Bovine Serum Albumin Conjugate Research Articles

Radioimmunoassay for 4-((5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)carbamoyl)benzoic Acid in Dog Plasma.

A specific and sensitive radioimmunoassay (RIA) was developed for 4-[(5, 6, 7, 8-tetrahydro-5, 5, 8, 8-tetramethyl-2-naphthyl)carbamoyl]benzoic acid (Am-80), a benzoylamide derivative possessing retinoid activity, in dog plasma. Antisera were prepared by immunizing rabbits with Am-80 and its haptenic derivative conjugated with bovine serum albumin (BSA) and keyhole limpet hemocyanin. Their properties were investigated by heterologous combination using 125I-labeled tracer. The antisera raised against BSA conjugate showed poor titers and high specificity, while the antisera from the other two conjugates showed high titers and reasonable specificity. The limit of quantification was 0.6ng/ml, when only 0.005ml of dog plasma sample was used per assay tube. The within- and between-assay variances were 1.2- 15.0% and 4.0-14.1 %, respectively. The direct RIA established here was highly specific for the determination of the unchanged form of Am-80 in dog plasma, enabling measurement of many samples at the same time.

Determination of P-Cresol (and Other Phenolics) Using a Conducting Polymer Based Electro-Immunological Sensing System

Abstract Antibodies to a p-cresol bovine serum albumin conjugate have been produced and incorporated into a conducting polymer to form an electro-immunological sensing system. Using flow injection analysis and pulsed electrochemical detection sensitive, reproducible and rapid responses to p-cresol and other phenolics can be obtained. The sensor is reusable.

Effect of heterologous paralytic shellfish poisoning toxin-enzyme conjugates on the cross-reactivity of a saxitoxin enzyme immunoassay

A polyclonal antiserum against saxitoxin (STX) was used in a competitive enzyme immunoassay for the detection of paralytic shellfish poisoning toxins. The extent of cross-reactions was determined from the amounts of neoSTX, decarbamoylSTX and gonyautoxin 2/3 (GTX2/3) that gave 50% inhibition in the assay. Horseradish peroxidase (HRP) conjugates of the toxins and a bovine serum albumin conjugate of STX (STX-BSA) were used. When compared with STX-BSA and STX as standard, the extent varied to which heterologous conjugates affected the binding values of the other toxins to the antibodies. The antibodies did not bind GTX2/3-HRP. By use of neoSTX-HRP or decarbamoylSTX-HRP as the labelled antigen instead of STX-HRP, the detection limit for neoSTX was improved to 100 pg ml-1.

Polyspecific and autoreactive IgA secreted by hybridomas derived from Peyer's patches of vomitoxin-fed mice: Characterization and possible pathogenic role in IgA nephropathy

A total of 122 immunoglobulin (Ig)A-producing hybridoma clones were isolated from the Peyer's patches of vomitoxin-fed BALB/c mice and the resultant antibodies were characterized for their antigenic specificity and pathogenic potential. When reactivity was tested against a panel consisting of DNA, sphingomyelin, thyroglobulin, collagen, casein, cardiolipin and bovine serum albumin conjugates of phosphorylcholine, inulin and trinitrophenol that were representative of self and non-self antigens, approximately 95% of the monoclonal IgAs bound to at least one of the panel antigens and 80% bound to more than one antigen. The polyspecificity of some of the monoclonal IgAs was further suggested by demonstrating the capacity of one antigen to inhibit binding of monoclonal IgA to another antigen. Protein staining and Western blotting of gradient native polyacrylamide gels, indicated that trimeric IgA predominated in the isolated monoclonal IgAs. Repeated injections of mice with representative monoclonal IgAs induced microhaematuria in three of four of the clones tested but not IgA deposition in the kidney glomerulus. In addition, three of the four monoclonal IgAs caused IgG and C3 deposition in the kidney mesangium. These and previous results suggest that dietary vomitoxin promotes the polyclonal activation and expansion of IgA-secreting B cells at the Peyer's patch level and that resultant polyspecific, autoreactive IgA may contribute to kidney pathogenesis.

