Synthesis and n.m.r. analysis of branched trisaccharide and pentasaccharide haptens of the beta-hemolytic streptococci group A and the preparation of synthetic antigens.

The synthesis of branched trisaccharide and pentasaccharide portions of the cell-wall polysaccharide of the beta-hemolytic Streptococci Group A is described. The key dissaccharide acceptors, allyl or 8-(methoxycarbonyl)ocytol 3-O-(3,4,6-tri-O-benzyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl)-4-O -benzyl - alpha-L-rhamnopyranoside, in conjunction with a selectively blocked alpha-L-rhamnopyranosyl chloride under Koenigs-Knorr conditions, afforded the branched trisaccharides in 81 and 62% yield, respectively. Analogously, glycosylation of the 8-(methoxycarbonyl)octyl disaccharide with a protected beta-D-GlcpNAc-(1----3)-alpha-L-Rhap-(1----3)-alpha-L-Rhap chloride gave the pentasaccharide in 43% yield. The key disaccharide acceptors were obtained, in turn, from the allyl or 8-(methoxycarbonyl)octyl rhamnoside acceptors and 3,4,6-tri-O-benzyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl chloride under Koenigs-Knorr conditions. The latter glycosyl donor has not been described previously. Removal of the protecting groups afforded the trisaccharide haptens as their 1-propyl and 8-(methoxycarbonyl)octyl glycosides and the pentasaccharide as its 8-(methoxycarbonyl)octyl glycoside. The compounds have been subjected to detailed analysis by two-dimensional n.m.r. methods. Preparation of the synthetic antigens followed coupling of the 8-(methoxycarbonyl)octyl glycosides to bovine serum albumin via the acyl azide intermediates.

Enzyme immunoassay with captured hapten. A sensitive gastrin assay with biotinyl-gastrin derivatives.

Two N-terminal biotinyl-gastrin derivatives were synthesized to investigate the effect of the length and chemical properties of the biotin-spacer on both the capture of the hapten by streptavidin or avidin adsorbed on polystyrene, and the antigenicity of the captured peptide. The observed full retainment of antibody binding capacity of the biotinyl-gastrins upon their immobilization, allowed to develop a sandwich-type ELISA with a sensitivity of one order of magnitude better than the standard ELISA with polystyrene-adsorbed gastrin. This hapten capture system reduces desorption particularly pronounced for low mass peptides, and avoids possible modifications or suppression of epitopes by the adsorption process with concomitant reduction of antibody binding affinity of the antigen. This new type of assay procedure may also represent a useful tool particularly for epitope mapping with relatively low mass synthetic protein fragments.

Fully synthetic immunogens. Part III. Synthesis of hinge-peptide/gastrin conjugates and their immunological properties.

As core molecule for the multiple attachment of antigenic peptides we have selected the human IgG1 hinge fragment 225-232/225'-232'. Two types of conjugates of this double-chain bis-cystinyl hinge-peptide were prepared i) by linking its C-termini to [NIe15]-human-little-gastrin-[2,17] and ii) by elongating the resulting hinge-peptide/[NIe15]-little-gastrin-[2-17] conjugate at the two N-termini with the human big-gastrin sequence 1-14 to produce the big-gastrin-[1-14]/hinge-peptide/little-gastrin-[2-17] conjugate. For the synthesis of these peptide structures both the route via the preformed double-chain bis-cystinyl peptide and the route via suitably protected monomeric bis-cysteinyl peptides were used. For the latter approach advantage was taken of the previous observation about the preferred oxidation of the bis-cysteinyl hinge-peptide 225-232 to the dimer in parallel alignment. Both synthetic routes led to identical products. Immunization experiments in guinea pigs with the synthetic hybrids led to surprisingly strong immune responses with anti-little-gastrin antibody titers comparable to those induced by the iso-1-cytochrome c/little-gastrin-[2-17] conjugate as carrier-hapten system. These findings show that the two gastrin constructs are fully competent immunogens. Additionally, the gastrin receptor-like specificity of the antibodies indicates that both the synthetic hybrids and the cytochrome c conjugate allow for expression of a little-gastrin-specific conformational epitope similar to the bioactive structure of this hormone. The usefulness of such synthetic hybrids is further confirmed by the observation that the bivalent immunogen, containing both the little-gastrin 2-17 and the big-gastrin 1-14 sequence, is capable of inducing an immune response against both antigenic sequences, although with different efficiency. These results fully confirm our expectations.

Immunoadsorbents with synthetic oligosaccharide hapten representing blood group A substances.

