Anti-bilirubin monoclonal antibody. III. Preparation and properties of monoclonal antibodies to unconjugated bilirubin-IX alpha

Anti-bilirubin-IX alpha monoclonal antibodies exclusively specific for unconjugated bilirubin-IX alpha are prepared and characterized. Using modified MBS (metamaleimidobenzoyl-N-hydroxysuccinimide ester) method, bilirubin-IX alpha was covalently coupled to bovine serum albumin (BSA) retaining its intramolecular hydrogen bonds as well as three-dimensional structure. Somatic cell fusion was performed between murine spleen cells immunized with the bilirubin-IX alpha-BSA and murine myeloma P3-X63-Ag8-U1 cells. After screening assay, 58 clones were identified which were producing antibodies not to albumin nor MBS, but to haptenic bilirubin-IX alpha. In inhibition analysis, the antibodies in the culture supernatant reacted only with bilirubin-IX alpha to a varying degree, but did not react with conjugated bilirubin-IX alpha, including bilirubin-IX alpha monoglucuronide, bilirubin-IX alpha diglucuronide, and ditauronic bilirubin-IX alpha.     

Production and characterization of monoclonal antibodies against aflatoxin M1.

By using an indirect enzyme-linked immunosorbent assay, four monoclonal antibodies were selected after fusion of mouse P3-NS1-Ag4-1 myeloma cells with spleen cells isolated from BALB/c mice that had been immunized with aflatoxin M1 (AFM1) conjugated to bovine serum albumin. Two of these antibodies were found to be specific for AFM1 and were designated AMW-1 and AMW-4. The specificities of AMW-1, which had higher affinity to AFM1, were determined by a competitive direct enzyme-linked immunosorbent assay with peroxidase-AFM1 as the marker. The relative cross-reactivity of each toxin (relative to AFM1) with AMW-1, as determined by the amount of aflatoxin necessary to cause 50% inhibition of enzyme activity, was 12, greater than 40, 12, and greater than 40 for B1, B2, G1, and G2, respectively.     

A novel method to determine total and free serum bilirubin.

The determination of total (unconjugated) and free serum bilirubin concentrations using a novel and sensitive method based on static fluorescence quenching of daneyl bovine serum albumin was developed. The method allowed the use of a sample of 5 μl or less to determine total bilirubin over a range of 10–200 μg/ml with good recovery (94.9 ± 2.2%). For the determination of total bilirubin, a denaturation medium containing 8 m urea, 10 mm dithloerythreitol, and 0.1 m Tris was employed to eliminate interference by human serum albumin itself. The method was also tested with patients' sera containing negligible conjugated bilirubin in order to compare it to a commonly used “diazo” method. The correlation between the two methods gave a practically linear relation (γ = 0.99). The effects of a number of potentially interfering substances were tested and the results showed the test was specific for bilirubin. Concentrations of free bilirubin were determined without adding a denaturation agent. The experimental values were in agreement with those calculated theoretically using the isotherm of a single binding site and an association constant of human serum albumin to bilirubin of 1.5 × 10⁸m^?1.     

Detection of ochratoxin A in porcine kidneys by a monoclonal antibody-based radioimmunoassay

By using an indirect enzyme-linked immunosorbent assay, eight monoclonal antibodies (MAbs) were selected. Mice were immunized with ochratoxin A that was conjugated to bovine serum albumin. The hybridoma cell line designated 10G2 was grown in tissue culture and as an ascites tumor. The MAb was characterized to be specific to ochratoxin A and of the immunoglobulin G (IgG) class. Subsequently, the ascites fluid of this hybridoma was used in a competitive solid-phase IgG radioimmunoassay on protein A-Sepharose CL-4B, with [14C]ochratoxin A as tracer. Porcine kidneys were extracted with 0.5% phosphoric acid in chloroform. A two-step cleanup was achieved on a Sep-Pak C18 cartridge and a Sep-Pak silica cartridge. Radioimmunoassay with MAbs coupled to protein A-Sepharose CL-4B allowed the detection of ochratoxin A in porcine kidneys at a concentration as low as 0.2 ng/g.     

Production and characterization of monoclonal antibodies to the macrocyclic trichothecene roridin A.

Two murine monoclonal antibodies to the macrocyclic trichothecene roridin A are described. Screening for antibody production was performed on absorbed anti-mouse immunoglobulin serum as double-antibody solid phase, and further characterization was done on affinity-purified anti-mouse IgG serum. The antibodies, designated 5G11 and 4H10, had affinity constants for roridin A of 9.25 X 10(7) and 1.7 X 10(7) liters/mol, respectively. In monoclonal antibody-based direct enzyme immunoassays, these IgG1 antibodies had detection limits for roridin A of 0.4 ng/ml (0.02 ng per assay) and 1.8 ng/ml (0.09 ng per assay), respectively. Both antibodies were most specific for the tested macrocyclic trichothecenes. The relative cross-reactivities of antibody 5G11 with roridin A, roridin J, verrucarin A, satratoxin G, and satratoxin H were 100.0, 43.8, 16.7, 3.7, and 18.9%, respectively; for antibody 4H10 they were 100.0, 6.3, 64.0, 4.4, and 4.9%, respectively.     

