Hemisuccinate-bovine Serum Albumin Research Articles

Development of Highly Specific Enzyme Immunoassay for Monitoring Serum Digitoxin Level in Patients

We previously developed radioimmunoassays (RIAs) for digitoxin and digoxin using antisera raised against digitoxin 3’-hemisuccinate-bovine serum albumin and digoxin 3’-hemisuccinate-bovine serum albumin conjugates, respectively. Very recently, we converted the RIA for digoxin to an enzyme immunoassay (EIA) system. Here, we aimed to convert the RIA for digitoxin to an EIA suitable for measuring serum digitoxin level in patients, using digitoxin 3’-hemisuccinate-β-D-galactosidase as an enzyme-labeled antigen. The developed EIA showed a quantification range of 1 to 70 ng/mL and exhibited high specificity for digitoxin, with low cross-reactivity to digitoxin metabolites. Compared with a commercial anti-digitoxin antiserum clinically used to monitor serum digitoxin level in patients, our antiserum showed much higher specificity for intact digitoxin. Intra- and inter-assay variations were less than 10.0% and 8.5%, respectively. Recovery was within the range of 93.7% - 107.5%. Mean digitoxin concentrations measured in serum samples (n = 26) from digitoxin-treated patients by EIA using our new antiserum and the commercial anti-digitoxin antiserum were 11.0 and 13.8 ng/mL, respectively. The present EIA, which is superior to RIA in terms of convenience and disposal of waste materials, is expected to be practically useful for clinical monitoring of intact digitoxin in serum.

Properties of Novel Anti-digoxin Antisera in Radioimmunoassay Using Homologous and Site Heterologous Tritium-Labeled Antigens Involving a [3H]-Leucine Moiety

The specificities of antisera against digoxin C-3' or C-3'' hemisuccinate-bovine serum albumin (BSA) conjugate were assessed by cross-reactivity studies with digoxin metabolites by radioimmunoassay (RIA) using the homologous and the site heterologous tritium-labeled antigens. One of the tracers used was digoxin 3'-hemisuccinyl-[3H]-leucine; the other was digoxin 3''-hemisuccinyl-[3H]-leucine, which had been prepared from digoxin 3''-hemisuccinate. When the tracer with [3H]-leucine at the C-3' position was used, antisera (I-1, I-3) elicited by digoxin 3'-hemisuccinate-BSA conjugate showed the following cross-reactivity: digoxigenin bisdigitoxoside (0.34%, 76%), digoxigenin monodigitoxoside (0.11%, 65%), digoxigenin (0.02%, 26%) and dihydrodigoxin (9.4%, 1.2%). However, when using the homologous antigen, antiserum (I-1) was highly specific against the digitoxose chain. When the site heterologous antigen, digoxin 3''-hemisuccinyl-[3H]-leucine was combined, this antiserum showed high cross-reactivity to digoxin degradation products. This digoxin RIA using antiserum (I-1) with the homologous antigen measures unmetabolized digoxin. On the other hand, the RIA system using antiserum (I-3) with the homologous antigen had cross-reactivity with the metabolites in accordance with their relative cardio-activities, so this system would be useful in therapeutic drug monitoring of digoxin.

Immunochemical Assay for Satratoxin G and other Macrocyclic Trichothecenes Associated with Indoor Air Contamination by Stachybotrys chartarum

Although satratoxin G (SG) is the primary macrocyclic trichothecene produced by Stachybotrys chartarum, a fungus frequently associated with outbreaks of indoor air illness, little is known about the actual quantities of this mycotoxin in suspect or normal building environments. Using antibodies produced against an SG hemisuccinate-bovine serum albumin conjugate, we devised an enzyme-linked immunosorbent assay that enabled the detection of as little as 100 pg/mL of SG and that could also detect other members of the satratoxin family. Detection by this assay of satratoxins in methanolic extracts of S. chartarum in rice cultures was comparable to that of high-performance liquid chromatography, indicating that this assay will be useful in detecting SG and other satratoxins in environmental samples.

Development of Radioimmunoassay for Measurement of Serum Digoxin in Digitalized Patients Using Novel Anti-digoxin Antiserum.