Effects of herbimycin A and ST638 on Fcϵ receptor-mediated histamine release and Ca 2+ signals in rat basophilic leukemia (RBL-2H3) cells

We examined the effect of the two protein tyrosine kinase inhibitors, α-cyano-3-ethoxy-4-hydroxy-5-phenylthiomethylcinnamide (ST638) and herbimycin A, on the activation processes of rat basophilic leukemia (RBL-2H3) cells by cross-linking of IgE receptors. RBL-2H3 cells sensitized with DNP-specific monoclonal IgE antibody were stimulated with multivalent antigen (DNP conjugate of bovine serum albumin). Analysis of phosphotyrosine-containing proteins in their lysates by SDS-PAGE and immunoblotting revealed that these two inhibitors efficiently inhibited the tyrosine phosphorylation of several proteins (32, 42, 56, 66, 72, 92, 150 kDa) including phospholipase C-γ1. The inhibitors also caused parallel inhibitions of the histamine release, the formation of inositol 1,4,5-trisphosphate, and the increase in cytosolic calcium ion concentration at the late sustained phase. A digital imaging fluorescence microscopic analysis of antigen-dependent calcium signals in individual cells showed that these two tyrosine kinase inhibitors inhibited the calcium influx from the external medium more powerfully than the mobilization of calcium ion from internal stores. In contrast, the inhibitors did not affect the increase in the cytosolic calcium ion concentration or the histamine release induced by the calcium ionophore A23187. Taken together, our results suggest that tyrosine phosphorylation following antigen stimulation regulates phosphatidylinositol hydrolysis and the influx of extracellular calcium.

Radioimmunoassay for 7-hydroxy metabolite of fluphenazine and its application to plasma level monitoring in schizophrenic patients treated long term with oral and depot fluphenazine.

Immunization of New Zealand white rabbits with a bovine serum albumin conjugate of 7-hydroxy-N-carboxyethyl-N-deshydroxyethylfluphenazine produced highly specific antisera for 7-hydroxyfluphenazine (7-OHFLU). A radioimmunoassay (RIA) was developed using antisera from one of the rabbits that enabled for the first time the determination of plasma levels of 7-OHFLU, an active metabolite of fluphenazine (FLU), in patients treated with oral FLU dihydrochloride or i.m. FLU decanoate. The assay method provided sufficient sensitivity to determine accurately 20 pg of 7-OHFLU in 200 microliters (0.1 ng/ml) of plasma with a coefficient of variation of < 10%. The antiserum used in the RIA for 7-OHFLU demonstrated negligible cross-reactivity with FLU and its metabolites such as FLU sulfoxide, N-deshydroxyethylFLU, FLU N4'-oxide, N-deshydroxyethyl-7-OHFLU, and 7-O-glucuronide of FLU and also with other antipsychotic agents and commonly coadministered drugs. The 7-OHFLU was present in measurable amounts in all plasma samples obtained at 4-week intervals from patients receiving a daily oral dose of 5 (n = 10), 10 (n = 13), or 20 (n = 14) mg of FLU dihydrochloride. Large interindividual variations in the plasma level of FLU and 7-OHFLU were noted and the mean plasma levels ratios of 7-OHFLU/FLU at these doses were 2.07 +/- 1.08, 2.07 +/- 1.13, and 2.02 +/- 0.82, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunohistochemical studies on localization of the extracellular polysaccharide produced by Xanthomonas oryzae pv. oryzae in infected rice leaves

