Characterization of morphine-specific monoclonal antibodies showing minimal cross-reactivity with codeine.

Six murine monoclonal antibodies against morphine were produced using N-(4-aminobutyl)normorphine as a hapten. Most of the antibodies obtained distinguished the substituents at the 3 and 6 positions of morphine. This property of the antibodies led to a reduction in cross-reactivity with codeine, morphine-3-glucuronide (M-3-G) and morphine-6-glucuronide (M-6-G) to negligible levels. However, one of the antibodies distinguished the substituent only at the 3 position of morphine, which cross-reacted with M-6-G, naloxone and naltrexone. In the competitive inhibition enzyme-linked immunosorbent assay, morphine was detected at concentrations as low as circa 100 pg/ml.

An approach for an immunoaffinity AIDS sensor using the conservative region of the HIV envelope protein (gp41) and its monoclonal antibody.

A monoclonal antibody for the conservative region of gp41, which is one of the HIV envelope proteins, was produced. The antigen determining site of gp41 was examined using the epitope mapping technique, followed by an enzyme linked immunosorbent assay. Some peptides had comparable affinities for the monoclonal antibody, but the peptide EGIEE, having a slightly weaker immunoaffinity than gp41, was the most preferable for the construction of an immunoaffinity AIDS sensor. For the detection of gp41, EGIEE was labelled with catalase and used as a mimic antigen; it was bound to the antibody present on an immuno-membrane and, due to the replacement reaction of the mimic antigen by gp41, indirect quantitative measurement of gp41 was possible using an oxygen electrode. Anti-gp41 antibody was also detected using a mimic antibody, which was chemically modified with polyethylene glycol. An immunoaffinity AIDS sensor was constructed using the mimic molecules which were tailored to have a suitable immunoaffinity for HIV antigen and/or antibody.

Super catalytic antibody [I]: decomposition of targeted protein by its antibody light chain.

Obtaining an antibody capable of destroying a targeted protein is the eventual goal in developing superior catalytic antibody. We established a monoclonal antibody recognizing a highly conserved sequence, RGPDRPEGIEEEGGERDRD, of gp41 of the HIV-1 envelope. The obtained antibody reacted with gp41 and gp160 of HIV-1. The isolated and purified light chain not only decomposed the above antigenic peptide but also destroyed the gp41 molecule, indicating a novel ability. The decomposition of the antigen is presumably started by scission of the peptide bond between Arg-Gly in the above sequence. The light chain did not decompose BSA and HSA at all, showing the high specificity to antigens. The antibody light chain is referred to as a super catalytic antibody.

Analysis of the antigen recognition sites of anti-methamphetamine monoclonal antibodies (II): unique feature of MA-3 antibody.

A monoclonal antibody against methamphetamine (MA-3 mAb) was found to be strongly bound to ephedrine. This feature was quite different from that of other fourteen mAbs against MA. Analyses of cDNA sequence and steric conformation by molecular modeling revealed that one hydrophilic pocket was generated in the heavy chain of MA-3 mAb involving CDRH-1 and CDRH-2. Asn33, Asn35, Asn50 and Asp52 were the main components of the unique pocket capable of binding to the hydroxyl group of ephedrine.

How and why 41S-2 antibody subunits acquire the ability to catalyze decomposition of the conserved sequence of gp41 of HIV-1.

It has become well known that antibodies obtained by immunization with the ground state of peptides can display proteolytic activity. Our antibody light chain produced by immunization with the peptide RGPDRPEGIEEEGGERDRD, a highly conserved sequence in envelope gp41 of HIV-1 showed the ability to cleave this peptide. Moreover, its heavy chain also decomposed the peptide, although this occurred at lower activity levels compared with the light chain, while the whole antibody did not show any catalytic activity. From molecular modeling, the light and heavy chains of the antibody were deduced to possess catalytic triads (Asp, His, and Ser) in their steric conformations, which may be responsible for the observed proteolytic activity.

Removal of catalytic activity by EDTA from antibody light chain.

Gp41 peptide antigen of the HIV-1 envelope (TP41-1:TPRGPDRPEGIEEEGGERDR, a highly conserved region) was enzymatically degraded by the antibody light chain 41S-2-L after an induction period. The peptide bond between Glu14 and Gly15 was cleaved early in the reaction. When EDTA was added in the induction period, it inhibited the degradation of TP41-1 thus ceasing the catalytic activity of 41S-2-L. In contrast, when EDTA was added after the induction period, only a small reduction in the catalytic activity was observed. These observations suggest that metal ions are important in stimulating catalytic activity early in the reaction.

Application of polymer-protected ultrafine platinum particles to the immunological detection of human serum albumin.

The immunological properties of ultrafine platinum particles protected with poly(methyl acrylate)-co-N-vinyl-2-pyrrolidone, having a group chemically reactive with protein, were examined. Polymer-protected ultrafine particles were able to chemically bond multiple human serum albumin (HSA) molecules on their surface. The particles behaved as a multi-epitope-type antigen for the immunoreaction. In an ELISA, the HSA-bound particles and HSA molecules competitively reacted against the anti-HSA monoclonal antibodies. The limit of detection of HSA was ca. 10 ng/ml in the assay. The particles were used in an immunodot analysis of anti-HSA polyclonal antibody. Within 30 min of the reaction, 1-10 micrograms/ml of HSA was detected. The protein fixed polymer-protected ultrafine particles are a useful new material in immunochemistry.