There is an antiserum elicited by digoxin 3'-hemisuccinate-bovine serum albumin (BSA) conjugate possessing high specificity for digoxin. Our study focused on development of RIA using this novel antiserum for measurement of digoxin in serum from digitalized patients. The property of the new antiserum was investigated by RIA with digoxin 3'-hemisuccinyl-[3H]leucine. The separation of bound and free fractions was performed using a dextran-coated charcoal suspension. The new antiserum bound approximately 50% of digoxin 3'-hemisuccinyl-[3H]leucine with a final dilution of 1:30000. The intra- and inter-assay coefficients of variation were <9% in the range of 0.52-4.17 ng/ml. The mean digoxin concentration in serum samples (n=35) from digitalized patients was estimated to be 0.68 ng/ml, which was lower than its measurement of digoxin with the commercial antidigoxin BSA serum and monoclonal anti-digoxin. It is apparent that the RIA described here has sufficient precision. The RIA system was available for the measurement of digoxin in serum from digitalized patients.

Preparation of specific antisera to digoxin by using digoxin C-3' and C-3" hemisuccinate-bovine serum albumin conjugates.

The preparation and antigenic properties of digoxin C-3' and C-3" hemisuccinate-bovine serum albumin (BSA) conjugates are described. The antisera were prepared by immunizing rabbits with each of the digoxin-BSA conjugates, and properties of the antisera were characterized by RIA with 3H-labeled digoxin. The antidigoxin antiserum from immunization with digoxin 3'-hemisuccinate-BSA conjugate possessed high specificity for digoxin, exhibiting fairly low cross-reactions with dihydrodigoxin (2.1%), digoxigenin monodigitoxoside (0.9%), digoxigenin bisdigitoxoside (0.6%), and digoxigenin (0.1%).

Preparation of monoclonal antibody against crocin and its characterization.

Three crocin-carrier protein conjugates were synthesized and their hapten numbers were determined by matrix-assisted laser desorption/ionization time of flight mass spectrometry. Three monoclonal antibodies against crocin were produced by hybridomas fused with the splenocytes immunized with crocin hemisuccinate-bovine serum albumin conjugate and HAT-sensitive mouse myeloma cell line, P3-X63-Ag8-653. They were identified as IgG2a and IgG2b possessing lambda light chain, respectively. Their wide reactivities against crocetin glycosides were discussed.

Production and characterization of monoclonal antibodies to dehydroepiandrosterone sulfate: Application to direct enzyme-linked immunosorbent assays of dehydroepiandrosterone sulfate and androsterone/epiandrosterone sulfates in plasma

Mice were immunized with 5-androstene-3β-ol-7,17-dione-7-CMO:bovine serum albumin (DHEA-7-O-CMO-BSA) or 5-androstene-3β-ol-17-one hemisuccinate-bovine serum albumin (DHEA-3HS-BSA) conjugates and monoclonal antibodies were produced, characterized, and selected for maximum DHEAS binding. Of these hybridomas, four clones from DHEA-3HS-BSA-immunized mice had acceptable criteria for the development of a competitive enzyme-linked immunosorbent assay (ELISA) for DHEAS in plasma. One hybridoma supernatant from DHEA-7-O-CMO-BSA-immunized mice showed 360% cross-reactivity to both androsterone sulfate and epiandrosterone sulfate. This allows the possibility of the direct determination of androsterone sulfate and epiandrosterone sulfate in plasma after correction for the DHEAS contribution. Both ELISAs employ a DHEA-3HS-thyroglobulin conjugate adsorbed to the wells of a standard 96-well microtiter plate. DHEAS in the standards or diluted plasma sample competes with immobilized DHEA-3HS-thyroglobulin for antibody-binding sites. Antibody is detected with anti-mouse-Ig peroxidase by further washing, adding o-phenylenediamine substrate, and reading the absorbance at 492 nm. The ELISAs are simple, reproducible, and reliable and, to our knowledge, they are the first tests employing monoclonal antibodies to DHEAS.

Measurement of the anti-HIV agent 2′,3′-didehydro-2′,3′-dideoxythymidine (D4T) by competitive ELISA

