Bovine Serum Albumin Conjugate Research Articles

Production of ultrasensitive antibodies against aflatoxin B 1

Aims: To produce specific antibodies against the haptenic fungal toxin aflatoxin B 1 (AFB 1 ) and apply these antibodies in immunochemical assays for aflatoxins. Methods and Results: Rabbits were immunized using an AFB 1 -bovine serum albumin conjugate and serum titres determined by double-antibody enzyme immunoassay. High titres of antibodies with very high affinity for AFB 1 were obtained 15 and 4 weeks after the initial immunization and the first booster immunization respectively. The antibodies were employed in enzyme immunoassay (EIA) and immunoaffinity chromatography (IAC) methods for aflatoxins. With a detection limit of 15.8 pg ml -1 for AFB 1 , the EIA employing these antibodies is the most sensitive test for AFB 1 described so far. In IAC columns, these antibodies provided high binding capacity for all major aflatoxins, including AFB 1 , AFB 2 , AFG 1 and AFG 2 . Conclusions: The antibodies described here are useful for the analysis of trace levels of aflatoxins. Significance and Impact of the Study: Polyclonal antibody-based EIA and IAC methods for aflatoxin analysis offer a suitable alternative to the more expensive monoclonal antibody-based methods.

ENZYME IMMUNOASSAY FOR CONJUGATED 7α-HYDROXY-3-OXO-4-CHOLENOIC ACID IN HUMAN URINE

A microplate enzyme immunoassay (EIA) was developed for the measurement of glycine- and taurine-conjugated 7α-hydroxy-3-oxo-4-cholenoic acids (CDCA-Δ4-3-one) in human urine. The antiserum was prepared by immunizing rabbits with N-(7α-hydroxy-3-oxo-4-cholen-24-oyl)-3-aminopropionic acid - bovine serum albumin conjugate. A colorimetric EIA was established using horseradish peroxidase-labeled antigen having a shorter bridge length than that of the immunogen, and 3, 3', 5, 5'-tetramethylbenzidine/hydrogen peroxide for the measurement of the enzyme activity. The reactivities of the antiserum for glycine and taurine conjugates of CDCA-Δ4-3-one was almost the same. The specificity of the antiserum was investigated by determining the cross-reactivities of various bile acids and related compounds. An appropriate dose-response curve for conjugated CDCA-Δ4−3-one was obtained in the range of 0.05–10 ng/well. This method was used for direct analysis of conjugated CDCA-Δ4−3-one in urine of healthy infants and patients with liver diseases.

Immune response to progesterone immobilized on Cu2+-induced amphifilic polyelectrolyte-protein complex: antigen specificity and affinity of hybridoma clones.

Cu2+-mediated complex formation between copolymers of acrylic acid with N-isopropyl-acyrlamide (CP1) and negatively charged covalent conjugate of bovine serum albumin with progesterone (BSA.P) was studied in neutral water in the presence of Cu2+. It was shown that under conditions where CP and BSA.P are negatively charged and incapable of binding to one another, the divalent Cu2+ act as "fasteners" promoting the formation of relatively stable water-soluble ternary polycomplexes. The immunogenic properties of ternary mixtures BSA.P-Cu2+-CP1 and BSA.P+IFA were investigated and the production of monoclonal antibodies (MAbs) against progesterone hormone was analyzed. Fusion following the two different immunization procedures resulted in the growth of comparable numbers of progesterone-specific MAbs with apparently similar antigen affinities. Thus, immunizations using antigens in BSA.P-Cu2+-CP1 appear to provide an efficient alternative to incomplete Freund's adjuvant.