An antibody reacting with extracellular polysaccharide from Xanthomonas oryzae pv. oryzae was used to detect the extracellular polysaccharide in infected leaf tissues of rice plants (Oryzae sativa cv. IR24) by immunofluorescence and immunoelectron microscopy. Using extracellular polysaccharide as a hapten and bovine serum albumin as a carrier, an extracellular polysaccharide – bovine serum albumin conjugate was prepared. After a rabbit was immunized against the conjugate, an antibody reacting with extracellular polysaccharide from X. oryzae pv. oryzae was isolated and purified. Using ELISA, the antibody was capable of detecting extracellular polysaccharide at concentrations above 0.1 μg/mL. Immunofluorescent antibody staining of infected rice leaves showed that extracellular polysaccharide produced by X. oryzae pv. oryzae was distributed in both xylem vessels and transverse veins but not in either sieve tubes or mesophyll tissues. This demonstrates that the distribution of extracellular polysaccharide coincided with that of bacteria in the infected leaf tissues. Immunoelectron microscopy showed that localization of extracellular polysaccharide was restricted to the area close to bacterial cells. The amount of extracellular polysaccharide may not be enough to plug xylem vessels of rice leaves infected with X. oryzae pv. oryzae.Key words: anti extracellular polysaccharide antibody, Xanthomonas oryzae pv. oryzae, immunofluorescent microscopy, immunoelectron microscopy, rice plant.

Preparation of conjugates of progesterone with bovine serum albumin in the reversed micellar medium

Progesterone-BSA (bovine serum albumin) conjugates which contain up to 47 steroid molecules linked to a BSA molecule have been prepared by the activated ester method, the conjugation step being carried out in reversed micellar solutions of sodium di(2-ethylhexyl) sulphosuccinate (AOT) ion octane. The number of incorporated steroid molecules increases on passing to increased water/AOT ratios at the given activated steroid/BSA ratio. The results show that the reversed micellar medium would be useful for preparation of conjugates of hydrophobic steroids with proteins in respect to simplicity and ease in obtaining conjugates with high steroid/protein ratios.

Reduction in the percentage of serotoninergic axons making synapses during the development of the superficial layers of the hamster's superior colliculus

Immunocytochemistry with an antibody directed against a serotonin (5-HT)-bovine serum albumin conjugate was combined with electron microscopy to assess the synaptic organization of the serotoninergic projection to the stratum griseum superficiale (SGS) of the superior colliculus of hamsters killed on postnatal days (P) 0, 3, 7, 12, 15, 20 and > 60. At least 200 5-HT-immunoreactive profiles were examined at each of these ages. In the newborn (PO) animals, 36.6% of the 5-HT-positive profiles made conventional synapses. This percentage remained fairly constant until P15 when only 17.3% of the profiles made conventional synaptic contacts. On P20, this value decreased to 10.5% and in the adult animals, it fell to 4.4% These results thus indicate a dramatic age-related change in the synaptic organization of the 5-HT input to the hamster's superior colliculus.

Temperature-responsive bioconjugates. 2. Molecular design for temperature-modulated bioseparations.

We have synthesized carboxyl semitelechelic oligo(N-isopropylacrylamide) (OIPAAm) using radical telomerization with 3-mercaptopropionic acid. This telomerization is also effective for the synthesis of carboxyl semitelechelic co-oligomers of IPAAm with butyl methacrylate (BMA) as hydrophobic or N,N-dimethylacrylamide (DMAAm) as hydrophilic comonomers. All co-oligomers are highly water-soluble at lower temperatures and exhibit phase separation with increasing temperature. Pure OIPAAm exhibits a lower critical solution temperature (LCST) at 32 degrees C, and the LCST for co-oligomers can be controlled to increase over 32 degrees C with increasing DMAAm composition and to decrease below 32 degrees C with increasing BMA composition. OIPAAm was grafted to bovine serum albumin (BSA) and bovine plasma fibrinogen (BPF) by activated ester-amine coupling. These OIPAAm-biomolecule conjugates maintain their temperature responses, are soluble in cold water, and precipitate over a range of temperatures related to oligomer content. Conjugates could be selectively precipitated and independently separated from conjugate solution mixtures with increasing temperature. In this case, the number of OIPAAm molecules attached to a conjugate affects the aggregate sizes of precipitated conjugates in mixtures. Both conjugate mixture ratios and solution concentrations influence the contamination of oligo(IPAAm-co-DMAAm)-BSA conjugates in precipitated oligo(IPAAm-co-BMA)-BPF conjugates. Furthermore, precipitated conjugates separated using centrifugation and filtration redissolve in water and maintain their biofunctionality, indicating the potential of strategy in reversible bioreactors and protein separations.

