Hapten Conjugates Research Articles

An antigenic HIV-1 peptide sequence engineered into the surface structure of transferrin does not elicit an antibody response

One novel approach for the biological delivery of peptide drugs is to incorporate the sequence of the peptide into the structure of a natural transport protein such as human serum transferrin (HST). However, a potential drawback is that the HST may increase the immunoreactivity of the peptide, in the same way that carrier proteins can be used to generate highly immunogenic peptide hapten conjugates. In this study we have generated a recombinant HST carrier protein that contains a peptide substrate of HIV-1 protease (VSQNYPIVL). The protein retained native HST function, and the peptide was surface exposed since it was immunoreactive in native dot blots, and was cleaved by HIV-1 protease. Immunisation of rabbits with the recombinant protein elicited only a very poor anti-peptide immune response. In contrast, strong anti-peptide immune responses were raised against both the peptide alone, and a chemical conjugate of the peptide with HST. These data demonstrate that it is possible to attenuate the immune response normally directed against an immunogenic peptide sequence by engineering into a surface exposed loop of HST. These findings may have an important impact on the future design of peptide delivery systems.

Analytical properties of immunosensors working in organic media.

The compatibility between organic solvents and immunoreagents was studied for the development of immunosensors for pesticides. On the basis on heterogeneous competitive enzyme formats, three assay types were used: immobilized antibodies (direct format), immobilized hapten conjugates (indirect format), and capture format based on immobilized protein A/G. In all cases, peroxidase enzyme label and fluorometric detection were employed. Initial findings were developed in batch working with the immunoreagents in different solvent systems (pure and mixed) to retrieve basic information about their performances. Monoclonal and polyclonal antibodies for carbaryl and 1-naphthol were used to develop sensors in different organic solvent mixtures. Polyclonal antibodies showed better sensitivity than monoclonal ones in comparable conditions. Sensitivity was better in the more polar solvents, methanol being the best. Comparison with an aqueous immunosensor showed lower sensitivity but better selectivity.

Improved Enzyme-Linked Immunosorbent Assay To Detect Didecyldimethylammonium Chloride, a Quaternary Ammonium Compound

Didecyldimethylammonium chloride (DDAC) is a commonly used wood protectant in Canada and must be monitored due to environmental concerns. Three new DDAC-like immunogens were synthesized and tested for their ability to produce antibodies that complex DDAC. The polyclonal antisera from rabbits immunized with the new hapten conjugates were compared by hapten homologous indirect competitive enzyme-linked immunosorbent assay (CELISA). The resulting antisera recognized DDAC to different degrees. CELISA based on anti-DDAC 3, 2, and 1 antisera had IC50s of 0.05, 16.0, and 17.2 μg/mL, respectively. All the antisera were more sensitive to DDAC than a previously reported polyclonal antiserum. The most sensitive and selective antiserum was produced from the hapten that most closely mimicked DDAC. The selectivity of the three immunoassays for compounds representing DDAC's different epitopes was tested. Each immunoassay exhibited the highest selectivity with compounds that most closely resembled the hapten. The effect of solvents and detergents was determined for the most sensitive immunoassay: CELISA based on anti-DDAC 3 antiserum. Recovery experiments were also carried out with this assay. This immunoassay (IC50 50 ppb) holds promise for the analysis of environmental samples since the regulatory discharge limit is 700 ppb. Keywords: Didecyldimethylammonium chloride; quaternary ammonium compounds; ELISA; immunoassay

Development of a Class-Specific Competitive ELISA for the Benzoylphenylurea Insecticides

