Determining and characterizing hapten loads for carrier proteins by MALDI-TOF MS and MALDI-TOF/RTOF MS.

The increasing number of bioconjugates used for bioanalytical purposes and in pharmaceutical industries has led to an increasing demand for robust quality control of products derived from covalently linking small molecules to proteins. Here we report, for the first time, a matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF)-based method to determine the quantity and location of the hapten zearalenone (ZEN) introduced to the carrier protein conalbumin (Con). This bioconjugate is of special interest because of its application in lateral flow immunoassays commercially available for fast testing of food and feed for the presence of ZEN, a common contaminant of all major cereal grains worldwide. Mass spectrometry (MS) analysis of the intact protein turned out to be highly reproducible allowing for the determination of the average hapten load of the carrier protein. In that way an easy and fast method to screen for changes in ZEN load after bioconjugate synthesis was established. For a more detailed hapten load characterization, measurements at the peptide level were of importance. Systematic studies, implementing post-source decay (PSD) and high- and low-energy collision-induced dissociation (CID), showed characteristic fragmentation pattern for three model peptides carrying between one and three lysines (the primary target for the ZEN modification) besides other, less obvious modification sites (serine, arginine and the N-terminus). By this, indicative reporter ions (m/z 203 and 316) and neutral losses (Δm/z 373 and 317) for the ZEN modification in general, plus immonium ions (m/z 87, 142 and 159) for the lysine modification in particular were identified. Based on these findings, proteolytic peptides, tentatively assigned to be modified, were unequivocally confirmed to be affected by bioconjugation. For a protein carrying on average only 2-3 modifications per molecule 29 Lys out of 59 potential modifications sites were actually modified. Considerations taking the protein structure into account showed that the affected Lys were predominantly located on the protein's surface.

Insight into the Mode of Action of Celangulin V on the Transmembrane Potential of Midgut Cells in Lepidopteran Larvae.

Celangulin V (CV) is the main insecticidal constituent of Celastrus angulatus. The V-ATPase H subunit of the midgut cells of lepidopteran larvae is the putative target protein of CV. Here, we compared the effects of CV on the midgut membrane potentials of Mythimna separata and Agrotis ipsilon larvae with those of the Cry1Ab toxin from Bacillus thuringiensis and with those of inactive CV-MIA, a synthetic derivative of CV. We investigated the changes in the apical membrane potentials (Vam) and basolateral membrane potentials (Vbm) of the midguts of sixth-instar larvae force-fed with the test toxins. We also measured the Vam and Vbm of larval midguts that were directly incubated with the test toxins. Similar to the effect of Cry1Ab, the Vam of CV-treated midguts rapidly decayed over time in a dose-dependent manner. By contrast, CV-MIA did not influence Vam. Meanwhile, the Vam of A. ipsilon larval midguts directly incubated with CV decayed less than that of M. separata larval midguts, whereas that of larvae force-fed with CV did not significantly change. Similar to Cry1Ab, CV did not affect the Vbm of isolated midguts. CV significantly inhibited V-ATPase activity in a dose-dependent manner. Therefore, CV initially inhibits V-ATPase in the apical membrane and affects intracellular pH, homeostasis, and nutrient transport mechanisms in lepidopteran midgut cells.

Immunogenic properties of mannose-containing ceramide tetrasaccharide from fresh-water bivalve, Hyriopsis schlegelii.

Antiserum against GlcNAc beta 1----2Man alpha 1----3Man beta 1----4Glc beta 1----1Cer (MlOse4Cer), a mannolipid isolated from spermatozoa of the fresh-water bivalve Hyriopsis schlegelii, was elicited in rabbits by repeated injection of a mixture of hapten-bovine serum albumin with Freund's adjuvant. The specificity of the affinity-purified antibody obtained from the serum was based on two forms of enzyme-immunodetection of its binding to structurally related glycolipids, either adsorbed to microtiter plates or chromatographed on thin-layer plates. The purified antibody exhibited a significant cross-reactivity with GlcNAc beta 1----2Man alpha 1----3(Xyl beta 1----2)Man beta 1----4Glc beta 1----1Cer, (MIXOse5Cer) containing a core structure closely related to MlOse4Cer, but almost unrelated to other glycolipids. Distribution of MlOse4Cer and MlXOse5Cer in various bivalve and snail glycolipid extracts were screened in thin-layer immunobinding assays by using this purified specific antibody. The presence of the glycolipid antigens was limited to certain taxonomic orders of shellfish species.