Measurement of plasma unbound unconjugated bilirubin

A method is described for measuring the unconjugated fraction of the unbound bilirubin concentration in plasma by combining the peroxidase method for determining unbound bilirubin with a diazo method for measuring conjugated and unconjugated bilirubin. The accuracy of the unbound bilirubin determination is improved by decreasing sample dilution, eliminating interference by conjugated bilirubin, monitoring changes in bilirubin concentration using diazo derivatives, and correcting for rate-limiting dissociation of bilirubin from albumin. The unbound unconjugated bilirubin concentration by the combined method in plasma from 20 jaundiced newborns was significantly greater than and poorly correlated with the unbound bilirubin determined by the existing peroxidase method (r = 0.7), possibly due to differences in sample dilution between the methods. The unbound unconjugated bilirubin was an unpredictable fraction of the unbound bilirubin in plasma samples from patients with similar total bilirubin concentrations but varying levels of conjugated bilirubin. A bilirubin-binding competitor was readily detected at a sample dilution typically used for the combined test but not at the dilution used for the existing peroxidase method. The combined method is ideally suited to measuring unbound unconjugated bilirubin in jaundiced human newborns or animal models of kernicterus.     

Production of polyclonal antibodies to the trichothecene mycotoxin 4,15-diacetylnivalenol with the carrier-adjuvant cholera toxin.

The trichothecene mycotoxin 4,15-diacetylnivalenol (DNIV) was conjugated to cholera toxin (DNIV-CT) for use as an immunogen and as an adjuvant for specific antibody production. Repeated intravenous injection of 7.5 micrograms of the conjugate was effective at generating specific antibodies to DNIV in rabbits as determined by enzyme-linked immunosorbent assay (ELISA). When small amounts (1 to 10 micrograms per animal) of DNIV-CT were used to immunize mice, polyclonal antibodies were observed as early as 4 weeks of immunization. The relative affinity of the antibodies to DNIV increased with the immunogen dose in mice. Antibodies were not detectable in either rabbits or mice that were injected with DNIV conjugated to the carrier protein bovine serum albumin or when DNIV-CT was blocked with glutaraldehyde. Competitive ELISA of mouse and rabbit serum revealed that the antibodies were most specific for DNIV but reacted to a small extent with fusarenone-X, deoxynivalenol, and nivalenol. No reactivity was observed with 3- or 15-acetyldeoxynivalenol. The results suggest that specific polyclonal antibodies can be prepared against a trichothecene when CT is used as an adjuvant and carrier protein. DNIV antibodies will be useful for monitoring the compound in food in conjunction with other trichothecene antibodies, detection of DNIV-producing cultures, and investigation of 8-ketotrichothecene biosynthesis.     

Production and characterization of monoclonal antibodies to the macrocyclic trichothecene roridin A

Two murine monoclonal antibodies to the macrocyclic trichothecene roridin A are described. Screening for antibody production was performed on absorbed anti-mouse immunoglobulin serum as double-antibody solid phase, and further characterization was done on affinity-purified anti-mouse IgG serum. The antibodies, designated 5G11 and 4H10, had affinity constants for roridin A of 9.25 X 10(7) and 1.7 X 10(7) liters/mol, respectively. In monoclonal antibody-based direct enzyme immunoassays, these IgG1 antibodies had detection limits for roridin A of 0.4 ng/ml (0.02 ng per assay) and 1.8 ng/ml (0.09 ng per assay), respectively. Both antibodies were most specific for the tested macrocyclic trichothecenes. The relative cross-reactivities of antibody 5G11 with roridin A, roridin J, verrucarin A, satratoxin G, and satratoxin H were 100.0, 43.8, 16.7, 3.7, and 18.9%, respectively; for antibody 4H10 they were 100.0, 6.3, 64.0, 4.4, and 4.9%, respectively.     