Enzyme immunoassay for 4-hydroxy-2-(4-methylphenyl)benzothiazole

A specific enzyme immunoassay (EIA) has been developed for 4-hydroxy-2-(4-methylphenyl)benzothiazole (KB-2714) (1), an active metabolite of 4-acetoxy-2-(4-methylphenyl)benzothiazole (KB-2683) (2) which is a promising anti-rheumatic agent. The EIA was based upon antiserum elicited against 5-(2-carboxyphenylazo)-4-hydroxy-2-(4-methylphenyl)benzothiazole (3)-bovine serum albumin conjugate and beta-galactosidase-labelled 5-amino-4-hydroxy-2-(4-methylphenyl)benzothiazole (5). The sensitivity of the EIA was significantly improved by the utilization of a bridge heterologous combination system. An appropriate dose-response curve of EIA for 4-hydroxy-2-(4-methylphenyl)benzothiazole was obtained in the range of 10 pg tube-1-20 ng tube-1. The specificity of EIA proved to be satisfactory in terms of cross-reactivities to 14 benzothiazole-related compounds including glucuronic acid and sulphuric acid conjugates. The proposed method was evaluated to be useful for the determination of 1 in urine and plasma with acceptable recovery and inter- and intra-assay precision.

The Mucosal and Systemic Response to Phosphorylcholine in Mice Infected with Trichinella spiralis

The presence of phosphorylcholine (PC) in Trichinella was confirmed by ELISA and Western blot experiments with the PC-specific myeloma TEPC-15. Anti-PC antibody production was detected in ELISA by cross-reaction with the PC-positive somatic polysaccharide of Aspergillus and the synthetic conjugate phosphorylcholine–bovine serum albumin conjugate and by inhibition with phosphorylcholine chloride (PCCl). The kinetics of the serum and mucosal anti-PC immunoglobulin response were determined following infection of CFW mice. Anti-PC IgA was a minor fraction of the serum response. In primary infections IgG binding to Trichinella antigen was partially inhibited by PCCl incubation, but by Day 6 following challenge infections, incubation with PCCl did not reduce IgG binding. PCCl incubation also reduced serum IgM binding to Trichinella antigen following primary infections, and in contrast to IgG, a reduction occurred following challenge infection as well. Following primary and challenge infections PCCl incubation also reduced bile IgA binding to Trichinella antigen. The kinetics and subclass distribution of the anti- Trichinella PC response were equivalent to the group I response reported for synthetic PC–protein conjugates. Anti-PC IgA production indicates that class switching occurred without maturation of the response. Immunization by feeding Trichinella antigen plus cholera toxin, in contrast to infection with larvae, did not affect anti-PC antibody production following infection. Since the response was not anamnestic and the serum IgG response was not downregulated, larval infection and antigen feeding differ in the anti-PC responses they induce. The anti-PC response does not appear to be protective in Trichinella infections in mice.

Immunoassay of podophyllotoxin.