Visualization of progesterone binding to plasma membrane ofxenopusoocytes

We have previously shown that oocyte maturation is induced by an immobilized progesterone, progesterone‐3‐carboxymethyloxime ‐ bovine serum albumin conjugate (P‐BSA) in Rana dybowskii. In this study, we confirmed the maturation inducing activity of P‐BSA on Xenopus oocyte and examined the binding character of the immobilized progesterone on the surface of Xenopus oocytes after removal of the vitelline layer. P‐BSA induced maturation of Xenopus oocytes but E‐BSA failed to do so as observed in Rana. Binding of the immobilized progesterone, fluorescein isothiocyanate‐labeled progesterone‐3‐O‐carboxymethyloxime‐BSA (P‐BSA‐FITC) on the devitellined oocytes surface was examined by fluorescence confocal microscopy. The binding affinity of P‐BSA‐FITC to the devitellined oocyte was higher than that of estrogen‐BSA‐FITC (E‐BSA‐FITQ or testo‐sterone‐BSA‐FITC (T‐BSA‐FITC). The binding disappeared in the presence of excess free progesterone but not in the presence of free estrogen. Maximum binding occurred after two‐h...

A Surface Plasmon Resonance-Based Immunosensors for Sensitive Detection of Heroin

A simple technique for sensitive detection of heroin based on surface-plasmon-resonance has been theoretically and experimentally investigated. The experiment was realized by using an anti-MO monoclonal antibody and a morphine (MO)-bovine serum albumin (MO-BSA) conjugate (antigen). The reason for using MO-BSA in the detection of heroine was also discussed. MO-BSA was immobilized on a gold thin film of SPR sensor chip by physical adsorption. The configuration of the device is allowed to be further miniaturized, which is required for the construction of a portable SPR device in the application of in-situ analysis.

Production of a Monoclonal Antibody for Sensitive Monitoring of Deoxycholic Acid Residues Anchored on Endogenous Proteins

Bile acid carboxy-linked glucuronides and adenylates have active ester groups, and thus may easily react with amino groups on endogenous tissue proteins resulting in physiological disorders. To identify such tissue-bound bile acids in vivo, we generated a monoclonal antibody which recognizes deoxycholic acid (DCA) residues anchored on endogenous proteins. The spleen cells from a BALB/c mouse, which was immunized with a conjugate of DCA and bovine serum albumin (BSA), were fused with P3/NS1/1-Ag4-1 myeloma to generate antibody-secreting hybridoma clones. The resulting monoclonal antibody (Ab#88; isotype γ1, λ) was specific to N-ε-deoxycholyl lysine as well as the nonamidated DCA, and enabled sensitive detection of DCA residues (270 femtograms) anchored on BSA molecules, introduced by the reaction of the DCA adenylate. The immunoaffinity column immobilizing the Ab#88 allowed selective extraction of the DCA-introduced substance P. The antibody will be useful for monitoring the formation and localization of tissue-bound DCA, which may be concern with the activity of DCA as a colon cancer promoter.

Peptide mimic of phosphorylcholine, a dominant epitope found on Streptococcus pneumoniae.

Even in the age of antibiotics, Streptococcus pneumoniae causes significant morbidity, especially in the young, the elderly, and the immunocompromised. While a carbohydrate-based vaccine exists, it is poorly immunogenic in the at-risk populations. In mice, antibodies directed against phosphorylcholine (PC), an epitope present on the cell wall C polysaccharide of all pneumococcal serotypes, protect against infection. However, PC itself is a poor vaccine candidate. We report here peptide mimics of PC based on the anti-idiotypic interaction of T15 anti-PC antibodies. T15 antibodies, the dominant and protective idiotype induced in mice by PC immunization, self-associate via a 24-amino-acid region in the PC binding site (ASRNKANDYTTEYSASVKGRFIVS; peptide 1). Peptide 1 has been shown to bind in the PC binding site. We demonstrated that amino acid sequences derived from peptide 1 starting at amino acid 9, 11, or 13 inhibit PC binding. Therefore, we immunized mice with bovine serum albumin (BSA) conjugates of peptide 1 or either of two selected 12-mers. The 12-mer peptides were not immunogenic. Mice immunized with peptide 1-BSA developed an anti-PC response consisting mainly immunoglobulin G1 and expressed the T15 heavy chain. Nonetheless, neither BALB/c nor CBA/N mice were protected from lethal pneumococcal infections by immunization with peptide 1-BSA. Preliminary data suggest that peptide 1-BSA is not able to elicit the canonical T15 light chain, explaining the absence of protection. This idiotype-derived mimotope of PC is a useful tool for understanding immunologic cross-reactivity and learning to design T-cell-dependent vaccines for S. pneumoniae.