2′,3′-didehydro-2′,3′-dideoxythymidine (D4T) is a thymidine analogue with potent anti-HIV activity in vitro and is currently being investigated therapeutically in patients with advanced HIV infection. We describe a first one-step competitive ELISA method developed for D4T measurement. Anti-D4T rabbit antibodies were raised against a D4T hemisuccinate-bovine serum albumin immunogen. A D4T-hemisuccinate-horseradish peroxidase conjugate and a monoclonal anti-rabbit IgG antibody insolubilized onto a microtiter plate were used as a tracer and capture system, respectively. The method was capable of detecting 2 ng/ml of D4T in cell cultures and 20 ng/ml of D4T in plasma samples previously separated in microconcentrator devices. Cross-reactivit y analysis showed that thymidine, D4T monophosphate, or azidothymidine, were weakly recognized by the ELISA and that thymine or other nucleosides were unreactive. The test was successfully used for the quantification of D4T in cell extracts from CEM or Molt 4 cell lines cultured with D4T and in the plasma of patients with advanced HIV infection, receiving D4T therapy. Moreover this ELISA could be used for the indirect quantification of D4T phosphorylated intracellular metabolites previously separated by reverse phase HPLC and hydrolyzed with alkaline phosphatase.

Development and validation of a monoclonal antibody enzyme immunoassay for measuring progesterone in saliva

A nonextraction, competitive, solid-phase enzyme immunoassay with a monoclonal antibody was developed and validated for measuring progesterone in saliva. The antibody was raised against 11 alpha-hydroxyprogesterone hemisuccinate-bovine serum albumin conjugate and was indirectly immobilized to the walls of microtiter wells. The labeled analyte (progesterone-horseradish peroxidase conjugate) was homologous with the immunogen. The lower detection limit (concentration equivalent to B0-3 SD) was 38 pmol/L of saliva sample. We validated the assay with studies to establish the independence of the concentration determined from the volume of saliva assayed, quantitative recovery of progesterone added to saliva, interference from possible cross-reactants, and agreement with a similar assay that incorporated an extraction step. In addition, we determined luteal-phase concentrations of salivary progesterone in normal women and compared the results with those of an older assay involving a polyclonal antibody.

Highly specific anti-digoxin antiserum from immunization with digoxin 3'-hemisuccinate-bovine serum albumin conjugate

Highly specific anti-digoxin antiserum from immunization with digoxin 3'-hemisuccinate-bovine serum albumin conjugate

The first specific antibody against cytotoxic polyacetylenic alcohol, panaxynol.

Antitumor polyacetylenic alcohol, panaxynol, was isolated and purified from a powder of the root of Panax ginseng C.A. Meyer. Panaxynol inhibited the growth of various kinds of cultured tumor cell lines in a dose-dependent manner. In this paper we demonstrated the first specific antibody production against panaxynol. Anti-panaxynol antibody was elicited in rabbits by immunization with panaxynol hemisuccinate-bovine serum albumin conjugate (panaxynol hemisuccinate-BSA conjugate). An enzyme immunoassay (EIA) for the determination of panaxynol was established using a double-antibody technique. The EIA was highly specific against panaxynol although the antibody showed a minimal cross-reactivity with other types of polyacetylenic alcohol, i.e. panaxydol (12.0%) and panaxytriol (0.77%). Panaxynol at a concentration as low as 6.4 ng/ml can be detected. Using this assay we reconfirmed the rapid consumption of panaxynol by target tumor cells in an in vitro-culture system. The anti-panaxynol antibody may be a valuable tool for studies of the biological properties of polyacetylenic compounds.

Direct Enzyme-Immunoassay for Plasma Androstenedione

Abstract A competitive and sensitive enzyme-linked immunoadsorbent assay (ELISA) allowing determination of androstenedione (A) in unextracted human plasma is described. A highly specific antiserum (anti-4-androsten-11α-ol-3,17 dione-11α hemisuccinate-bovine serum albumin) was employed. Horseradish peroxidase (HRP) was used as the label enzyme; free-bound separation was carried out by adsorbing purified IgG of antiserum on polystyrene balls. Plasma protein binding capacity was reduced by adding 8-anilinonaphtalene-1-sulfonic acid ammonium salt (ANSA). After 3h-incubation, enzyme activity was measured by a colorimetric reaction using o-phenylenediamine dihydrochloride as chromogen and hydrogen peroxide as substrate. The sensitivity of the assay was 5 pg/tube. In order to compare ELISA with radioimmunoassay (RIA) A estimations, plasma samples from thirty women with different endocrine disorders were assayed. A good correlation was found between the two techniques: r = 0.930, p. > 0.001. The accuracy, reprodu...

Preparation of specific antiserum to digitoxin by using digitoxin hemisuccinate-bovine serum albumin conjugate at the digitoxose C-3' position

Preparation of specific antiserum to digitoxin by using digitoxin hemisuccinate-bovine serum albumin conjugate at the digitoxose C-3' position