A competitive enzyme-linked immunosorbert assay (ELISA) was developed for the quantitative detection of five benzophenylurea insecticides in soil (90% methanol extraction) and water. No significant matrix effects were observed. Conjugates of diflubenzuron derivatives with proteins were prepared by attachment at either the urea nitrogen of diflubenzuron or the 4-position of the aniline ring, wherein bridging groups of various lengths were used between the hapten and the protein carrier. All combinations of antiserum to each immunogen and enzyme tracer were examined, and an assay was developed using an antibody developed to a hapten conjugate coupled through a succinyl chain at the 4-aniline position. The assay gave 50% inhibition of antibody binding (IC50) at 0.6 ppb for diflubenzuron, 5 ppb for teflubenzuron, 10 ppb for flufenoxuron, 31 ppb for lufenuron, and 45 ppb for chlorfluazuron, with detection limits in the range of 0.05−2.3 ppb for these compounds. The basis for the superior cross-reaction of diflubenzuron was examined by molecular modeling, which suggested that the planarity of the molecules, electron-withdrawing groups, and steric effects of chlorines attached to the phenyl ring may be critical factors affecting antibody binding. Keywords: Benzoylphenylurea; polyclonal antibody; ELISA; molecular modeling

Development of an Enzyme-Linked Immunosorbent Assay for the Detection of the Pyrethroid Insecticide Fenpropathrin

A competitive enzyme-linked immunosorbent assay (ELISA) was developed for the quantitative detection of fenpropathrin [(RS)-R-cyano-3-phenoxybenzyl-2,2,3,3-tetramethylcyclopropanecarboxylate]. Polyclonal antisera were isolated from rabbits immunized with two different fenpropathrin hapten conjugates. One hapten contained an amino function; the other contained a carboxyl group for conjugation to carrier proteins. Mollusk hemocyanins, thyroglobulin, and fetuin were used as carrier proteins. The antisera varied greatly in their affinities for fenpropathrin. A homologous assay system using the coating antigen format was the most sensitive. The IC50 for fenpropathrin was 20 Ig/L, and the lower detection limit was 2.5 Ig/L. Pyrethroids, such as phenothrin, permethrin, resmethrin, fenvalerate, deltamethrin, cyfluthrin, and cypermethrin, and the pyrethroid metabolites, 3-phenoxybenzoic acid and fenpropathrin acid, did not cross-react significantly in this assay. Ten percent acetone or methanol and a pH of 4 were determined to be optimum assay conditions. Various cationic, anionic, and nonionic detergents had no significant effect on the assay.

In vitro analysis of xenoestrogens by enzyme linked receptor assays (ELRA).

Receptor binding assays are a modern approach to the effects-related analysis of xenoendocrines. Human estrogen receptors are used to develop an analytical tool for the detection of estrogenic substances in environmental samples. A sensitive Enzyme Linked Receptor Assay (ELRA) was developed in a microwell plate format. The receptor assay is based on similar principles as competitive immunoassays (ELISA). However, receptor binding always implies a biological effect, either agonistic or antagonistic. The choice of suitable tracers or hapten conjugates is an important step in assay development. For this purpose and for the determination of receptor-affinities of relevant xenoestrogens, a surface plasmon resonance (SPR) biosensor (BIAcore) was used for binding studies with immobilized receptors. Results with commercially available hapten-conjugates (estradiol-BSA) show a direct correlation between the amount of immobilized estradiol receptor and the amount of bound hapten-conjugate. Based on these BIAcore experiments an ELRA was developed. The calibration curves show a detection limit of 0.1 microgram/l for 17 beta-estradiol. Cross-reactivities of different steroids and xenoestrogens are reported.

Long-lasting unresponsiveness to polyclonal T cell-binding immunoglobulins.

