Low Molecular Weight Haptens Research Articles

Immunologic Responses to Isocyanates in Sensitized Asthmatic Subjects

Isocyanates, highly reactive compounds used extensively in industry, are the most commonly reported cause of occupational asthma in industrial populations.1 Sensitized individuals may develop immediate and/or late asthmatic responses following isocyanate exposure.2 The mechanisms by which isocyanates cause asthma are not well defined. The limited information available suggests that isocyanate asthma is mediated via predominantly non-IgE mechanisms, and that isocyanates may act like a foreign low molecular weight hapten, inducing antigen-specific T-cell responses and airway inflammation.

Crystal structure of a catalytic antibody Fab with esterase-like activity.

Antibodies with catalytic properties can be prepared by eliciting an antibody response against 'transition state analog' haptens. The specificity, rate and number of reaction cycles observed with these antibodies more closely resemble the properties of enzymes than any of the many other known enzyme-mimicking systems. We have determined to 3 A resolution the first X-ray structure of a catalytic antibody Fab. This antibody catalyzes the hydrolysis of a p-nitrophenyl ester. In conjunction with binding studies in solution, this structure of the uncomplexed site suggests a model for transition state fixation where two tyrosines mimic the oxyanion binding hole of serine proteases. A comparison with the structures of known Fabs specific for low molecular weight haptens reveals that this catalytic antibody has an unusually long groove at its combining site. Since transition state analogs contain elements of the desired product, product inhibition is a severe problem in antibody catalysis. The observation of a long groove at the combining site may relate to the ability of this catalytic antibody to achieve multiple cycles of reaction.

10] Coupling of carbohydrates to proteins by diazonium and phenylisothiocyanate reactions

Haptens are small molecules that are incapable of stimulating antibody formation unless they are chemically linked to a macromolecule. In the process of diazotization, low molecular weight haptens may be rendered immunogenic by covalent conjugation to protein. This chapter describes the coupling reactions of carbohydrates to proteins using either the diazotization reaction, or the phenylisothiocyanate procedure. A host of Salmonella typhimurium O-antigen oligosaccharides has been generated from their respective polysaccharides, converted to the corresponding p -phenylisothiocyanto glycosides, and coupled to various proteins to be used as synthetic vaccines. This chapter discusses the phenylisothiocyanate procedure to couple p -isothiocyanatophenyl β -D-glucopyranoside, p -isothiocyanatophenyl β -L-glucopyranoside, and p -isothiocyanatophenyl α -D-mannopyranoside to bovine serum albumin (BSA). However, the method may readily be extended to a variety of noncarbohydrate haptens and to other natural or synthetic macromolecules. For example, pisothiocyanatophenyl-derivatized haptens may be reacted with solid matrices to afford solid column absorbents for affinity chromatography.

Identification and characterization of a monoclonal antibody that neutralizes ricin toxicity in vitro and in vivo.

We wanted to identify and characterize MAbs with specificity for the toxic lectin ricin, which could serve as detection reagents in elucidating mechanisms and tissue distribution. Neutralizing MAbs could be developed into immunotherapeutics to reverse clinical intoxications from immunotoxin or to counteract the use of ricin as a terrorist or biological warfare weapon. Two hybridomas, UNIVAX 70 and 138, producing MAbs against ricin were identified by Western blot strip analysis. The antibodies were IgG1 and were specific for the ricin A chain with no ricin B chain cross-reactivity. The MAbs neutralized ricin in vitro in an EL-4 mouse leukemia cell assay and in an in vivo mouse model. The two antibodies recognized the same epitope or overlapping epitopes, based on a competition with one another. All further characterization proceeded on the assumption that they were the same. The MAb UNIVAX 70/138 was characterized in vivo by titrating it against an 18 micrograms/kg (> six LD50) i.v. challenge and by titrating the i.v. toxin challenge against a constant dose of 100 micrograms of passive antibody per mouse. A 4:1 molar ratio of MAb to ricin led to neutralization of > or = 90% of the toxin in vitro. The MAb recognized ricin toxoid prepared by formaldehyde treatment and after conjugation of low molecular weight haptens (based on ELISA) equally as well as it recognized ricin and ricin A chain. The affinity and specificity of UNIVAX 70/138 give it excellent reagent potential, and the toxin-neutralizing capacity makes it at least a log and a half better than the next best candidate immunotherapeutic.

Immunometric assay of low molecular weight haptens containing primary amino groups.