The purpose of our research was to design and develop a sensitive immunoassay for the natural Podophyllum lignan, podophyllotoxin [1], which is the precursor of the anti-cancer drugs etoposide [2] and teniposide [3]. In order to obtain a specific antiserum, bovine serum albumin and keyhole limpet hemocyanin conjugates of succinyl-podophyllotoxin [5] were prepared as antigenic hapten carrier proteins. These antigens were used to develop polyclonal antibodies to 1 in rabbits. The antisera were used for the development of an immunoassay for the detection of 1 and its 4'-demethyl derivative 4. As little as 0.16 nmol of 1 is detectable with this ELISA. The assay was applied to the direct analysis of 1 in plants for the screening of potential drug plants which allowed quantitation in less than 0.2 mg of plant tissue.

Preparation and characterization of monoclonal antibodies against ornithine decarboxylase

In order to develop a method for the immunocytochemical detection of ornithine decarboxylase (ODC), EC 4.1.1.17, we have prepared and characterized monoclonal antibodies (MAbs) against ODC. The primary structure of rat ODC ( Rattus Norvegicus) was used for the selection of an epitope by computer calculations. The epitope (P16), a hexadecapeptide representing ODC-(345–360), was synthesized by means of solid phase peptide synthesis and coupled to a carrier protein. A bovine serum albumin conjugate of the P16 peptide was used as the immunogen for the production of MAbs in mice. Hybridoma clones were screened and the specificity of the monoclonal antibodies was tested in an ELISA utilizing a thyroglobulin conjugate of the hexadecapeptide. Two hybridoma cell lines were developed, i.e., MP16-2 and MP16-3. The epitope specificity of the MAbs produced by these cell lines was characterized in an ELISA using a set of small peptides representing parts of the P16 hexadecapeptide chain. MP16-2 recognized the ODC-(355–360) portion whereas MP16-3 reacted with the ODC-(345–350) part of the hexadecapeptide. Further studies showed that both MAbs also recognized native ODC but not the inhibited (i.e., ODC labelled with 3H-DFMO) enzyme indicating that the selected epitope was associated with the active site of ODC or a locus in its direct vicinity.

Salmonella typhi O:9,12 polysaccharide-protein conjugates: characterization and immunoreactivity with pooled and individual normal human sera, sera from patients with paratyphoid A and B and typhoid fever, and animal sera.

Polysaccharide of O:9,12 specificity purified from Salmonella typhi was conjugated to tetanus toxoid or bovine serum albumin in order to obtain defined antigenic material that would contain O chain free of other S. typhi antigens and that would be suitable for characterizing host humoral response to only S. typhi O-chain antigens. These artificial conjugates were strongly reactive in immunodots with 18 pooled and 3 individual serum samples from patients with typhoid fever and with rabbit anti-Salmonella O antiserum (group D, factors 1, 9, and 12). They reacted weakly with one serum sample from one human with paratyphoid A. These results suggest that the periodate oxidation and the reductive amination used in the conjugation conserved the immunogenicity of the O chain and allowed its absorption to nitrocellulose. They also suggest that the bovine serum albumin conjugate could be used in the diagnosis of S. typhi infections as normal sera may react with the protein molecule of the tetanus toxoid conjugate.

Immunogenicity of various beta-lactam antibiotic-protein conjugates and cross-reactivity of the antibodies produced in guinea pig.

Immunogenic properties of bovine serum albumin (BSA) conjugates of various beta-lactam antibiotics (ABs) and cross-reactivity of the produced antibodies were investigated in guinea pigs. Each AB-BSA conjugate was immunized with complete Freund's adjuvant. Following the second injection, AB-specific antibodies were detected with high titers. Produced antibodies showed concentration-dependent inhibition by the corresponding AB. Cross-reactivity between the produced antibodies and ABs was evaluated with two methods, i.e. by testing for reactivity with the various AB-ovalbumin conjugates, and by assaying for inhibition of enzyme-linked immunosorbent assay (ELISA) by the various ABs. The latter method could detect minor cross-reactions more sensitively than the former one. The results suggested that similarity of the acyl side chain of a cephalosporin nucleus or a penicillin nucleus contributes to immunological cross-reactivity.