We have shown that mice after a single injection of anti-T cell antibody followed by multiple injections of a second xeno-, allo- or syngeneic anti-T cell antibody differing from the former in species origin developed specific, long-lasting tolerance to the second antibody. To characterize the mechanism of this anti-antibody unresponsiveness and the modalities accompanying the preinjection step, we injected mice with anti-pan T, anti-CD4 or anti-CD8 monoclonal antibodies, followed by multiple injections of polyclonal rabbit anti-mouse thymocyte globulin (RbATG). Our observations indicate that: (i) Depletion of CD4+ cells is the most important factor for tolerance induction to subsequently injected RbATG. Non-depleting mAb were less effective, and antibody-induced CD4 modulation or blockade were inconsequential. (ii) The prevention of anti-antibody responses involves specific B cell tolerance, as shown by suppression of anti-RbATG but not anti-bovine serum albumin (BSA) antibodies after challenge of tolerant mice with RbATG-BSA conjugates. (iii) Suppression of anti-antibody responses involves T cell unresponsiveness rather marginally, as demonstrated by in vitro spleen cell restimulation with RbATG and in vivo antibody response to RbATG-fluorescein isothiocyanate hapten conjugate. (iv) Analysis of isotypes of anti-RbATG antibodies does not suggest alterations in Th1/Th2 tuning as being responsible for this kind of tolerance. (v) Over 150-day survival of fully allogeneic skin grafts (CBA-to-C57BL/6) was observed in mice preinjected with anti pan-T or anti-CD4 mAb followed by RbATG. Mice treated with anti-CD4 mAb alone rejected allografts within 21 days and induced anti-antibodies. Taken together, our experiments suggest B cell tolerance as main mechanism in this type of acquired long-term humoral unresponsiveness to foreign, polyclonal, T cell-binding immunoglobulins.

Derivation and Properties of Recombinant Fab Antibodies to the Phenylurea Herbicide Diuron

The Fab domain sequences from messenger RNA of a mouse hybridoma cell line secreting a monoclonal antibody (MAb) specific for the phenylurea herbicide diuron were amplified and inserted into the M13 phagemid vector pComb8. Phage displaying diuron-specific Fab fragments were selected by binding to magnetic beads coated with diuron hapten conjugates, and eluted with diuron. Soluble rFab in E. coli lysates bound diuron with half-maximal inhibition (I50) of 1.6−12 ppb (ng/mL) in indirect and direct competition enzyme immunoassays (EIAs). The optimal competing hapten was different for indirect EIAs with proteolytic or recombinant Fabs and the intact MAb. Selectivity of rFabs for phenylureas was similar to that of the original MAb and its proteolytic Fab fragment and was not significantly affected by exchange of heavy and light chains among rFab clones, indicating that a single antibody species had been cloned. Keywords: Immunoassay; recombinant antibodies; monoclonal antibodies; diuron; herbicide; phenylurea; arylurea

Induction of an epitope-specific humoral immune response by lipopeptide-hapten conjugates: enhancement of the anti-melittin response by a synthetic T helper (Th)-cell epitope.

Lipopeptides of bacterial origin constitute potent immunoadjuvants when combined with antigens. After the immunization with lipopeptides covalently coupled to non-immunogenic low-molecular-mass antigens or haptens, a hapten-specific humoral immune response can often be obtained. The response against synthetically prepared melittin fragments was further enhanced by the additional introduction of a T helper (Th)-cell epitope into the lipopeptide-hapten conjugate. The Th-cell epitope applied, which is presented by the MHC class II molecule of the BALB/c (H-2d) haplotype, consisted of a synthetic 16-amino-acid oligopeptide derived from sperm whale myoglobin. The immune-enhancing effect was most pronounced for the melittin-derived peptide fragments [Mel(1-16)] and [Mel(17-26)-CONH2]. Antibodies obtained after 3 immunizations with the conjugates recognized the synthetic as well as the native melittin molecule. Our results show that it is possible to markedly enhance a weak hapten-specific immune response by coupling the haptens to a lipopeptide conjugated to a haplotype-specific T helper-cell epitope. The novel conjugates are well suited for the optimization of immunization procedures, and for the development of novel synthetic vaccines.

