Antibodies directed against the thiomannose moiety of a glycoconjugate of 2-imino-2-methoxyethyl 1-thio-alpha-D-mannopyranoside and bovine serum albumin

Anti-thiomannose antibodies were induced in rabbits immunized with a glycoconjugate of 2-imino-2-methoxyethyl 1-thio--d-mannopyranoside (Man-S) and bovine serum albumin (BSA). Also anti-BSA antibodies directed against the BSA moiety of the glycoconjugate were detected in low concentrations in the immune serum. However, antibodies against the combinatorial epitope of the hapten group and the carrier protein were not detected. The anti-thiomannose and the anti-BSA antibodies were isolated in pure forms by affinity chromatography on Sepharose 4B-bearing thiomannosyl-BSA ligands or BSA ligands. The anti-thiomannose antibodies constituted the major fraction of the antibodies, and these antibodies were isolated in pure form for the first time. The specificity of the thiomannose antibodies was established from data of experiments of periodate oxidation, perpropionic acid oxidation, hapten inhibition, and agar diffusion. Isoelectrofocusing showed that the anti-thiomannose antibody preparation consisted of at least six isomeric proteins, all of which exhibited antibody activity against the glycoconjugate of thiomannose and BSA.     

Determination method of 1-methyl-1,2,3, 4-tetrahydroisoquinoline,an endogenous parkinsonism-preventing substance,by radioimmunoassay

To develop a sensitive and simple assay method for 1-methyl-1,2,3,4-tetrahydroisoquinoline (1MeTIQ), an endogenous parkinsonism-preventing substance, we designed two kinds of 1MeTIQ-bovine serum albumin (BSA) conjugates to recognize the methyl group at the 1 position of 1MeTIQ since this is the critical structural difference between 1MeTIQ and parkinsonism-inducing substances. These hapten antigens were synthesized from 1MeTIQ analogues and BSA. A specific antiserum against 1MeTIQ was obtained from a rabbit immunized with one of the hapten antigens. To utilize this antiserum for radioimmunoassay, detailed studies were carried out to establish optimum conditions. The antiserum recognized 1MeTIQ and showed little cross-reactivity with endogenous 1MeTIQ analogues and proteins. It was confirmed to be suitable for radioimmunoassay, and a standard curve was prepared in the range of 0.5 to 100 pmol of 1MeTIQ. This method was sensitive enough to measure endogenous 1MeTIQ in rat brain. This method should be applicable for evaluation of the progression or prognosis of Parkinson's disease (PD).     

Antibodies directed against the thiomannose moiety of a glycoconjugate of 2-imino-2-methoxyethyl 1-thio-α-d-mannopyranoside and bovine serum albumin

Anti-thiomannose antibodies were induced in rabbits immunized with a glycoconjugate of 2-imino-2-methoxyethyl 1-thio-α-d-mannopyranoside (Man-S) and bovine serum albumin (BSA). Also anti-BSA antibodies directed against the BSA moiety of the glycoconjugate were detected in low concentrations in the immune serum. However, antibodies against the combinatorial epitope of the hapten group and the carrier protein were not detected. The anti-thiomannose and the anti-BSA antibodies were isolated in pure forms by affinity chromatography on Sepharose 4B-bearing thiomannosyl-BSA ligands or BSA ligands. The anti-thiomannose antibodies constituted the major fraction of the antibodies, and these antibodies were isolated in pure form for the first time. The specificity of the thiomannose antibodies was established from data of experiments of periodate oxidation, perpropionic acid oxidation, hapten inhibition, and agar diffusion. Isoelectrofocusing showed that the anti-thiomannose antibody preparation consisted of at least six isomeric proteins, all of which exhibited antibody activity against the glycoconjugate of thiomannose and BSA.     

A structurally diversified linker enhances the immune response to a small carbohydrate hapten

The tether employed to covalently attach β-mannan disaccharide glycoconjugates influences the specificity of rabbit antibodies that protect against Candida albicans. Two glycoconjugates containing (1?→?2)-β-mannan disaccharides linked to chicken serum albumin (CSA) either via a structurally uniform or via a stereodiversified spacer were prepared and evaluated in immunization trials in mice and rabbits. Immunization with conjugate vaccine possessing a structurally diversified linker induced higher IgG titers against Candida albicans cell wall phosphomannan than a conjugate with a structurally uniform linker. These results suggest that affinity maturation and the specific antibody response can be shifted towards recognition of the desired hapten by employing a linker with diversified configuration.     