Role of apolipoprotein D in the transport of bilirubin in plasma

Apolipoprotein D (apo D) is a 30-kDa glycoprotein of unknown function that is associated with high-density lipoproteins (HDL). Because unconjugated bilirubin has been shown to bind apo D with a 0. 8:1 stoichiometry, we examined the contribution of this protein to transport of bilirubin in human plasma. Density gradient centrifugation analysis using physiological concentrations of [(14)C]bilirubin reveals that 9% of unconjugated bilirubin is associated with HDL, with the remaining pigment bound primarily to serum proteins (i.e., albumin). The percentage of total plasma bilirubin bound to HDL was found to increase proportionally with bilirubin concentration. Affinity of human apo D for bilirubin was determined by steady-state fluorescence quenching, with Scatchard analysis demonstrating a single binding site for unconjugated bilirubin with an affinity constant (K(a)) of approximately 3 x 10(7) M(-1). Incorporation of apo D into phosphatidylcholine vesicles had no effect on K(a), suggesting that a lipid environment does not alter the affinity of the protein for bilirubin. Using stopped-flow techniques, the first-order rate constant for bilirubin dissociation from apo D was measured at 5.4 s(-1) (half-time = 129 ms). Our findings indicate that HDL is the principal nonalbumin carrier of bilirubin in human plasma and further support the proposition that the affinity of HDL for bilirubin is primarily the result of binding to apo D.     

In vitro permeability and metabolic stability of bile pigments and the effects of hydrophilic and lipophilic modification of biliverdin

Bile pigments, including bilirubin and biliverdin are tetrapyrrolic, dicarboxylic acids capable of forming conjugates at their propionic acid groups via ester or amide bonds. They possess substantial antioxidant and anti-mutagenic activities and therefore their intestinal absorption might influence the development of cardiovascular disease and cancer. The aim of this study was to investigate whether altering the physico-chemical properties of bile pigments would improve their permeability in an in vitro assay of absorption. Native and synthetically modified bile pigments were tested for gastrointestinal permeability and metabolic stability using the Caco-2 cell line. In addition, a gross measure of their toxic effects was tested in a red blood cell co-incubation assay. The apparent permeability of unconjugated bilirubin (1), bilirubin ditaurate (2) and biliverdin (3) through Caco-2 cell monolayers was determined to be 10.4+/-1.2x10(-7), 35.2+/-3.4x10(-7) and 37.0+/-1.6x10(-7) cm/s (mean+/-SD), respectively, while biliverdin diglucosamine (4), and biliverdin dioctylamine (5) were impermeable. Unconjugated bilirubin, biliverdin, bilirubin ditaurate and biliverdin diglucosamine did not decompose when incubated in Caco-2 cell homogenates, whereas biliverdin dioctylamine decomposed over time. Only unconjugated bilirubin showed toxicity towards red blood cells (> or = 1000 microM), an effect that was abolished by the addition of 40 g/L serum albumin. The data presented here suggest that bile pigments are absorbed across the Caco-2 cell monolayer and that conjugation of biliverdin to hydrophilic or lipophilic moieties decreases their absorption and can reduce their metabolic stability. In summary, exogenous bilirubin and biliverdin supplements could be absorbed across the intestinal epithelium in vivo and potentially increase circulating concentrations of these antioxidant compounds.     

Inaccuracies in measurement of conjugated and unconjugated bilirubin in bile with ethyl anthranilate diazo and solvent-partition methods.

A criticial evaluation was made of the ethyl anthranilate diazo and two solvent-partition methods for the determination of conjugated and unconjugated bilirubin in human and rat bile. The ethyl anthranilate diazo reagent, which reacts only with conjugated bilirubin in serum, also diazotized a variable proportion of unconjugated bilirubin in bile and thus overestimated the concentration of monoconjugates. With the Weber-Schalm and modified Folsch solvent-partition methods applied to human or rat bile, 4--9% of added 14C-labelled unconjugated bilirubin partitioned with the conjugated bilirubin in the upper phase, and 4--9% of added 14C-labelled conjugated bilirubin partitioned into the lower phase. With dog bile, the spill-over of 14C-labelled bilirubin into the lower phase was 9--11%. Analysis of azopigments from the Weber-Schalm partition confirmed that over two-thirds of the bilirubin in the lower phase represents monoconjugates, principally the less-polar monoxylosides and monoglucosides. These solvent-partition methods thus overestimate the concentration of unconjugated bilirubin in bile.     

Determinant analysis and interaction studies on monoclonal antibodies to bovine IgG1 and IgG2.