Dual-label time-resolved fluoroimmunoassay of psychopharmaceuticals and stimulants in serum

A new method to measure two different drugs simultaneously by time-resolved fluoroimmunoassay (TR-FIA) has been developed. In the TR-FIA reported here, psychopharmaceuticals [chlorpromazine (CPZ) and desipramine (DSP)] and methamphetamine (MA) contained in serum are assayed by a combined use of a new europium (Eu) chelate and a samarium (Sm) chelate, as labels. The drug concentrations were determined by the competition between a labeled antigen with Eu 3+ or biotin and a sample antigen. A microtiter plate coated with a mixture of rabbit IgGs (anti-MA and anti-CPZ or anti-MA and anti-DSP) was used. In the assay of MA and CPZ, Eu 3+ labeled MA–bovine serum albumin conjugate (MA–BSA) and biotinylated CPZ–BSA were added to the well with their non-labeled standard solutions or samples. MA was assayed by measuring the fluorescence intensity of Eu 3+ at 615 nm. After incubation of the Sm 3+ labeled streptavidin, CPZ was assayed by measuring the fluorescence of Sm 3+ at 643 nm. In the assay of MA and DSP, Eu 3+ labeled DSP–BSA and biotinylated MA–BSA were used. In our dual-assay, the minimum detection limits of these drugs were 1ng/ml for MA, 10 ng/ml for CZP and 10 ng/ml for DSP. Since the simultaneous detection of different drugs by TR-FIA is time and sample saving, the method can be employed in rapid and sensitive screening tests.

Tissue Transglutaminase, Coagulation Factor XIII, and the Pro-polypeptide of von Willebrand Factor Are All Ligands for the Integrins α9β1 and α4β1

We previously reported that MOLT-3 human lymphocyte-like leukemia cells adhere to tissue-type transglutaminase (tTG) through the integrin alpha(4)beta(1). We now report that G-361 human melanoma cells also adhere to tTG, although they do not express alpha(4)beta(1). G-361 cells utilize two additional integrins, alpha(9)beta(1) and alpha(5)beta(1) to adhere to tTG. Furthermore, blood coagulation factor XIII (FXIII), another member of the transglutaminase family that is highly homologous to tTG, and propolypeptide of von Willebrand factor (pp-vWF) also promoted cell adhesion through alpha(9)beta(1) or alpha(4)beta(1) in G-361 or MOLT-3 cells, respectively. In the case of pp-vWF, alpha(9)beta(1) and alpha(4)beta(1) both bind to the same site, comprised of 15 amino acid residues and designated T2-15. Moreover, SW480 human colon cancer cells stably transfected to express alpha(9)beta(1), but not mock transfectants, adhered to tTG, FXIII, pp-vWF, and T2-15/bovine serum albumin conjugate. These data identify tTG, FXIII, and pp-vWF as shared ligands for the integrins alpha(9)beta(1) and alpha(4)beta(1). This report is the first to unambiguously show that these two integrins share the same cell adhesion site within one protein and provides strong support for classifying alpha(9)beta(1-) and alpha(4)-integrins as functionally related members of an integrin subfamily.