Allergens of Parietaria judaica pollen--I. Purification and characterization of a hapten and a low molecular weight allergenic peptide.

A low mol. wt allergen (Pj-2) and a hapten (Pj-H3) were purified from Parietaria judaica pollen by means of long-term aqueous extraction, dialysis and gel filtrations. The yield of the Pj-2 allergen was 0.94% (w/v) of the total protein present in the aqueous extract of the pollen, while its allergenic activity was about 60% of the total dialyzable activity, as verified by skin prick tests, ELISA- and RAST-inhibition experiments. The homogeneity of this allergen was demonstrated by one single sharp peak on HPLC, one single band on PAGE-SDS and by one single arc on IEF. Its mol. wt, estimated by HPLC and amino acid composition, was 10,400. The amino acid analysis showed 73 amino acid residues, and lysine was predominant, with 20 residues. The hapten Pj-H3 was 0.2% (w/v) of the total protein found in the pollen aqueous extract. It was inactive in skin prick tests even at a protein concn of 2 mg/ml, while it was capable of inhibiting by 60% in ELISA- and RAST-inhibition experiments, suggesting an immunochemical relationship with both IgE and allergens specific to P. judaica. The homogeneity was demonstrated by one single sharp peak on HPLC and one single band on PAGE-SDS. The amino acid analysis showed 10 amino acid residues, with no specific traits, and the mol. wt determined by gel filtration and amino acid composition was 1000. An immunochemical relation between the allergen and the hapten was also suggested by the results of an ELISA-inhibition test, and by the ability of the hapten to partially inhibit the precipitin line between rabbit antibodies to whole P. judaica pollen extract and the Pj-2 allergen. The allergen and the hapten described above, purified at homogeneity and in an antigenically active state, both provide adequate material for further structural and immunological characterizations.

Peptide conjugated chitosan foam as a novel approach for capture-purification and rapid detection of hapten--example of ochratoxin A.

A novel bioassay for the detection and monitoring of Ochratoxin A (OTA), a natural carcinogenic mycotoxin produced by Aspergillus and Penicillium fungi, has been developed and applied for the screening of red wine. Here we report the immobilization and orientation of NOF4, a synthetic peptide, onto 3-D porous chitosan supports using a N-terminal histidine tag to allow binding to M(++) ions that were previously adsorbed onto the high surface area biopolymer. Three divalent cations (M(++)=Zn(++), Co(++), Ni(++)) were evaluated and were found to adsorb via a Langmuir model and to have binding capacities in the order Zn(++)>Co(++)>Ni(++). Following Zn(++) saturation and washing, C-terminus vs. the N-terminus His-tagged NOF4 was evaluated. At 1000 µg L(-1) OTA the N-terminus immobilization was more efficient (2.5 times) in the capture of OTA. HRP labeled OTA was added to the antigen solutions (standards or samples) and together competitively incubated on biospecific chitosan foam. The chemiluminescence substrate luminol was then added and after 5 min of enzymatic reaction, light emission signals (λmax=425 nm) were analyzed. Calibration curves of %B/B0 vs. OTA concentration in PBS showed that half-inhibition occurred at 1.17 µg L(-1), allowing a range of discrimination of 0.25 and 25 µg L(-1). In red wine, the minimum concentration of OTA that the system can detect was 0.5 µg L(-1) and could detect up to 5 µg L(-1). Assay validation was performed against immunoaffinity column (IAC) tandem reversed-phase high pressure liquid chromatography with fluorescence detection (HPLC-FLD) and provided quite good agreement. The association of chitosan foam and specific peptide represents a new approach with potential for both purification-concentration and detection of small molecules. In the future this assay will be implemented in a solid-sate bioelectronic format.

Universal reagent immunosorbent assay (URI) for haptens.

Preliminary data on the use of a universal reagent, 125I-labelled second antibody, for the measurement of small molecules using immunosorbent techniques are described. The results showed that for two chemically unrelated haptens, norethisterone and diphenoxylic acid, the procedure could be optimised to measure these molecules at concentrations similar to those measurable using conventional liquid-phase or solid-phase techniques. No significant loss of norethisterone-antibody specificity could be demonstrated.

A glass-fiber filtration assay for solution phase hapten-antibody binding. Effect of surface treatment with a polycation.