Induction of an epitope-specific humoral immune response by lipopeptide–hapten conjugates: enhancement of the anti-melittin response by a synthetic T helper (T h)-cell epitope

Lipopeptides of bacterial origin constitute potent immunoadjuvants when combined with antigens. After the immunization with lipopeptides covalently coupled to non-immunogenic low-molecular-mass antigens or haptens, a hapten-specific humoral immune response can often be obtained. The response against synthetically prepared melittin fragments was further enhanced by the additional introduction of a T helper (T h)-cell epitope into the lipopeptide–hapten conjugate. The T h-cell epitope applied, which is presented by the MHC class II molecule of the BALB/c (H-2 d) haplotype, consisted of a synthetic 16-amino-acid oligopeptide derived from sperm whale myoglobin. The immune-enhancing effect was most pronounced for the melittin-derived peptide fragments [Mel(1–16)] and [Mel(17–26)-CONH 2]. Antibodies obtained after 3 immunizations with the conjugates recognized the synthetic as well as the native melittin molecule. Our results show that it is possible to markedly enhance a weak hapten-specific immune response by coupling the haptens to a lipopeptide conjugated to a haplotype-specific T helper-cell epitope. The novel conjugates are well suited for the optimization of immunization procedures, and for the development of novel synthetic vaccines.

Down-regulation of poison ivy/oak-induced contact sensitivity by treatment with a class II MHC binding peptide:hapten conjugate.

Immune regulation of contact sensitivity to the poison ivy/oak catechol was studied at the level of class II MHC-restricted T cell recognition of hapten:peptide conjugates. In this study we have shown that 1) T cells from C3H/HeN (H-2k) mice, immunized with a synthetic I-Ak binding peptide coupled to 3-pentadecyl-catechol (PDC; a representative catechol in urushiol), recognized peptides derived from syngeneic cells linked to the same catechol; 2) T cells from draining lymph nodes of C3H/HeN mice skin-painted with PDC proliferated in response to a peptide carrier:PDC conjugate only when it was linked at the 7th, but not the 4th or the 10th, position on the peptide carrier; and 3) tolerization studies confirmed down-regulation of PDC-induced delayed-type hypersensitivity following treatment with a single I-Ak binding peptide carrying PDC covalently bound to a lysine residue at the middle (7th) TCR contact position. Tolerization with peptide:PDC conjugate resulted in abrogation of hapten-specific T cell proliferative responses that correlated with diminished IL-2 secretion. On the basis of these data we propose that it may be sufficient to couple the hapten at a single, well-chosen position on a carrier peptide to target a relevant population of T cells involved in contact sensitivity.

Production and characterization of monoclonal antibodies against two haptenic derivatives of 1 alpha,25-dihydroxyvitamin D3 conjugated with bovine serum albumin through the C-3 or C-24 position.

To develop the immunochemical methods for determining 1 alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3] in clinical samples, a variety of monoclonal anti-1,25(OH)2D3 antibodies have been generated. Two kinds of hapten-carrier conjugates, 25-hydroxyvitamin D3 3-hemisuccinate (hapten 3-HS) and 1 alpha-hydroxy-25,26,27-trinorvitamin D3 24-oic acid (hapten 24-OA) conjugated with bovine serum albumin, were used for immunization. Spleen cells from SD rats or BALB/c mice, each immunized with the conjugate of hapten 3-HS or 24-OA, were fused with P3/NS1/1-Ag4-1 myeloma cells. After screening by ELISA employing beta-galactosidase-labeled haptens, seven kinds of hybridomas secreting anti-1,25(OH)2D3 antibodies were established. Binding characteristics of these antibodies (Ka 0.73-20 x 10(9) M-1) were investigated by an RIA using tritium-labeled 1,25(OH)2D3. The data suggested that the rat monoclonal antibody 3R-1 derived from the hapten 3-HS and the mouse monoclonal antibody 24M-3 from the hapten 24-OA would be available for developing practical analytical systems.

5455032 Use of phosphocholine hapten conjugates in vaccines : Kenny James J; Long Dan L Rockville, MD, United States assigned to The United States of America as represented by the Department of Health and Human Services

5455032 Use of phosphocholine hapten conjugates in vaccines : Kenny James J; Long Dan L Rockville, MD, United States assigned to The United States of America as represented by the Department of Health and Human Services

N epsilon-cyclosuccinyllysine: synthesis and formation from hemisuccinylated polylysine and its hapten conjugates with sulfamethoxazole during acid hydrolysis.