Direct determination of naturally occurring biologically active compound–serum albumin conjugate by matrix‐assisted laser desorption/ionization mass spectrometry

Opium alkaloids were conjugated with bovine serum albumin (BSA) to give individual antigen conjugates which were analyzed using BSA as an internal standard by matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry. It became clear that 9 molecules of thebaine were conjugated in a thebaine–BSA conjugate. Codeine and morphine contents in individual conjugates were determined to be 12 and 6 molecules, respectively. Using a relative molecular mass of BSA, 11.5 molecules of forskolin conjugated with BSA. Tetrahydrocannabinolic acid (THCA)–carrier protein conjugates using β‐alanine or without β‐alanine as a linker were analyzed. From these results it became evident that the hapten molecules of THCA–β‐alanine–BSA, THCA–BSA, THCA–β‐alanine–human serum albumin (HSA) and THCA–HSA were 17.2, 12.7, 27.8 and 54.4, respectively. Crocin–HSA conjugates were synthesized by two methods using succinate and NaIO4 cleavage of sugar moiety. A broad molecular peak of crocin–HSA conjugate appeared at around m/z 98,342 resulting in 30 molecules of crocin‐hemisuccinate conjugated with HSA. The crocin–HSA synthesized via NaIO4 oxidation showed a sharp molecular peak around 69,504 compared to that of hemisuccinate preparation. The number of crocin combined with HSA was, however, smaller 3.1 molecules.Analysis of monoclonal antibody by MALDI mass spectrometry determined the exact molecular weight and their purities.

Competitive immunoassay method for 5-methyl-2′-deoxycytidine

The paper describes the preparation of monoclonal antibodies against 5-methyl-2′-deoxycytidine (5MedCyd) and its utility in the development of a competitive immunoassay system for 5MedCyd. The hybridoma was constructed by fusion of the spleen cells removed from mouse immunized with 3′-succinyl-2′-deoxycytidine keyhole-limpet hemocyanin conjugate with SP2/0-Ag14 myeloma cells. Three high titer clones that secreted anti 5MedCyd antibodies were selected. The antibodies from the three clones recognize 5MedCyd; however, small cross reactivity was observed with 5-methylcytidine and 5-methylcytosine, which possess 5-methylcytosine moiety in their structure. The clone that showed the lowest cross reactivity with these compounds was designated as 8C-4. 8C-4 antibody, which had very slight cross reactivity with the other adenosine and guanosine analogues tested in our investigation particularly the methylated ones. This monoclonal antibody was used for establishment of an immunoassay method for 5MedCyd. In this method, 8C-4 antibody was allowed to react with the free 5MedCyd in a standard solution or a urine sample in competition with 3′-succinyl-2′-deoxycytidine bovine serum albumin conjugate immobilized on microtiter plate wells. Bound antibody was quantified with alkaline phosphatase-labeled goat anti-mouse secondary antibody and p-nitrophenylphosphate substrate solution. A linear relationship with good correlation coefficient was obtained over the range 1–40 μM. The specificity, reproducibility and accuracy of the proposed method was proved. There is a good correlation between the results obtained by this method and by liquid chromatography over the entire linear range. By this system, evaluation of the urinary 5MedCyd level as a biological marker for leukemic patients will be possible.

Selectively conjugated melittins for liposome time-resolved fluoroimmunoassay of theophylline in serum.

Theophylline (Th) has been selectively conjugated to the four amino groups of melittin (Mel) by solid phase peptide synthesis. The cytolytic activity of the resultant Th-Mel compounds was tested on liposomes trapping the bovine serum albumin (BSA) conjugate with 4,7-bis(chlorosulfophenyl)-1,10-phenanthrol ine-2,9-dicarboxylic acid (BCPDA). The loss of lytic activity was the highest for Th-K7-Mel. Th-G1-Mel retains almost the same lytic activity as Mel. A homogeneous liposome time-resolved fluoroimmunoassay (LITRFIA) of Th in serum has been carried out with Th-G1-Mel between 5 ng and 10 microg.