Radioligands bound to murine monoclonal and polyclonal antibodies in solution are rapidly and effectively separated from free ligand by filtration through either untreated or polyethylenimine (PEI)-pretreated glass-fiber filters. Ligand binding to selected anti-morphine monoclonal antibodies, determined by filtration through untreated filters, was significantly greater (1.5-3.0-fold) than the binding activities obtained by gel filtration. However, radioactivity retained by filters that were pretreated with PEI (pH 4) was essentially the same as that obtained using the Sephadex G-25 short-column assay. The pH dependence of the retention on the coated fibers suggests that the mechanism of binding of antibodies to glass is different from that to the PEI-treated surfaces. The quantitative aspects of the assay are reported. The method should prove useful where quantitative, rapid and inexpensive binding radioassays need to be performed.

Isolation of a human spermatozoal hapten "II2.2' and its reaction with naturally occurring human sperm-immobilizing sera from infertile patients.

An extract of human spermatozoa was prepared using Hyamine 2389 and Triton X-100. With gel and ion-exchange chromatography several fractions were obtained of which are reacted specifically with sperm-immobilizing antibodies of infertile females and males in an immune inhibition test. This fraction showed haptenic properties, ahd a molecular weight of 1600 and was excluded as an anticomplement factor. After conjugation with cytochrome c the hapten II2.2 formed precipitation reactions with 3 out of 4 sperm-immobilizing sera. The titer reduction in sperm-immobilizing sera after adsorption with II2.2 may represent an in vitro model for a possible treatment of infertility in cases of a humoral sensitization against spermatozoa. On the other hand, the hapten might easily be synthesized and could, after conjugation to an appropriate carrier, serve as a contraceptive vaccine.

Dogger bank itch. 4. An eczema-causing sulfoxonium ion from the marine animal, Alcyonidium gelatinosum [Bryozoa].

Repeated exposure to the marine bryozoan, Alcyonidium gelatinosum, frequently provokes an eczematous allergic contact dermatitis known as the "Dogger Bank Itch". The dermatitis, representing a severe occupational disease, is especially widely distributed among trawlermen working in the Dogger Bank area in the North Sea. The allergy is shown to belong to the type of cell-mediated hypersensitivity. The hapten has been identified as the (2-hydroxyethyl)dimethylsulfoxonium ion. The isolation, structure determination and synthesis are discussed.

Isolation of a novel lipid hapten, cytolipin S, from rat spleen.

Antisera against rat erythrocytes contain agglutinins directed against unknown lipid determinants. Complement-fixation shows more reactivity with lipid extracts of rat spleen than of other rat tissues. The isolation of the reactive lipid from rat spleen, cytolipin S, is described. Cytolipin S is a glycosphingolipid containing glucose, galactose, and galactosamine with molar ratios of 1:2:1. It migrates on TLC like asialo GM1 (more slowly than cytolipin R, a ceramide tetrasaccharide, or cytolipin F, a ceramide pentasaccharide). Asialo GM1 and cytolipin S, when properly combined with auxiliary lipids, react very similarly with anti-rat erythrocyte sera by complement-fixation. However, cytolipin S is much more effective than asialo GM1 in inhibiting the hemagglutination reactions. It is concluded that cytolipin S and asialo GM1 are ceramide tetrasaccharides having different chemical structures and that the structural differences probably will be found in the carbohydrate linkages other than that between the terminal and penultimate residues.

Allergic contact dermatitis to laurel (Laurus nobilis L.): isolation and identification of haptens.

Two methods, using methylenelactone dimethylamino adducts, were used to remove selectively alpha-methylene gamma-butyrolactones from Laurus nobilis L. extracts. Isolated lactones were identified and "treated" extracts recovered. Two guinea-pig groups were sensitized to crude extracts and "treated" extracts, respectively, and tested with primary sensitizer and with different lactones. Only the first group showed strong skin reactions to crude extracts and to the lactones. Treated extracts were shown to be anallergic.

[Erythrocyte diagnostica made from salmonellal phagolysates]. / Eritrotsitarnye diagnostikumy iz fagolizatov sal'monell.