Immunoadsorbents with a synthetic oligosaccharide hapten representing human blood group A specific substances are prepared. The synthetic hapten, known as A-trisaccharide, which carries a space arm, is chemically attached to various solid supports, either directly through a suitable functional group at the end of the spacer arm or indirectly via a protein conjugated to the hapten. The preparation involves simple and mild procedures for the activation and/or derivatization of the supports. The latter includes naturally occurring polyhydroxy materials such as agarose, cellulose, or cellulose derivatives, and other particulate materials such as inorganic diatomites and a synthetic organic copolymer. The methods used for the coupling concern specifically the preparation of controlled-capacity and high-efficiency immunoadsorbents, with limited incorporations, which may be prepared easily and used for the selective removal, or affinity chromatographic separation, of specific antibodies from plasma environment or blood. It has been found that while hapten incorporation to the support may be varied rather easily, the physical nature of the support as well as the form of the hapten is important in determining the efficiency of an immunoadsorbent.

Measurement of the class III antidysrhythmic drug, UK-68,798, in plasma by radioimmunoassay.

A sensitive radioimmunoassay (RIA) for the specific determination of 1-(4-methanesulphonamidophenoxy)-2-[N-(4-methanesulphonamido -phenethyl)-N- methylamino]ethane (UK-68,798), a novel class III antidysrhythmic agent, in human plasma is described. Specific antisera were raised in sheep using desmesyl-UK-68,798-succinate-ovalbumin conjugate as the antigenic hapten carrier protein. The antisera produced exhibited high specificity for UK-68,798 compared with known metabolites from animals, other antidysrhythmic agents and co-administered drugs. Good correlation was found in a comparison of the RIA method with a high-performance liquid chromatography (HPLC) method (r = 0.997) and a 10-fold lower limit of determination was observed for the RIA method compared with the HPLC method (0.05 and 0.5 ng ml-1, respectively). The RIA method was applied to the analysis of UK-68,798 in plasma obtained from human volunteers receiving the compound.

New strategies for the design of catalytic antibodies.

A new approach is advanced which is aimed at expanding the scope of antibody catalysis. It entails the design and synthesis of haptens that will elicit antibodies to catalyze acyl transfer reactions by a methodology which we term "bait and switch" catalysis. What we have done involves the placement of a point charge on the hapten in close proximity to, or in direct substitution for, a chemical functional group we wish to transform in the respective substrate. The haptenic charge should induce a complementary charge (on an amino acid residue) at the binding site. The substrate will lack this charge but will retain a similar overall structure. The monoclonal antibodies that bind these substrates now have the potential to act as general acids/bases for substrates having hydrolyzable functional groups.

Immunoassay for methamphetamine with a new antibody.

p- and o-Aminomethamphetamine were synthesized as haptens to be coupled with carrier protein at the benzene ring of methamphetamine. Immunogens were prepared by the glutaraldehyde method or the MBS (N-(m-maleimidobenzoyloxy)succinimide) type cross-linking reagent method. In particular, immunization with p-aminomethamphetamine-bovine serum albumin (BSA) conjugate prepared by the glutaraldehyde method gave an anti-methamphetamine antiserum having a low cross-reactivity with methylephedrine. With the antiserum, three kinds of immunoassays for methamphetamine were established. An enzyme immunoassay (EIA) and an enzyme-linked immunosorbent assay (ELISA) were developed with alkaline phosphatase (ALP) as a label enzyme. The amount of antibody bound ALP conjugate was determined by its activity in dephosphorylating p-nitrophenyl phosphate in EIA and nicotinamide adenine dinucleotide phosphate (NADP+) in ELISA. The range of methamphetamine measurable by ELISA was 0.025-0.5 ng/well and its sensitivity was superior to that of EIA (0.3-300 ng/tube). A latex agglutination inhibition reaction test (LAIRT) was also developed for the mass screening method of urine samples. The sensitivity of this method for methamphetamine was 0.1 micrograms/ml urine.

The rational design and synthesis of haptens having specific activity as full agonists or full antagonists at the benzodiazepine receptor.

The use of computer graphics hardware, in conjunction with molecular modeling software, has allowed for a structural analysis of compounds that bind to the benzodiazepine receptor (BZR) in the nM range. The definition of additional binding requirements together with steric and/or hydrophobic limitations has been directly correlated with profiles of in vivo activity, both for full agonists and full antagonists. This information has been used for the rational design of haptens that contain the antigenic determinants necessary for the production of antibodies specific for either full agonists or for full antagonists at the BZR. The synthesis of these novel compounds has been completed.

Synthesis of 7 alpha- and 7 beta-carboxymethyl-9(11)-ene derivatives of estrone and estradiol.

As part of a search for derivatives designed to conjugate to amino groups, either of a protein carrier for antibody production or of an immobilized side-chain on a polymer for affinity chromatography, functionalized estrone and estradiol analogues were prepared. These modified steroids were obtained via the introduction of a carboxymethyl side-chain at the C-7 alpha and C-7 beta position on an adrenosterone derivative and were then aromatized on the A ring. These new compounds are unsaturated at the C-9 (11) position, which could be useful for a second modification.