A monoclonal antibody-based enzyme-linked immunosorbent assay of ursodeoxycholic acid 3-sulfates in human urine

Sulfation of the 3-hydroxy group is assumed to be a major metabolic route of ursodeoxycholic acid (UDCA) which is used for treating various hepatobiliary diseases. We have developed a sensitive enzyme-linked immunosorbent assay (ELISA) for determining the total amount of nonamidated, glycine- and taurine-amidated ursodeoxycholic acid 3-sulfates (UDCA 3-Suls) using a newly established monoclonal antibody. In this study, 12 kinds of antibody-secreting hybridoma clones were generated by a fusion experiment between P3/NS1/1-Ag4-1 myeloma cells and the spleen cells from a BALB/c or an A/J mouse which had been immunized with a conjugate of nonamidated UDCA 3-Sul and bovine serum albumin. One of the monoclonal antibodies, Ba-10 (γ2a, κ), had suitable binding properties for clinical application, which was group-specific to the UDCA 3-Suls, and showed negligible cross-reactivities with various related bile acids including potentially interfering compounds, namely, the unconjugated UDCA, UDCA 7- N-acetylglucosaminide, the 3-sulfates of cholic acid, chenodeoxycholic acid and deoxycholic acid. The antibody Ba-10 allowed us to develop a sensitive competitive ELISA system whose measurable range was approximately 2–200 pg per assay. A serial dilution study indicated that the ELISA enables the direct measurement of the UDCA 3-Suls in human urine before and after the administration of exogenous UDCA. The daily urinary excretion rate of UDCA 3-Suls from healthy male volunteers ( n = 5) was determined to be a mean of 131 ± 61.2 (SD) μg as the nonamidated UDCA 3-Sul equivalent.

Development of a One Step Strip Test for the Detection of (Dihydro)streptomycin Residues in Raw Milk

The one step strip test described is a competitive immunoassay in which the detector reagent consists of colloidal gold particles coated with affinity purified monoclonal anti(dihydro)streptomycin antibodies. The capture reagent in the assay is a streptomycin- bovine serum albumin conjugate which is immobilised on the lateral flow membrane of the test device. In the test procedure, three drops of raw milk are brought into the sample well of the test device and allowed to migrate over the membrane. The more analyte present in the sample, the more effectively it will compete with the streptomycin immobilised on the membrane for binding to the limited amount of antibodies of the detector reagent. A sufficient amount of (dihydro)streptomycin in the sample will thus prevent the binding of the detector reagent to the streptomycin immobilised on the membrane, resulting in disappearance of the test line in the read out zone. Using raw milk samples, spiked with either streptomycin or dihydrostreptomycin, complete disappearance of the test line was obtained with 160 ng ml−1 and 190 ng ml−1, respectively. This demonstrates that the test is applicable for screening raw milk samples for the presence of (dihydro)streptomycin residues at MRL level (EU: 0.2 mg (dihydro)streptomycin kg−1 milk). The major advantages of the one step strip test are that results can be obtained within 10 min and that all reagents are included in the test device.

Synthesis of haptens and conjugates for ELISAs of phytoestrogens. Development of the immunological tests.

Seven carboxylic acid haptens of isoflavonoids were synthesized, with the spacer arm on the oxygen atom at the C7 position for one series, with formononetin, daidzein, equol, biochanin A, and genistein, and at the C8 position for a second series, with only formononetin and daidzein. The different haptens were coupled to bovine serum albumin (BSA) and to swine thyroglobulin (Thyr). Polyclonal antibodies were generated against the BSA conjugates. Enzyme-linked immunosorbent assays (ELISAs) were developed based on competition between free phytoestrogens and the Thyr-hapten conjugates for specific antibodies. IC(50) values of the standard curves ranged between 0.8 and 20 ng/mL that is, 0.3 and 9.2 pmol/well. The antibodies obtained should be useful for assays in vegetable matter as well as in biological fluids after a separation step. These ELISAs should be valuable also in the food industry to control phytoestrogen concentrations prior to and after processing.

Antigen binding characteristics of antibodies against hydroxyl radical modified thymidine monophosphate

Reactive oxygen species (ROS) are formed in living organisms during normal metabolic reactions as well as under different environmental stresses. In this study, thymidine monophosphate (TMP) was exposed to hydroxyl radical (OH) and challenged in rabbits. TMP and OH-modified TMP were found to be nonimmunogenic. The TMP was linked to bovine serum albumin (BSA) by carbodiimide reaction, and then modified with the OH. The neoantigens, TMP–BSA, and ROS–TMP–BSA conjugates induced highly specific antibodies against immunogens. Induced antibodies exhibited appreciable cross-reactivity with various polynucleotides and nucleic acids. In this respect, the induced antibodies resembled the diverse antigen-binding characteristics of naturally occurring systemic lupus erythematosus (SLE) anti-DNA autoantibodies.