Radioimmunoassay for medroxyprogesterone acetate (Provera) using the 11alpha-hydroxy succinyl conjugate

The development of a radioimmunoassay for medroxyprogesterone acetate (MPA) employing rabbit antibodies made against a bovine serum albumin (BSA) conjugate of the hemisuccinate ester of 11alpha-hydroxy MPA are described. The 11alpa-hydroxyl group was introduced into MPA by a series of chemical and microbiological transformations. The acid succinate MPA was coupled to BSA by reacting it with N,N'-carbonyldiimidazole. Cross-reactivities of several MPA analogs were compared suing rabbit antisera developed against the C-11 conjugate of MPA and goat antiserum developed against a C-3 conjugate of MPA. Antibodies formed against the C-11 conjugate showed increased specificity to steroid analogs which changes in the C-21 side chain. The profiles of serum MPA levels vs time, measured with both the C-3 and C-11 antisera, after oral drug administration to the monkey were similar. After intramuscular drug administration to dogs, serum MPA levels measured with the C-3 antisera were consistently greater as compared with those with C-11 antisera. Factors affecting precipitation of the antibody-antigen complex, assay precision, and assay sensitivity were evaluated.     

Stimulation by D-glucose of mitochondrial oxidative events in islet cells.

Antibodies were elicited to FAD by using the hapten N-6-(6-aminohexyl)-FAD conjugated to the immunogenic carrier protein bovine serum albumin. Cross-reactivity was determined by Ouchterlony double-diffusion analysis with N-6-(6-aminohexyl)-FAD coupled to rabbit serum albumin. Anti-FAD IgG was partially purified by (NH4)2SO4 precipitation followed by DEAE-cellulose/CM-cellulose and bovine serum albumin-agarose chromatography. The partially purified anti-FAD IgG fraction failed to inhibit the catalytic activities of the flavin-containing enzymes nitrate reductase, xanthine oxidase and succinate dehydrogenase, whereas enzyme activity could be inhibited by addition of antibodies elicited against the native proteins. However, the partially purified anti-FAD IgG fraction could be used as a highly sensitive and specific probe to detect proteins containing only covalently bound flavin, such as succinate dehydrogenase, p-cresol methylhydroxylase and monoamine oxidase, by immuno-blotting techniques. Detection limits were estimated to be of the order of femtomolar concentrations of FAD with increased sensitivity for the 8 alpha-N(3)-histidyl linkage compared with 8 alpha-O-tyrosyl substitution.     

Affinity electrophoresis and its applications to studies of immune response

Affinity electrophoresis (AEP) is a useful technique for separation of biomolecules such as plasma proteins, enzymes, nucleic acids, lectins, receptors, and extracellular matrix proteins by specific interactions with their ligands in electric fields and for the determination of dissociation constants for those interactions. Two-dimensional affinity electrophoresis (2-D AEP), which was newly developed by a combination of isoelectric focusing with AEP, has been used for studies on immune response to haptens. Antihapten antibodies, which were induced by immunization of a mouse with the hapten-conjugated bovine serum albumin, were separated by 2-D AEP into a large number of groups of IgG spots with a few microliters of antiserum. Each group of spots showed an identical affinity for the hapten but different isoelectric points as in the case of monoclonal antibodies specific to the hapten. This enabled us to study the diversification and affinity maturation of antihapten antibodies in the course of immunization of a single mouse. Furthermore, effects of a carrier and a hapten array on the production of antihapten antibodies and the cause of charge heterogeneity of monoclonal antibodies were also examined to understand the molecular basis of the immune response in vivo.     

Immunochemical characterization of polyclonal and monoclonal Streptococcus group A antibodies by chemically defined glycoconjugates and synthetic oligosaccharides.

Synthetic oligosaccharides of increasing complexity that represent different epitopes of the Streptococcus Group A cell-wall polysaccharide were used as haptens and glycoconjugates of bovine serum albumin (BSA) and horse hemoglobin (HHb) to characterize polyclonal and monoclonal antibodies. Rabbits were immunized with the BSA glycoconjugates of a linear trisaccharide, branched trisaccharide, and branched pentasaccharide. The binding specificities of the polyclonal antisera were determined by a series of inhibition ELISA studies in which disaccharide through pentasaccharide haptens were used as inhibitors of antibody-glycoconjugate binding. Monoclonal antibodies derived from mice immunized with a killed bacterial vaccine were selected for their binding to native polysaccharide antigen coupled to BSA and the BSA glycoconjugates of the di- and linear tri-saccharides. Polyclonal antibodies were moderately specific for the oligosaccharide epitope of the immunizing glycoconjugate and only those antibodies raised to the branched pentasaccharide antigen showed cross-reaction with the bacterial antigen. The behaviour of selected monoclonal antibodies parallels the binding profile of polyclonal antibodies in that the two highest-titre antibodies were directed toward an epitope displayed by the branched pentasaccharide.     