Antibodies against arylcyclohexylamines and their similarities in binding specificity with the phencyclidine receptor.

Rabbit antibodies were generated against five unique epitopes of phencyclidine (PCP)-like molecules to determine the molecular requirements for arylcyclohexylamine binding to the PCP receptor. Three of the haptens contained the three ring structures of PCP. A fourth hapten was synthesized from a derivative of the highly potent PCP analog, 1-[1-(2-thienyl)cyclohexyl]piperidine. The fifth hapten, 5-[N-(1'-phenylcyclohexyl)amino]pentanoic acid, was used as a haptenic model for N-ethyl-1-phenylcyclohexylamine, one of the most potent arylcyclohexylamines. These haptens were bound covalently to bovine serum albumin and were then used as antigens to immunize rabbits. The affinities and cross-reactivity patterns of the resulting five antibodies were studied in a [3H]PCP radioimmunoassay using standard curves of various arylcyclohexylamines. The dissociation constants ranged from 1.9 to 51.6 nM. From the average IC50 values of the radioimmunoassay dose-response curves, the relative potency of each ligand to PCP was determined. Least-squares linear regression was used to correlate these data with relative potency data from two [3H]PCP receptor binding assays and a PCP drug discrimination assay in the rat. Only relative potency data from the anti-5[N-(1'-phenylcyclohexyl)amino]pentanoic acid antibody showed a significant correlation with data from the three pharmacological studies (r2 = 0.80, 0.57 and 0.78, respectively; p less than .05 in all cases). These data indicated the 5-[N-(1'-phenylcyclohexyl)amino]pentanoic acid hapten contained the pharmacologically active features needed for arylcyclohexylamine binding to the PCP receptor.

Polypeptide fluoroacetate derivatives. Synthesis and 19F n.m.r.

The synthesis and characterization of monofluoroacetyl (MFAc) functionalized haptens are described. These were covalently bound to polypeptide carriers (bovine serum albumin and poly-D-lysine) by primary amine/succinimide ester or primary amine/acid chloride coupling. Epitopic densities of the resulting antigens were determined by both 19F n.m.r. and picryl sulfonic acid assays. 19F n.m.r. experiments defining the stability of MFAc ester and amide linkages as a function of media (including in vitro), time, and temperature are presented. These results indicate that MFAc functionalized antigens are well suited for further immunologic studies.

Rapid drug haptenization procedure: application to gentamicin and quinidine.

A rapid and simple drug conjugation procedure which utilizes the heterobifunctional reagent N-hydroxysuccinimidyl 6-(4'-azido-2'-nitrophenylamino)hexanoate is described. With this protocol two drugs of diverse structure and solubility have been successfully haptenized. Rabbits immunized with these conjugates yielded drug-specific antibodies as judged by a solid phase radioimmunoassay. Furthermore, using this technique, no cross-reactivity could be detected. Such haptens also elicited an immune response in mice thus further extending their potential for the production of drug specific monoclonal antibodies.

The use of a monosaccharide linkage group in a heterologous solid-phase enzyme immunoassay for phenytoin.

The preparation is described of monosaccharide-hapten derivatives containing a galactosyl linkage group which bears little structural resemblance to the straight-chain hydrocarbons commonly used for attaching haptens to proteins for use as immunogens. These derivatives are readily synthesised and are coupled to enzymes under mild conditions to produce bridge-heterologous enzyme immunoassays. The use of beta-galactosidase-monosaccharide-phenytoin derivatives in the development of sensitive phenytoin enzyme immunoassays is described and the assays are compared with hapten-heterologous, site-heterologous and homologous phenytoin enzyme immunoassays prepared using other phenytoin derivatives. This technique has application in the development of immunoassays for haptens which have only one functional group to which linkage groups can be attached for covalent coupling of the hapten to proteins.

Triprolidine radioimmunoassay: disposition in animals and humans.

A hapten derivative of triprolidine, bearing an acrylic acid side chain ortho to the pyridine ring nitrogen atom, was synthesized and coupled to bovine serum albumin. Immunization of New Zealand White rabbits with the resulting drug-protein conjugate resulted in the production of antisera capable of binding a radioiodinated tyramine conjugate of the triprolidine hapten derivative at high antiserum dilutions (1:70,000-1:150,000). These antisera were used to develop a radioimmunoassay (RIA) for triprolidine in human plasma with a sensitivity limit of 0.1 ng/mL (0.01 ng of actual mass). The known hydroxymethyl and carboxyl metabolites of triprolidine cross-reacted weakly (less than 2 and less than 0.05%, respectively) with this antiserum. The RIA could be used for the direct analysis of triprolidine in human and rabbit plasma, but not for rat or dog plasma, presumably due to the presence of other interfering substances (possibly metabolites). The validity of the RIA procedure in human plasma was demonstrated by comparative analysis of a number of samples by quantitative TLC (r = 0.985, slope = 1.076). The assay was employed to describe the pharmacokinetics of triprolidine in the rabbit (t 1/2, beta = 1.7 h). The assay had adequate sensitivity to detect low circulating drug concentrations in humans after therapeutic oral doses and also substantiated previous disposition experiments with triprolidine in humans (t 1/2, beta = 2.27 h). TLC analysis demonstrated that the absolute oral bioavailability of triprolidine (1-mg/kg dose) in the dog was low (4%). A comparison of triprolidine pharmacokinetic parameters in dogs, rabbits, rats, and humans revealed considerable similarity in elimination characteristics in these species.