The novel amino acid N epsilon-cyclosuccinyllysine (4) forms as a side reaction during acid hydrolysis of N epsilon-hemisuccinylated succinylsulfamethoxazole-polylysine conjugates. The presence of 4 in hydrolysates can be obscured in amino acid analysis. Identification of 4 was based on chromatographic and electrophoretic comparisons with authentic N epsilon-cyclosuccinyllysine, which was synthesized in high yield by acid-catalyzed ring closure, under anhydrous conditions, of N epsilon-hemisuccinyl-L-lysine. The latter was prepared by an improved route, starting from N-alpha-(tert-butyloxycarbonyl)-L-lysine. The amount of 4 formed is influenced by the extent of succinylation and the conditions of hydrolysis and has reached 30 mol % of lysine. Both the hemisuccinyllysyl and the haptenized succinyllysyl residues participate in cyclization. This was confirmed by the synthesis and hydrolytic behavior of hemisuccinylated polylysine (n = 16) and N4,N epsilon-succinyl(sulfamethoxazole)(lysine). Formation of 4 can result in error in the estimation of the degree of ligand substitution based on lysine content in synthetic hemisuccinylated skin test antigens and peptide immunogens, especially when the degree of epitope substitution is low. 4 appears to be sufficiently stable for trial as an amino acid surrogate.

PS-51-2 Isokinetic muscle strength in long-term IDDM patients in relation to neuropathy

A method is reported for the solid-state fluorodensitometric determination of haptens in hapten—protein conjugates. The applicability of the method is shown using the bovine serum albumin conjugate of cleistanthin B, a toxic glycoside of the plant Cleistanthus collinus. This non-destructive technique, which is relatively simple, sensitive, rapid and versatile, can be used for the determination of protein conjugates of fluorogenic haptens.

Immunochemical techniques: Antibody production for pesticide analysis. A review

Immunochemical techniques supplement traditional analytical methods in an ideal way because they are extremely sensitive, simple, and inexpensive. The most important types presently used for environmental analysis are immunoassays, immunosensors, immunochromatography, and immunolabeling. Representative examples are given. Standardized immuno-chemical methods for environmental monitoring profit from monoclonal antibodies because of defined selectivities and affinities toward the analyte. A critical step in the production of monoclonal antibodies is the development of efficient screening procedures to identify suitable hybridoma lines. Immunomagnetic beads offer an alternative to conventional screening with immunoassays because the problems of being time-consuming, escaping detection or loosing valuable cells can be solved. This technique is based on tagging surface receptors of hybridoma cells with binding properties of the secreted antibodies by magnetic beads, which are coated with hapten conjugates. In spite of the considerable progress in hybridoma technology, antibody development has entered an entirely new domain because of the potential of recombinant antibodies. Recombinant technology will eventually allow faster production of antibodies with new binding properties (mutant antibodies) and also a stepwise abandonment of animal experiments required for antibody production. The production of phage antibodies for s-triazines is described.

Taxane-Specific Monoclonal Antibodies: Measurement of Taxol, Baccatin III, and "Total Taxanes" in Taxus brevifolia Extracts by Enzyme Immunoassay

Three monoclonal antibodies with either specificity to taxol or baccatin III, or cross-reactivity with several common taxanes have been prepared and used to develop sensitive competitive-inhibition enzyme immunoassays. The hybridomas producing these monoclonal antibodies were obtained by fusing P3X63Ag8.653 plasmacytoma cells and splenocytes from mice hyperimmunized with keyhole limpet hemocyanin-7-succinyltaxol or -7-succinylbaccatin III conjugates. Direct and indirect competitive inhibition enzyme immunoassays were developed with these monoclonal antibodies and microtiter plates coated with bovine serum albumin conjugates of the complementary hapten. Detection limits for the direct competitive inhibition enzyme immunoassays, conducted in buffer containing 10% MeOH, were 0.6 nM taxol for 3C6 (anti-taxol); 1.1 nM baccatin III for 3H5 (anti-baccatin III); and 0.6 nM taxol or baccatin III for 8A10 (anti-taxane). The immunoassays accurately detected taxol, baccatin III, and "total taxanes" in crude MeOH extracts of Taxus brevifolia bark and in hplc fractions of these extracts.