Precipitation of polyribosomes with pepsin digested antibodies

Polyribosomes prepared from rabbit reticulocytes were precipitated with a “γG” fraction prepared from goat antiserum to rabbit hemoglobin (specific). A similar preparation from rabbit antiserum to rat hemoglobin (control) precipitated the same proportion of the polyribosomes. The non-specificity of the precipitation was significantly reduced by pre-digestion of the antibodies with pepsin. After pre-digestion of either whole antiserum or of the “γG” fraction, the precipitation of polyribosomes by the specific antibodies was increased slightly while precipitation of polyribosomes by the control antibodies was virtually eliminated.     

Purification, specificity, and hypervariable region sequence of anti-pneumococcal polysaccharide antibodies elicited in a single rabbit.

Four homogeneous antibodies to type VIII pneumococcal polysaccharide (S8) were isolated from the serum of a single rabbit (3322) by affinity chromatography on an S8 immunoadsoebent by utilizing gradient elution with cellobiose and NaCl. The binding properties of these antibodies were determined by a radioimmunoassay with 125I-bovine gamma-globulin-S8. Cellobiose (a disaccharide unit of S8) was the immunodominant group of each of the four antibodies, but each antibody bound to this disaccharide with different relative affinities. The amino acid sequences (positions 0-40) of three of the four antibody light chains were each different both in framework and first hypervariable region sequences. The fourth antibody light chain has a blocked amino terminus. These findings indicate that antibodies elicited by a relatively simple antigen and examined at one time during the course of immunization in a single rabbit may exhibit common specificities for an oligosaccharide determinant, yet have different binding affinities for that determinant as well as different primary structures in the complementarity (hypervariable) regions and framework regions.     

Synthetic guinea pig insulin B-chain C-terminal decapeptide: a novel immunogen for generating immunocytochemical-grade antisera

Antisera to guinea pig insulin are not commonly available, largely because of the short supply and limited immunogenicity of the intact hormone. To overcome these problems we have employed a novel reagent, synthetic guinea pig insulin B-chain C-terminal decapeptide, as a hapten for raising antibodies that react with intact guinea pig insulin. The decapeptide, coupled to bovine serum albumin, was successfully used as an immunogen in rabbits. The resulting anti-serum was employed for immunocytochemical staining of guinea pig insulin in pancreatic sections. The specificity of the staining was verified by both pre-absorption and pre-immune serum controls. The utility of this new antiserum for investigations of guinea pig insulin physiology is discussed.     

Core alpha1,3-fucose is a key part of the epitope recognized by antibodies reacting against plant N-linked oligosaccharides and is present in a wide variety of plant extracts

Carbohydrates have been suggested to account for some IgE cross- reactions between various plant, insect, and mollusk extracts, while some IgG antibodies have been successfully raised against plant glycoproteins. A rat monoclonal antibody raised against elderberry abscission tissue (YZ1/2.23) and rabbit polyclonal antiserum against horseradish peroxidase were screened for reactivity in enzyme-linked immunosorbent assay against a range of plant glycoproteins and extracts as well as neoglycoproteins, bee venom phospholipase, and several animal glycoproteins. Of the oligosaccharides tested, Man3XylFucGlcNAc2(MMXF3) derived from horseradish peroxidase was the most potent inhibitor of the reactivity of both YZ1/2.23 and anti- horseradish peroxidase to native horseradish peroxidase glycoprotein. The reactivity of YZ1/2. 23 and anti-horseradish peroxidase against Sophora japonica lectin was most inhibited by a neoglycoconjugate of bromelain glycopeptide cross-linked to bovine serum albumin, while the defucosylated form of this conjugate was inactive as an inhibitor. A wide range of plant extracts was found to react against YZ1/2.23 and anti-horseradish peroxidase, with particularly high reactivities recorded for grass pollen and nut extracts. All these reactivities were inhibitable with the bromelain glycopeptide/bovine serum albumin conjugate. Bee venom phospholipase and whole bee venom reacted weakly with YZ1/2.23 but more strongly with anti-horseradish peroxidase in a manner inhibitable with the bromelain glycopeptide/bovine serum albumin conjugate, while hemocyanin from Helix pomatia reacted poorly with YZ1/2.23 but did react with anti-horseradish peroxidase. It is concluded that the alpha1, 3-fucose residue linked to the chitobiose core of plant glycoproteins is the most important residue in the epitope recognized by the two antibodies studied, but that the polyclonal anti-horseradish peroxidase antiserum also contains antibody populations that recognize the xylose linked to the core mannose of many plant and gastropod N-linked oligosaccharides.      