A new enzyme immunometric assay of small haptens containing primary amino groups (thyroxine, MW 777; substance P, MW 1347; endothelin, MW 2492) is described. The procedure involves different sequential steps: (1) immunocapture of the haptens (standard or sample) by monoclonal anti-hapten antibodies coated on 96-well microtiter plates; (2) cross-linking of haptens via their amino groups to the wells using homobifunctional reagents (glutaraldehyde or disuccinimidyl suberate); (3) denaturing treatments (HCl or methanol); (4) measurement of linked epitope using the same monoclonal anti-hapten antibodies labeled with acetylcholinesterase. A minimal detectable concentration in the 4-10 fmoL/mL range was observed. Each assay appeared to be 70-200 times more sensitive than conventional competitive enzyme immunoassay using the same monoclonal antibody-coated plate technology and acetylcholinesterase-hapten conjugates as enzymatic tracers. Precision and specificity were very satisfying. Good correlation was noted between this assay and the competitive assays performed for different biological samples (plasma, tissues, or supernatant cell culture).

Monoclonal antibodies in hapten immunoassays.

This review deals with the potency of monoclonal antibodies (MAbs) to haptens in immunoassays. Specificity and affinity of MAbs to haptens are the major determinants to be considered. Specificity of MAbs depends on the selection of the hapten coupling site to the carrier protein and the antigen used for the screening of MAbs. Nevertheless, cross-reactivity can occur with compounds related to the hapten. This polyspecificity may be circumvented with the use of many MAbs, as has been demonstrated for MAbs to cyclosporine. Affinity of MAbs to haptens is often lower than that of corresponding polyclonal antibodies (PAbs), thereby limiting assay sensitivity. Low affinity is more frequently observed with low molecular weight (100-300) haptens than with larger haptens, such as digoxin or cyclosporine. Affinity enhancement by increasing resemblance to the immunogen can be effective in resolving the lack of sensitivity. With suitable selection strategies. MAbs exhibit real advantages over classical PAbs to haptens because large amounts of worldwide standardized reagents can be prepared.

A rapid whole‐blood immunoassay system

A new immunoassay system has been developed which allows the detection of circulating antigens, antibodies or drugs in whole blood without specialized personnel or equipment. This is achieved by the use of bispecific reagents, which comprise specific antibodies or antigens that are coupled to a non-agglutinating antierythrocyte antibody. Within two minutes, these reagents cause specific agglutination of a patient's own red cells in samples that contain the relevant analyte. Levels of low molecular weight haptens also can be measured by the use of an indirect, agglutination-inhibition assay. This simple immunoassay method would fulfil the needs of many physicians and Third-World countries and also has mass-screening and veterinary applications.

Intestinal transport of sulfanilic acid in rats immunized with protein-sulfanilic acid conjugate.

Intestinal transport of sulfanilic acid was examined by means of an in vitro everted sac technique in rats immunized with a bovine gamma-globulin-sulfanilic acid conjugate. At a low concentration of sulfanilic acid, the intestinal transport of sulfanilic acid was decreased in rats immunized with bovine gamma-globulin-sulfanilic acid conjugate. This phenomenon was dose dependent and antigen specific, since there was no difference in the transport of sulfanilic acid at a high concentration and of an unrelated hapten. These results suggested that parenteral immunization impaired not only the intestinal transport of macromolecular antigens, as previously shown, but also the transport of the low molecular weight hapten, sulfanilic acid.

Iodoacetylated and biotinylated liposomes: effect of spacer length on sulfhydryl ligand binding and avidin precipitability

Because of the sustained interest in liposomes as immunogens and vehicles for drug delivery, the present investigation was designed to reevaluate the iodoacetyl group as a means of binding sulfhydryl-containing substances to liposomes in thioether linkage, and to develop an alternative method by which liposomes with bound ligand can be conveniently and rapidly separated from free ligand. For the purpose of the first goal, we synthesized a homologous series of dimyristoylphosphatidylethanolamine (DMPE) derivatives in which the iodoacetyl (IA) function was separated from the phospholipid amino group by either 0, 1, or 2 aminoethylthioacetyl (AETA) spacers. Results show that liposomes prepared with IA-DMPE can not bind 125I-radiolabeled rabbit IgG which had been thiolated by reaction with S- acetylmercaptosuccinic anhydride . Significant IgG attachment was, however, obtained with liposomes containing either IA-AETA-DMPE or IA-(AETA) 2-DMPE, and the amount bound was directly related to spacer length. In contrast, spacer length had no effect on the covalent binding of a low molecular weight hapten, N- dinitrophenylcysteine . Other parameters (incubation time, IgG concentration, density of IA-(AETA) 2-DMPE, sulfhydryl inhibitors) were also examined. To achieve the second objective, biotinyl-(AETA) 2-DMPE was incorporated into the same liposomal bilayers that contained the iodoacetylated derivatives. Thus, liposomes with bound ligand could be readily precipitated by avidin, and washed free of unreacted IgG by low speed centrifugation. Comparative experiments with liposomes containing biotinyl-DMPE revealed that spacer length also had a pronounced effect on the avidin precipitability of liposomes in the presence of proteins that may be non-covalently absorbed or covalently bound to the model membrane surface.

Cross-reactivity between sesquiterpene lactones related to parthenin in parthenin-sensitized guinea pigs.

