Dwane E. Wylie

METAL BINDING PROPERTIES OF A MONOCLONAL ANTIBODY DIRECTED TOWARD METAL-CHELATE COMPLEXES

A monoclonal antibody that recognizes cadmium-EDTA complexes has been produced by the injection of BALB/c mice with a metal-chelate complex covalently coupled to a carrier protein. The ability of purified antibody to recognize 16 different metal-EDTA complexes was assessed by measuring equilibrium binding constants using a KinExA™ immunoassay instrument. The antibody bound to cadmium- and mercury-EDTA complexes with equilibrium dissociation constants of 21 and 26 nM, respectively. All other metal-EDTA complexes tested, including those of Mn(II), In(III), Ni(II), Zn(II), Co(II), Cu(II), Ag(I), Fe(III), Pb(II), Au(III), Tb(III), Ga(III), Mg(II), and Al(III) bound with affinities from 20- to 40,000-fold less than that determined for the cadmium-EDTA complex. With the exception of mercury and magnesium, the binding of divalent metal-chelate complexes was well-correlated with the size of the metal ion. The amino acid sequences of the heavy and light chain variable regions were deduced from polymerase chain reaction-amplified regions of the corresponding genes and subsequently used to construct molecular models of the antigen binding region. The key residue for cadmium binding in the model for 2A81G5 appeared to be histidine 96 in the heavy chain.

Detection of mercuric ions in water by ELISA with a mercury-specific antibody

An immunoassay that detects mercuric ions in water at concentrations of 0.5 ppb and above is described. The assay utilizes a monoclonal antibody that binds specifically to mercuric ions immobilized in wells of microtiter plates. Within the range of 0.5-10 ppb mercury, the absorbance in the enzyme-linked immunosorbent assay (ELISA) is linear to the log of the mercuric ion concentration. The quantitation of mercury by ELISA correlates closely with results from cold-vapor atomic absorption. Other divalent metal cations do not interfere with the assay, although there is interference in the presence of 1 mM chloride ions. The optimum pH for mercury detection is 7.0, although 2 ppb mercury can be detected over a wide pH range. The assay is as sensitive as cold-vapor atomic absorption for mercury detection and can be performed with only 100 microliters of sample.

Immunoaffinity chromatography utilizing monoclonal antibodies: Factors which influence antigen-binding capacity

Differences in antigen-binding capacity of a monoclonal antibody coupled to Sepharose under varying conditions were explored. The extent of cyanogen bromide activation, and the pH of the coupling reaction had a profound effect upon the rate of antibody coupling, but only small differences in antigen-binding capacity were observed if the antibody coupling reaction was terminated when 80-90% of the antibody was covalently coupled to Sepharose. However, if antibody was incubated with activated resin until 100% coupling was attained, the antigen-binding capacity of the resulting immunoadsorbent decreased significantly. Monoclonal antibody coupled to Sepharose via an N-hydroxysuccinimide ester linkage and approximately half the antigen-binding capacity of antibody coupled by CNBr activation. Concentrations of monoclonal antibodies as high as 13 mg/ml of packed resin could be used without noticeable steric hindrance.

16Cys encoded by the RHce gene is associated with altered expression of the e antigen and is frequent in the R0 haplotype

Serological observations have suggested that numerous D, many e (especially in Blacks), several E, and rare c variants exist within the Rh blood group system. The molecular basis for expression of many of these variants has been elucidated. This study describes five unrelated Caucasians whose red blood cells reacted with polyclonal anti‐e but did not react with some monoclonal anti‐e, which suggested that they carried a variant e antigen. Molecular investigation revealed the presence of a 48G→C change (encoding cysteine instead of tryptophan at amino acid 16) in their RHce gene. No other differences were found, which suggests that amino acid residues located in the first transmembrane region can affect expression of the e antigen, whose critical residues are on the predicted fourth external loop of the protein. This polymorphism has not previously been observed because polyclonal anti‐e does not distinguish this variant from wild type. This position is polymorphic in RHce alleles and the presence of the 48C nucleotide is often found in the R0 (Dce) haplotype.

Research reportImmunoaffinity chromatography utilizing monoclonal antibodies: Factors which influence antigen-binding capacity

Differences in antigen-binding capacity of a monoclonal antibody coupled to Sepharose under varying conditions were explored. The extent of cyanogen bromide activation, and the pH of the coupling reaction had a profound effect upon the rate of antibody coupling, but only small differences in antigen-binding capacity were observed if the antibody coupling reaction was terminated when 80-90% of the antibody was covalently coupled to Sepharose. However, if antibody was incubated with activated resin until 100% coupling was attained, the antigen-binding capacity of the resulting immunoadsorbent decreased significantly. Monoclonal antibody coupled to Sepharose via an N-hydroxysuccinimide ester linkage and approximately half the antigen-binding capacity of antibody coupled by CNBr activation. Concentrations of monoclonal antibodies as high as 13 mg/ml of packed resin could be used without noticeable steric hindrance.

Evidence supporting the requirement for two proline residues for expression of c

BACKGROUND: In humans, c antigen expression is associated with a proline residue at amino acid position 103 in the second extracellular loop of the CE protein. Comparison of nonhuman primate Rh proteins suggested that c reactivity might actually involve two proline residues. It has been shown that the RBCs of New World capuchin monkeys (Cebus apella) react with anti‐c. To further define the amino acid residues involved in c expression, Rh cDNA from the capuchin was analyzed.