A characterization of the BALB/c lambda and kappa class antibodies to alpha (1,3) glycosidic linkages

The avidity relationships of kappa and lambda antibodies with specificity for the glucosyl-alpha (1,3)-glucose disaccharide epitope were compared using a solid-phase hapten inhibition assay. The kappa class antibodies, induced by the T-cell-dependent antigen, nigerosyl-keyhole limpet hemocyanin (N-KLH), and the lambda class antibodies, induced by the T-cell-independent antigen, Dextran B1355, were only marginally distinguishable (approximately equal to 10-fold) on the basis of hapten inhibition using the free disaccharide, nigerose, as inhibitor. Analysis of the binding site sizes of antibodies induced by N-KLH through inhibition with oligosaccharides of different sizes showed that the disaccharide protein antigen did not select for antibodies with smaller binding site sizes. The cellular basis for these findings is discussed.     

A radioactive hapten-binding assay for measuring antibodies to the pentapeptide determinant of peptidoglycan.

A major portion of the humoral immune response to peptidoglycans is directed against the non-cross-linked pentapeptide side chains of these ubiquitous bacterial antigens. At present, no specific and sensitive assay for pentapeptide antibody determination is available. Therefore, a radioimmunoassay has been developed which employs the synthetic pentapeptide hapten L-Ala-gamma-D-Glu-L-Lys-D-Ala-D-Ala, labeled by the active ester method of Bolton and Hunter to high specific activities (6.74 to 18.18 muCi/mug) with 125I, and used as a reagent for measuring pentapeptide antibody. A-variant streptococcal antisera, known to contain pentapeptide antibodies as shown by quantitative precipitation, would bind more than 95% of the radiolabeled hapten in contrast to 2 to 3% by preimmune rabbit sera. Specificity of the binding reaction was demonstrated by inhibition experiments imploying various synthetic oligopeptides related or unrelated to the pentapeptide in the radioimmunoassay. Binding curves established with serial dilutions of peptidoglycan antiserum were linear from 15 to 500 mug/ml of antibody permitting pentapeptide antibody measurement within this range. Comparative data on pentapeptide antibody determinations by quantitative precipitation and radioimmunoassay are given and the time course of the production of this antibody in 14 rabbits hyperimmunized with A-variant streptococcal vaccine is reported.     

Fluorescein and tetramethyl rhodamine as haptens in enzyme immunohistochemistry

Summary Fluorescein (Fl) and tetramethyl rhodamine (Rh) were evaluated as possible candidates for a double hapten sandwich system in enzyme immunohistology. Monoclonal antibodies were raised against Fl and Rh. Their fine-specificity was tested with a competition-like assay. A pair of Mab's was selected for immunohistology in which they functioned as a bridge between Fl/Rh conjugated antibodies and Fl/Rh labeled peroxidase and alkaline phosphatase, respectively. The binding of fluorescein labeled antibodies could be successfully demonstrated in histological slides. A large variability in the efficacy of staining was observed in the case of rhodamine labeled antibodies. The phenomenon is explained by assuming that tetramethyl rhodamine isothiocyanate reacts preferentially with lysine residues near to, or embedded in, hydrophobic regions in a protein. This condition may reduce the accessibility of the Rh moiety for anti-Rh antibodies.Abbreviations AEC 3-amino-9-ethylcarbazole - AP Alkaline phosphatase - BSA Bovine serum albumin - CGG Chicken gamma globulin - c-IF Cytoplasmic immunofluorescence - DAB 3,3-Diamino benzidine HCl - ELISA Enzyme linked immunosorbent assay - Fl Fluorescein - F/P ratio Number of fluorochrome molecules per molecule of protein - Ig Immunoglobulin - Mab Monoclonal antibody - OPD Ortho-phenylenediamine-dihydrochloride - PBS Phosphate buffered saline - PBS-T PBS with 0.2% Tween-20 - PO Peroxidase - RAM/PO Peroxidase labeled rabbit antiserum directed against mouse immunoglobulins - Rh Tetramethyl rhodamine - RT Room temperature In honour of Prof. P. van Duijn     

Isolation and characterization of sets of anti-l -fucose and anti-bovine serum albumin antibodies directed against a glycoconjugate ofl -fucose and bovine serum albumin