Influence of the hapten-fluorophore bridge on binding parameters in a fluoroimmunoassay for carbamazepine.

Raising antibodies to a hapten (drug or steroid) requires that it be coupled to a carrier protein through a bridge such that the hapten has maximum exposure with minimum changes in its configuration. In raising antisera in six sheep against carbamazepine (CBZ) coupled to bovine albumin, we found that some subpopulations of the antibodies recognized the bridge linking the drug to the carrier protein. To study the influence of the bridge on a fluoroimmunoassay for CBZ, we prepared four tracers by linking the carbamyl nitrogen of CBZ to fluorescein via four alkyl bridges of different lengths and structures. We calculated various binding parameters--including antibody affinity, binding capacity, and heterogeneity index--for each tracer and chose for the final fluoroimmunoassay the tracer that gave the best displacement with CBZ. We then optimized and validated the assay for direct measurement of CBZ in serum or plasma. The antibodies are coupled to magnetizable particles, which greatly facilitates separation and ensures removal of endogenous interferents.

Radioimmunoassay for psychotropic drugs. III: Synthesis and properties of haptens for trifluoperazine and fluphenazine.

For the development of radioimmunoassay procedures for trifluoperazine and fluphenazine, three haptens, N-(2-carboxyethyl)desmethyltrifluoperazine, N-(4-carboxybutyl)desmethyltrifluoperazine, and 10-[3-(4-carboxyethylpiperazinyl)-3-oxopropyl]-2-trifluoromethyl-+ ++10H- phenothiazine, were synthesized and characterized. Each hapten was coupled to bovine serum albumin, and the number of hapten residues per mole of bovine serum albumin was calculated by UV spectrophotometric methods. Antibodies to each hapten-protein conjugate were developed in rabbits, and titers of the antisera were checked by evaluating their binding characteristics to the tritiated drug.

Specific antibodies directed against toxins of Ptychodiscus brevis (Florida's red tide dinoflagellate).

Specific antibodies directed against Ptychodiscus brevis 'brevetoxins' have been produced in a goat. The haptenic toxin T34 was chemically reduced to toxin T17, covalently-linked to succinic acid via anhydride coupling, and coupled to bovine serum albumin using standard carbodiimide condensation procedures. The hapten coupling efficiency ranged from 10.4 to 13.5 moles of toxin bound per mole of protein. Antibody titers were directly related to the frequency of immunization, and weekly intervals appeared optimum for maintaining adequate titers. [3H]Brevetoxin T17 is displaced in a competitive manner from the antibody-antigen complexes by unlabeled toxin, but the antibodies do not distinguish between T17 and T34. The sensitivity achieved, using purified brevetoxins as competitive inhibitors of [3H]T17 binding, was 600 picograms. The assay linearity ranged from 1.5 to 48 ng.

Monoclonal antibodies to a nitroxide lipid hapten.

The isolation and characterization of a hybridoma cell line producing a monoclonal IgG1 antibody against a spin-label nitroxide group is described. The antibody recognizes a synthetic hapten containing linked dinitrophenyl and 2,2,6,6-tetramethylpiperidinyl-1-oxy groups, having an affinity of 3.6 +/- 1.0 . 10(6) M-1 for the soluble hapten at 25 degrees. The antibody binds to phospholipid vesicles containing 2 mol% of spin label-derivitized lipid (lipid hapten) with an affinity of 1.5 +/- 0.2 . 10(8) M-1. This monoclonal IgG1 mediates the binding of hapten-bearing lipid vesicles to mouse macrophage RAW264 cells bearing Fc receptors. The cellular responses to this binding are similar to those observed previously using polyclonal rabbit anti-hapten IgG. As with the heterogeneous antibodies, the monoclonal IgG1 is more efficient in mediating cellular uptake when the vesicles are in the "fluid' physical state (dimyristoylphosphatidylcholine at 37 degrees C) compared to "solid' (dipalmitoylphosphatidylcholine at 37 degrees C). Despite the enhanced binding of "fluid' phospholipid vesicles to cells, only the "solid' vesicles triggered a significant respiratory burst in Raw264 macrophages.