An antibody with dual catalytic activity

Antibodies raised to the bovine serum albumin conjugate of the adduct of acetoxybutadiene with N-(4-carboxybutanoyl)maleimide have been shown to catalyse the Diels-Alder cycloaddition of acetoxybutadiene with N-benzyl- and N-ethyl-maleimide. For one antibody, designated H11, the reaction was selective for 1-acetoxybutadiene; 1-methoxybutadiene and penta-2,4-diene were not substrates. Product inhibition was not observed but the reaction was found to be pH dependent showing a maximum rate at pH 8.5. Analysis of the products of cycloaddition catalysed by H11 indicated that the expected acetoxy adduct was obtained but underwent further reaction catalysed by H11 to afford the corresponding alcohol. This unexpected discovery of a dual catalytic activity associated with an antibody is discussed in the context of hydrolysis reactions catalysed by antibodies.

Immune responses to colophony, an agent causing occupational asthma.

Inhalation of fumes from heated colophony (pine resin) is a recognised cause of occupational asthma, although the mechanisms by which colophony produces symptoms are unclear and specific immune responses to colophony have not been reported in sensitised workers. A study was carried out to determine whether colophony is antigenic. The immune responses to colophony were studied in C57BL/6 mice and Dunkin Hartley guinea pigs after intraperitoneal injection of colophony conjugated to bovine serum albumin (BSA) or human IgG by a mixed anhydride procedure. Colophony and dinitrofluorobenzene were also compared in an assay of dermal sensitisation. Mice immunised with the colophony conjugates produced antibodies which recognised conjugates of both BSA and human IgG irrespective of which had been used as the immunogen. Solutions of unconjugated colophony inhibited the binding of antibodies to the BSA-colophony and BSA-abietic acid conjugates, confirming that the antibodies recognised one or more components in the colophony. Portuguese colophony also abrogated the antigen binding of serum from guinea pigs immunised with the BSA-colophony conjugate. Spleen cells from immunised mice proliferated in the presence of the conjugates. Although there was some cross reactivity in these responses, it was not as marked as in the antibody assays. Unconjugated colophony failed to induce an immune response when injected intraperitoneally with adjuvant. Skin sensitisation could not be induced in mice by topical application, or by subcutaneous or intradermal injection of unconjugated colophony. Colophony components have the potential to act as haptens and an immune component could be involved in the pathogenesis of occupational asthma in workers exposed to colophony. Colophony is not readily immunogenic unless conjugated ex vivo to proteins.

Production and specificity of antisera raised against 25-hydroxyvitamin D 3-[C-3]-bovine serum albumin conjugates

In order to obtain specific antisera for use in the enzyme immunoassay of 25-hydroxyvitamin D 3, three hapten-carrier conjugates having different lengths of bridges at the C-3 position were prepared from 25-hydroxyvitamin D 3 by coupling with bovine serum albumin using the active ester method. The specificity of anti-25-hydroxyvitamin D 3 antisera elicited in rabbits was tested by a cross-reaction study with closely related secosterols and by measuring the plasma levels of 25-hydroxyvitamin D 3 by means of radioimmunoassay using tritium-labeled antigen. The results indicated that the specificity of the antisera obtained is higher than that of vitamin D-binding protein, and that some of these antisera are suitable for enzyme immunoassay.

A direct radioimmunoassay for 5 alpha-androstane-3 alpha,17 beta-diol 17-glucuronide.

Synthesis of the 11 alpha-hemiglutaryl derivative of 5 alpha-androstane-3 alpha,17 beta-diol 17-glucuronide (androstane-diol-17G) starting from androsta-4,9(11)-diene-3,17-dione through a 10-step sequence and the preparation of its bovine serum albumin conjugate is described. By using this conjugate, antiserum was raised in rabbits which proved to be very specific for androstanediol-17G. A direct radioimmunoassay using a double antibody procedure is described for the measurement of androstanediol-17G from plasma without prior chromatography.