A set of anti-carbohydrate antibodies and a set of anti-protein antibodies were isolated from the serum of rabbits immunized with a glycoconjugate ofl-fucose and bovine serum albumin. The sets were separated by affinity chromatography by a two-column method on adsorbents withl-fucose or bovine serum albumin ligands. Isoelectrofocusing results showed that the anti-carbohydrate antibodies consisted of 11 molecular species and the anti-bovine serum albumin antibodies consisted of seven molecular species. The anti-carbohydrate antibodies are all of the IgG type while the anti-protein antibodies contain three types of globulin molecules, IgA, IgG, and IgM. The former antibodies should be useful as markers for unique glycoproteins of diseased cells and the latter antibodies may be useful for investigating the mechanism of simultaneous synthesis of three types of immunoglobulins.     

Separation of anti-histone antibodies from nonimmune histone-precipitating serum proteins, predominantly alpha2-macroglobulin.

Calf thymus histories formed precipitates with human, rabbit, guinea pig, mouse, goat, or sheep sera regardless of whether the subjects were immunized with histones. Their order of activity in precipitation was: H4 >H3 >H2a >H2b ? H1. Precipitation occurred with more than one serum protein fraction, but not with purified IgG, IgM, or crystallized albumin. The major nonimmune precipitant had the size and charge properties of α2-macroglobulin. Serological analysis with anti-α2-macroglobulin antiserum confirmed the presence of this protein in the precipitating fractions, and its selective depletion from histone-precipitated serum. Specific anti-histone antibodies were separated from the nonimmune precipitants by DEAE-cellulose chromatography.     

Antibodies specific to a deoxyribodinucleotide sequence.

Antibodies were raised in rabbits against the bovine serum albumin conjugate of dpApT. Analysis by double diffusion in agar gel and quantitative precipitation test showed the presence of antibodies specific to the hapten in the antisera. Quantitative data on the specificity of the antibodies were obtained by studying the inhibition of the binding of 3H-dpApT to the antisera by various nonradioactive mono- and oligonucleotides, using a nitrocellulose membrane binding assay. The antibodies were found to be highly specific for the dinucleotide sequence dpApT. The antibodies were able to bind to synthetic oligonucleotides containing the sequence dpApT and to denatured calf thymus DNA.     

Isolation and characterization of sets of anti-l-fucose and anti-bovine serum albumin antibodies directed against a glycoconjugate ofl-fucose and bovine serum albumin

A set of anti-carbohydrate antibodies and a set of anti-protein antibodies were isolated from the serum of rabbits immunized with a glycoconjugate ofl-fucose and bovine serum albumin. The sets were separated by affinity chromatography by a two-column method on adsorbents withl-fucose or bovine serum albumin ligands. Isoelectrofocusing results showed that the anti-carbohydrate antibodies consisted of 11 molecular species and the anti-bovine serum albumin antibodies consisted of seven molecular species. The anti-carbohydrate antibodies are all of the IgG type while the anti-protein antibodies contain three types of globulin molecules, IgA, IgG, and IgM. The former antibodies should be useful as markers for unique glycoproteins of diseased cells and the latter antibodies may be useful for investigating the mechanism of simultaneous synthesis of three types of immunoglobulins.     

Selective antibodies to methadone enantiomers: synthesis of (R)- and (R,S)-methadone conjugates and determination by an immunoenzymatic method in human serum

Selective antibodies to (R)-methadone (Mtd) and to its racemate were produced in rabbits by immunization with conjugates of (R)- or (R,S)-hemisuccinyl-methadol-bovine serum albumin, respectively. A hapten was first prepared by reduction of (R)- or (R,S)-Mtd with sodium borohydride, followed by esterification with succinic anhydride. The conjugation of hapten with albumin was achieved by the mixed anhydride method. After immunization of rabbits, the titers and specificity of each antibody were determined by ELISA. The antibodies obtained were tested with (R)-, (S)-, (R,S)-Mtd, its major metabolite (EDDP), and some drugs of abuse (morphine, codeine, cocaine). The sensitivities of antibodies to (R)- and (R,S)-Mtd were about 1 and 2 ng/ml, respectively. Selective (R)-antibodies recognized (R)-Mtd about 40 times more avidly than the (S)-isomer, while an antiserum against (R,S)-Mtd recognized (R)- and (S)-isomers to about the same degree. Both selective antibodies showed little interference (about 0.5%) with EDDP metabolite and no crossreactivity with morphine, codeine, and cocaine. These two selective antibodies were used to develop an immunoenzymatic method (ELISA) for the determination of (R)- and (R,S)-Mtd in serum samples of patients under maintenance treatment for narcotic addiction.