adducts, but without a carrier protein, for usein a specificity study on an antibody against a GA-conjugated hapten compound: Histamine monoclonal antibody (AHA-3) as a model

In our recent study on monoclonal antibodies (mAbs AHA-1-5) against glutara ldehyde (GA)-conjugated histamine (HA), we identified one mAb (AHA-S) which can detect neuronal HA in the rat brain with an immunocytochemistry method (ICC) [Fujiwara ct at (1999) J. Biochem. 126, 503-509], In the present stu dy 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 dis tinguished alterations in the chemical structure of the molecule, showing d ecreased immunoreactivity with all the GA-adducts of (R)-(-)-alpha-methylhi stamine, 1- and 3-methylhistamine, L-histidine, and 1- and 3-methyl-L-histi dine. On the other hand, AHA-1 mAb only reacted with GA-adducts of 3-MeHA ( 3-MeHA-GA) and HA (HA-GA), to most the same degree, in relatively high conc entration ranges. AHA-3, 4, and 5 mAbs reacted about 10- times more strongl y with 1-MeHA-GA than with HA-GA, but reacted very little or not at all wit h the other analogs. These results may suggest that ABA-2 mAb recognized bo th 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 wit h NaBH4, and especially the imidazole group more strictly than the other mA bs, This may partly explain why AHA-S, among the five AHA mAbs, can detect neuronal HA with an ICC method. The present ELISA method for GA-hapten addu cts should be applicable to other antibodies against GA-conjugated biologic ally active amines or amino acids, thus allowing the study of antibody spec ificity for ICC more easily and accurately than was previously possible wit h hapten-protein conjugates as antigens.