For the first time O antigens obtained from phagolysates were proved to be suitable for use as material for the production of highly specific erythrocyte diagnostic preparations. O antigens obtained from Salmonella by two methods, i.e. phage disintegration and Grasset's method, were subjected to comparative chemical analysis and found to have no essential difference. Nevertheless, the sensitizing potency of O antigens obtained from phagolysates were experimentally shown to be 3 times greater than that of O antigens obtained by Grasset's method. The optimum sensitizing doses established in the passive hemagglutination test for O antigens obtained by both methods indicated that these antigens were highly sensitive and specific.

[The immunogenic and serological properties of the O-specific polysaccharide (L-hapten) in Shigella]. / Immunogennye i serologicheskie svoistva O-spetsificheskogo polisakharida (L-gaptena) shigell.

O-specific polysaccharide (L-hapten) was isolated earlier (Zh. mikrobiol. epidemiol. immunobiol., 1989, No. 11, pp. 8-11). In this paper L-hapten was shown to be unable, even at high concentrations (up to 2,000 micrograms/ml), to sensitize sheep red blood cells for passive hemagglutination by O-antibodies. At the same time classical LPS and heat-activated LPS were active at concentrations ot 32 and 8 micrograms/ml respectively. The O-antibody-neutralizing activity of L-hapten was lower than that of LPS 10(3)-10(4) times in the passive hemagglutination test and 25-50 times in competitive ELISA. The immunogenicity of isolated L-hapten was very weak: primary response in mice to the i.v. injection of 1-10 micrograms of L-hapten was similar to the effect produced by 10(-3)-10(-4) micrograms of LPS. No protective activity of L-hapten was noted in mice when the challenge dose of virulent shigellae was 16 LD50 or more, and only a weak protective effect was observed with a low challenge dose (8 LD50). The molecular basis of low serological and biological activity of L-hapten is discussed. The most probable explanation of the results obtained in this study is that L-hapten contains some nonspecific carbohydrates, inserted in or complexed with the O-side chain. Despite its low immunogenicity, L-hapten can be an important component of effective bacterial vaccines provided it is included into a suitable delivery system as is the case with Shigella ribosomal vaccine.

[O-specific L-hapten in the composition of Shigella sonnei]. / O-spetsificheskii L-gapten v sostave Shigella sonnei.

Along with classical lipopolysaccharide (LPS), O-specific material not precipitated by ultracentrifugation has been isolated from the water-phenol extract of S. sonnei avirulent strain 9090 possessing complete antigenic properties. The purification of O-antigen contained in the supernatant fluid has been carried out by the gel filtration of the fluid, previously treated with ribonuclease, in a column packed with Sephadex G-100. The polysaccharide nature of O-antigen thus obtained, the absence of lipid A and KDO and the low content of hexoses, or core-specific saccharides of S. sonnei LPS, in this antigen make it possible to classify this material with O-components of microbial cells, described by different authors as "native protoplasmic polysaccharide" or "L-hapten" and formed by polymers of LPS O-side chains. The content of this component in S. sonnei strains under study is, on the average, 2.5% of the weight of dry microbial substance. L-hapten preparations obtained in the course of our investigations have been found to contain two O-specific antigens detected by immunoelectrophoresis and immunodiffusion, as well as by sedimentation in saccharose gradient, where they form peaks corresponding to 4.3 S and 10.8 S. This polysaccharide O-antigen is supposed to be capable of interaction with ribosomal particles and suitable for use as a component of ribosomal dysentery vaccines.

Preparation and characterization of diethoxy- and monoethoxy phosphylated ('aged') serine haptens and use in the production of monoclonal antibodies.

In this study, the first mechanism-based monoclonal antibodies have been produced that recognize and differentiate diethoxy- and monoethoxyphosphorylated serine residues. Haptens were synthesized as the stable phosphonate form of phosphoserine esters to improve the immunoresponse. Following condensation with a glutaric anhydride to link the phosphoserine moieties to carrier protein, the hapten densities attached to bovine serum albumin and keyhole limpet henocyanin were determined by partial trypsin digestion and MALDI mass spectrometry, and confirmed using a fluorescent assay (FITC) to quantify unmodified lysine residues. The conjugation reactions were pH optimized to improve hapten density. Screening of subclones led to the identification of two monoclonal antibodies: (a) N257/25.11 that specifically recognizes (EtO)2P(O)-Ser as the phosphylated or inhibited form, and (b) N262/16 that recognizes (EtO)(HO)P(O)-Ser as the 'aged' form. Analysis of blood samples treated with paraoxon (EtO)2P(O)-OPhNO2 showed a concentration dependent recognition of the phosphylated form.