Engineering a unique glycosylation site for site-specific conjugation of haptens to antibody fragments.

A natural N-linked glycosylation site (Asn-Val-Thr) at amino acid positions 18-20 (Kabat's numbering) was identified in the framework-1 (FR-1) region of the light chain variable (V kappa) domain of a murine anti-B cell lymphoma Ab, LL-2. Our earlier studies demonstrated that no contact between the V kappa-appended oligosaccharide and the Ag binding site was evident, because glycosylation at this site did not affect the Ag binding property of the Ab. By using the murine LL-2 F(ab')2 fragment (which is devoid of constant region-appended oligosaccharide) as substrate, as much as five bifunctional chelator molecules per F(ab')2 fragment could be site specifically conjugated at the V kappa-appended carbohydrate moiety with no reduction in immunoreactivity. The resulting conjugates labeled efficiently with both 90Y and 111In, with no significant effect on Ab affinity. In contrast, conjugation of less than five chelates/Ab fragment randomly at lysine residues resulted in a three- to fivefold reduction in affinity. By a single Arg to Asn mutation, an N-linked glycosylation site similar to that of LL-2 was introduced in the FR-1 segment of a nonglycosylated, humanized anti-carcinoembryonic Ag (CEA) Ab, MN-14 (hMN-14). Glycosylation at the engineered carbohydrate-addition site was demonstrated by SDS-PAGE analysis. Neither glycosylation nor site-specific conjugation of chelate at the V kappa-appended carbohydrate moiety resulted in the loss of immunoreactivity. The glycosylated hMN-14 conjugate labeled efficiently with 90Y.

Modified Immunoassays for Polyether Toxins: Implications of Biological Matrixes, Metabolic States, and Epitope Recognition

Polyether marine toxins are responsible for the seafood intoxication phenomena known as neurotoxic shellfish poisoning (due to brevetoxins), ciguatera (due to ciguatoxin), and diarrheic shellfish poisoning (due to okadaic acid). Using traditional techniques of hapten (pure toxin) conjugation to protein to create complete antigen, animal immunization and antibody isolation, and specific antibody subpopulation purification, discriminating antibodies have been isolated that detect brevetoxins and ciguatoxin, but not okadaic acid, in a dose-dependent fashion. Using microorganic chemistry and purified toxins, a unique set of tools has been created for the study of polyether ladder toxin accumulation; depuration; and specific site localization in tissues, food sources, and clinical samples. Developed test protocols can detect toxin in dinoflagellate cells, in extracts from food sources, in seawater and culture media, and in human serum samples. Enzyme-linked immunosorbent assay protocols developed for eventual collaborative testing have been successful in limited applications within the laboratory (correlation coefficient of 0.92 excluding 2 outliers), and alternative formats are being developed to optimize the basic test for use in research laboratories, regulatory laboratories, and field inspections.

Monoclonal antibodies specific for the organophosphate pesticide azinphos‐methyl

2‐(2‐Mercapto‐5‐methyl‐1,3,2‐dioxaphosphorinan‐5‐yl,2‐sulphide)methoxyacetic acid has been synthesized and used to prepare an azinphos hapten and protein conjugates. Monoclonal antibodies of high affinity against the pesticide azinphos‐methyl were prepared from mice immunized with the hapten‐ovalbumin conjugate. The best monoclonal antibody could be used in an indirect enzyme‐linked immunosorbent assay (ELISA) to quantify azinphos‐methyl at concentrations from 50 pg ml‐1 to 2 ng ml‐1 (2.5–100 pg/assay). The corresponding limit of detection of the pesticide was less than 40 pg ml‐1. Only the closely related pesticide azinphos‐ethyl showed any appreciable cross‐reactivity in this assay.