A Fluorescent Biosensor for Detection of Zearalenone

ABSTRACT A fluorescent biosensor was developed on a KinExATM flow spectrofluorimeter for the near real-time detection of soluble zeaalenone. Briefly, solutions of zearalenone and a monoclonal antibody directed against a protein conjugate of zearalenone, were incubated for thirty minutes to permit equilibrium binding to occur. The reaction mixture was then passed over a packed column of small beads (98 μm) whose surfaces were coated with a covalent conjugate of zearalenone and bovine serum albumin (BSA). Following a short wash with buffer to remove excess unbound primary reagents, the packed beads were subjected to a brief contact with fluorescein isothiocyanate-labeled polyclonal secondary antibody directed against the primary monoclonal, once again followed by a short wash. As this assay depends on the ability of soluble antigen to compete with immobilized antigen, increasing concentrations of zearalenone result in decreasing fluorescence observed on the bead pack. This assay is rapid (≅ 60 minutes) and can be adapted to various other analytes of interest.

Formation of monoclonal antibody against a major ginseng component, ginsenoside Rg1 and its characterization. Monoclonal antibody for a ginseng saponin.

The ratio of hapten and bovine serum albumin in an antigenconjugate was determined by matrix-assisted laserdesorption/ionization time-of-flight mass spectrometry. Ahybridoma secreting monoclonal antibody against ginsenosideRg(1) was produced by fusing sprenocytes immunized with aginsenoside Rg(1)-bovine serum albumin conjugate withHAT-sensitive mouse myeloma cell line, P3-X63-Ag8-653. A very smallcross-reaction appeared with ginsenoside Re. The full measuringrange of the assay extends from 0.3 mug ml(-1) to 10 mug ml(-1) ofginsenoside Rg(1).

Enzyme-linked immunosorbent assay for 2-deoxycytidine

An enzyme-linked immunosorbent assay (ELISA) method that can be used to quantify 2′-deoxycytidine (dCyd) in plasma is described. In this method, 3′-succinyl-dCyd bovine serum albumin conjugate (3′sdCyd–BSA) adsorbed on the microtiter plate wells is bound to anti-dCyd monoclonal antibody in inverse proportion to free dCyd in the sample or standard. Bound antibody is quantified with alkaline phosphatase-labeled anti-immunoglobulin (Alp-IgG) antibody and p-nitrophenylphosphate (PNPP) substrate solution. A linear relationship with good correlation coefficients was obtained in the linear range of 10–1000 μ M. There was no cross-reactivity from structurally related compounds with the antibody. In addition, the constituents of plasma do not affect the assay. The intra- and inter-assay reproducibilities are satisfactory at 2–3% relative standard deviation. A short period of incubation at 37°C is required for equilibration of the antigen–antibody reaction. There is a good correlation between the results obtained with the proposed ELISA and a liquid chromatographic method over the entire linear range. The analytical procedure is convenient, and one can analyze 150 samples per working day, facilitating the processing of serial samples. The present method is expected to contribute to further elucidation of the role of dCyd in various biological and biochemical systems.

Lectin-Mediated Drug Targeting: Discrimination of Carbohydrate-Mediated Cellular Uptake between Tumor and Liver Cells with Neoglycoconjugates Carrying Fucose Epitopes Regioselectively Modified in the 3-Position.

The circumvention of efficient "carbohydrate traps" in the liver is required for targeting glycoconjugates on tumor cells. As shown in the model system of bovine serum albumin (BSA) conjugates, the nature of R(1)-R(3) of the fucose epitope plays an important role in the discrimination of cellular uptake between tumor and liver cells as well as in the cytotoxic activity.