The naturally‐occurring sesquiterpene lactone, parthenin, is responsible for a high incidence of allergic contact dermatitis in India. Cross‐sensitivity tests on guinea pigs sensitized to parthenin indicate a relatively high immunological specificity for this low molecular weight hapten. Coronopilin and damsin, sesquiterpene lactones most similar to parthenin, gave positive reactions but other structurally related lactones such as dihydroisoparthenin, tetra‐hydroparthenin, tetraneurin‐A, hysterin, helenalin, and tenulin, or hymenin, a diastereoisomer of parthenin, were negative.

Distance between two binding sites of the same antibody molecule: A neutron small-angle scattering study of pig anti-Dnp antibody complexed with mono-Dnp-dextran

Although the general features of the antibody mole- cules are known, considerable uncertainty still exists as to their shape when present in immune complexes. Small-angle X-ray scattering analysis employed [ 1,2] showed that the radii of gyration of rabbit antibodies decrease upon binding low-molecular weight haptens. With the use of neutron small-angle scattering, we have shown [3]. that pig antibodies which occur early and late during an immune response to Dnp-antigens and differ by their ability to precipitate antigen [4], differ by their radii of gyration as well; when reacting with a low-molecular weight hapten, the radii of gyra- tion of the early and late antibodies decrease by 4.6% and 7.5%, respectively. This paper reports on neutron small-angle scattering experiments aimed at determining the distance between the two binding sites of the same antibody molecule employing complexes of anti-Dnp antibody with an antigenically univalent, high molecular weight ligand. The contrast variation method offers a possibility to measure the distance between two ligands bound to the same molecule when the condition is fulfilled that the ligands have a sufficiently large volume and that their neutron scattering density differs from that of the antibody. To this end, a high molecular weight

Neutron small-angle scattering study on two different precipitin types of pig Anti-Dnp antibodies

Anti-Dnp antibodies elicited in pigs at an early phase of the immune response were previously shown to react with polyvalent high molecular weight Dnpantigens in a different way than antibodies elicited at a later phase [l] . While the early antibodies form mostly insoluble complexes (i.e., precipitates) when interacting with heavily Dnp-substituted proteins, the late antibodies mostly form soluble complexes with the same antigens. It has been suggested that early and late antibodies differ in their shapes and/or the flexibilities of their F, arms [l] . Neutron smallangle scattering experiments were carried out to test this assumption. The present paper reports that the early and late antibody molecules do differ by their radii of gyration. Moreover, it is shown that the radii of gyration change upon complexing the antibodies with a low molecular weight hapten, the extent of change being approximately the same for both the early and late antibodies.

Rat thoracic duct cells as a substitute for T cells and carrier in the antibody response of mouse spleens deficient in thymus cells.

The immune response of mouse spleen cells to sheep red blood cells (SRBC) in vitro depends on the presence of thymus derived helper lymphocytes (T cells). This has been demonstrated by depleting spleen cells of T cells by treatment with complement and anti-θ serum1 and showing that the in vitro immune response can be restored by addition of antigen sensitized syngeneic or allogeneic T cells2. Although un-sensitized syngeneic T cells usually fail to restore the immune response of spleens deprived of T cells (B spleens), unsensitized allogeneic thymus cells do so3. This “allogeneic effect” can act through factors released in the culture supernatant of allogeneic lymph cell mixtures4. We show here that xenogeneic (rat) thoracic duct cells (RTDC), whether antigen sensitized or not, can restore an in vitro immune response of thymus-depleted mouse spleen cells. They stimulate antigen specific T cell responses by two mechanisms. In one, they substitute both for the carrier and the carrier-specific T cells and thus stimulate T cell response to a low molecular weight hapten. In the other, they stimulate T cells through amplification of the effect of a small number of carrier-specific mouse T cells.

Evaluation of some extraction methods for the preparation of bacterial lipopolysaccharides for structural analysis.

The extraction of lipopolysaccharides (LPS) from smooth strains of Salmonella typhimurium for structural studies using methylation analysis is preferably done by hot phenol‐water method on a cell‐wall preparation obtained by mechanical disintegration of γ‐irradiated bacteria. The LPS obtained by this method was less contaminated than LPS extracted by hot phenol‐water from acetone dried bacteria or bacteria pretreated with 1 per cent formaldehyde. LPS for structural analysis from rough mutants is, due to its lipophilic character, preferably extracted with a mixture of phenol, chloroform and petroleum ether. The presence of the precursor of the O side‐chain, LI‐hapten, in the different LPS‐ and supernatant fractions was assessed by gel precipitation tests. The LPS forms a line near the antigen well whereas the low molecular weight hapten forms a line near the antiserum well. The hapten was not formed in excess in the smooth strain S. typhimurium LT2. In the rough mutant studied, S. typhimurium SL 733, O‐antigenic material was found in both the LPS‐preparation (probably as a covalently linked O side‐chain) and the supernatant (as a hapten).