Investigation of the RH Locus in Gorillas and Chimpanzees

The human Rh blood-group system is encoded by two homologous genes,RhD andRhCE. TheRH genes in gorillas and chimpanzees were investigated to delineate the phylogeny of the humanRH genes. Southern blot analysis with an exon 7-specific probe suggested that gorillas have more than twoRH genes, as has recently been reported for chimpanzees. Exon 7 was well conserved between humans, gorillas, and chimpanzees, although the exon 7 nucleotide sequences from gorillas were more similar to the humanD gene, whereas the nucleotide sequences of this exon in chimpanzees were more similar to the humanCE gene.The intron between exon 4 and exon 5 is polymorphic and can be used to distinguish the humanD gene from theCE gene. Nucleotide sequencing revealed that the basis for the intron polymorphism is anAlu element inCE which is not present in theD gene. Examination of gorilla and chimpanzee genomic DNA for this intron polymorphism demonstrated that theD intron was present in all the chimpanzees and in all but one gorilla. TheCE intron was found in three of six gorillas, but in none of the seven chimpanzees. Sequence data suggested that theAlu element might have previously been present in the chimpanzeeRH genes but was eliminated by excision or recombination.Conservation of theRhD gene was also apparent from the complete identity between the 3′-noncoding region of the human D cDNA and a gorilla genomic clone, including anAlu element which is present in both species.The data suggest that at least twoRH genes were present in a common ancestor of humans, chimpanzees, and gorillas, and that additionalRH gene duplication has taken place in gorillas and chimpanzees. TheRhCE gene appears to have diverged more thanRhD among primates. In addition, theRhD gene deletion associated with the Rh-negative phenotype in humans seems to have occurred after speciation.

Db-binding peptides from influenza virus: Effect of non-anchor residues on stability and immunodominance

Relative affinities were determined for the interaction of H-2Db with all the peptides from the A/PR/8/34 strain of influenza virus that contained the Db-binding motif. The results indicated that, even though 23 peptides with the appropriate motif were identified and analysed, binding of only five of them could be detected at peptide concentrations lower than 10(-7) M. Of these five, only one, TGICNQNII, bound with better affinity than the nucleoprotein-derived natural epitope, ASNENMETM. The origin of the higher binding peptide was the influenza neuraminidase, a protein for which little cytosolic processing would be expected since it is a surface glycoprotein. To establish why many of the influenza-derived peptides did not bind, the role of non-anchor residues on Db-peptide interactions was analysed, using a scheme where QDIENEEKI, a non-binding peptide from the influenza virus polymerase 1, was sequentially converted to ASNENMETI, which binds to Db with an affinity similar to that of ASNENMETM. Although all positions examined influenced peptide binding, peptide residue no. 2 (P2) was of particular importance. Therefore, each of the 20 naturally occurring amino acids were inserted at this position to investigate their effects on peptide-MHC interaction. The results indicated that amino acids having side chains with charged or ring structures were deleterious, while non-polar and polar residues were either neutral or facilitated binding to different degrees. Our data also indicated that every residue of the peptide contributes to the stability of the MHC-peptide complex, and the final affinity is dependent on the nature of the amino acids at each position, not just on those at a small number of anchor positions. The results also suggested that increased stability, as indicated by the half-life of the peptide-MHC class I complex, might play an important role in selecting the immunodominant epitope.

The JAL antigen (RH48) is the result of a change in RHCE that encodes Arg114Trp

BACKGROUND: The JAL antigen (Rh48) was discovered more than 30 years ago when it caused hemolytic disease of the fetus and newborn in an African American family. A decade later it was found to cause hemolytic disease of the fetus and newborn in a Caucasian family. The presence of the same low‐prevalence antigen in two different ethnic groups is rare, but additional JAL+ in both groups was subsequently identified. This study was undertaken to investigate the RH gene(s) responsible for expression of JAL and to determine the structural relationship between JAL and other Rh antigens.

ROLE OF NON-ANCHOR RESIDUES OF DB-RESTRICTED PEPTIDES IN CLASS I BINDING AND TCR TRIGGERING

To understand better, the role of non-anchor residues of class I restricted T cell epitopes in class I binding and TCR stimulation, a panel of peptides was synthesized in which each of the non-anchor positions of the Db-restricted influenza peptide, ASNENMETM, was changed to each of the 20 natural amino acids (AAs). The relative affinity of all the peptides for Db was determined and their ability to stimulate anti-ASNENMETM cytotoxic T cell hybridomas was also assessed. The results illustrated that for Db binding, the AAs with the most solvent exposure had the smallest effect on binding. Changes at other positions affected binding to different degrees. Results for the recognition by the T cell hybridomas indicated that a peptide-MHC complex represents a multitude of epitopes, as each hybridoma recognized a different subset of peptides. Most changes in the highly solvent-exposed residues negatively affected recognition by all hybridomas while changes in other positions affected each hybridoma differently, independent of the direction of the side chain of the AA at that position. Furthermore, the use of saturating concentrations of low and high binding peptides showed that, as long as the class I-peptide complex is formed, the T-cell receptor does not differentiate between high and low binding peptides. This indicates that, although the stability of the class I-peptide complex is highly dependent on peptide affinity, the class I MHC conformation induced by low affinity peptides does not necessarily differ significantly from that induced by high affinity peptides. The results of peptide-class I recognition by one ASNENMETM-specific hybridoma was used to construct a peptide that differed from ASNENMETM at four of the nine residues, yet stimulated the hybridoma to a level comparable to ASNENMETM. In addition, peptides bearing the canonical Db-binding motif but unable to bind to the class I molecule with high affinity could be made to bind Db, by changing unfavorable AAs to favourable ones at appropriate positions. The extended motif determined was used to identify more accurately the peptides derived from Coxsakie b3 virus that would bind Db. It was also shown that some of the canonical characteristics of the peptide motif could be obviated and still obtain high affinity binding, provided optimal AAs, were present at secondary anchor positions.