Two mouse monoclonal antibodies SKb1 and SKb6 were prepared by fusion of myeloma cells with spleen cells of female Balb/c mouse immunized with a mixture of bovine IgG1 and IgG2. In radioimmunoassay, SKb1 bound specifically to IgG2 but SKb6 reacted with both IgG1 and IgG2 molecules. In the competition experiments, heavy chain isolated from bovine IgG could inhibit the binding of 125I-IgG1 and 125I-IgG2 to SKb6, while it failed to inhibit the binding of 125I-IgG2 to SKb1. The epitope reacting with SKb1 was found to be present not only on bovine IgG2 but also on goat IgG and was not present on IgG molecules isolated from the serum of rabbit, rat, sheep, horse, human and monkey. Similarly, the epitope reacting to SKb6 was found to be present on bovine IgG1 and IgG2 and also on IgG molecules isolated from goat and sheep serum but was absent in the IgG molecules isolated from the serum of rabbit, rat, horse, human and monkey. The association constants of the interactions of SKb1 with 125I-IgG2 and of SKb6 with 125I-IgG1 and 125I-IgG2, determined by Scatchard analysis, Steward-Petty plot and Sips plot, were found to be in the order of 10(8)-10(10) L/M. The association constants were determined at varying temperatures to obtain the thermodynamic parameters. The enthalpy (delta H0) and entropy (delta S0) values for the above antigen-antibody interactions were in the range of 9.15-15.96 kcal/mole and 36.96-41.15 eu/mole respectively. The heterogeneity indices for similar interactions determined by Sips equation were consistent with the expected values for binding of monoclonal antibodies with homogeneous protein determinants.     

Studies on neurosteroids XIV. Levels of dehydroepiandrosterone sulfate in rat brain and serum determined with newly developed enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay (ELISA) of dehydroepiandrosterone sulfate (DHEAS), one of the neurosteroids, has been developed for measuring its brain and serum levels in rats without deconjugation. 11 alpha-Hemiglutaryloxy-DHEAS was newly synthesized, conjugated with bovine serum albumin (BSA), and immunized to rabbits for the production of anti-DHEAS antibodies. A bridge-heterologous ELISA system employing the sequential saturation method exhibited a high sensitivity with a midpoint of 100 pg. Although the antibody significantly cross-reacted with epiandrosterone sulfate, it easily discriminated the unconjugated steroids and pregnenolone sulfate, which is reported to exist in the brain at a much higher level when compared with DHEAS. The brain homogenate or serum was treated with hexane to remove the lipophilic compounds and purified with an OASIS HLB cartridge. The DHEAS levels were then determined by ELISA. The overall recovery rate through the pretreatment was a satisfactory and constant (81.8 +/- 3.4% for brain, 89.3 +/- 3.0% for serum, mean +/- standard deviation). This ELISA afforded a satisfactory serial dilution study and recovery test. The intra- and inter-assay coefficients of variation were lower than 13%, which showed the precision of the proposed method. The applied method showed that DHEAS was not detected in some brain samples and its levels were much lower than those previously reported and than its serum levels.     

Medroxyprogesterone acetate in human serum

Conjugates of medroxyprogesterone acetate (MPA) in human serum are investigated using chromatography and techniques (equilibrium dialysis, gel filtration, and polyacrylamide gel electrophoresis) previously described for studying the binding of MPA. 17 serum samples were obtained from 7 women at various times after the intramuscular injection of 150 mg Depo-Provera. Mean concentration of MPA in the unconjugated fraction of serum was 3.9 mg/ml (range 0.8-10.7 ng/ml); in the conjugated fraction, the value was 2.7 ng/ml (range 0.6-11.4 ng/ml), a mean value of 81.7% (range 18.4-286%) of that in the unconjugated fraction. The conjugate appears to be mainly a glucuronide since solvolysis released only small amounts of MPA. MPA metabolites were also detected in blood. The MPA levels in blood measured by radioimmunoassay were generally lower when serum was extracted with an organic solvent rather than when the assay was carried out directly in the serum. This finding suggests the presence in blood of either MPA in a conjugated form or metabolites interacting with the antiserum which were not extracted by the solvents used. Equilibrium dialysis showed that undiluted plasma bound 85.8% of triated hydrogen-MPA; with increasing dilution of the plasma, the amount of bound triated hydrogen-MPA decreased. The apparent association constant calculated according to the method of Vermeulen and Verdonck was 2.6 x 10 4 1/mol. MPA appeared to be loosely bound to albumin in blood but there was no specific binding protein for the steroid. MPA conversion to the glucuronide may be 1 of the factors regulating the level of the unconjugated but presumably biologically active steroid in blood.     

Locations of herpes simplex virus type 2 glycoprotein B epitopes recognized by human serum immunoglobulin G antibodies.

Herpes simplex virus type 2 (HSV-2) glycoprotein B (gB-2) gene segments were expressed as recombinant proteins in Escherichia coli. gB-2 recombinant proteins were reacted with human serum immunoglobulin G (IgG) antibodies in Western immunoblot assays. Initially, samples were tested for the presence of HSV-1-specific antibodies and HSV-2-specific antibodies by using HSV-infected cell lysates as antigen targets in Western blot assays. Serum samples that contained HSV-2-specific IgG (n = 58), HSV-1-specific IgG (n = 33), or no detectable HSV antibodies (n = 31) were tested for reactivities with the gB-2 recombinant proteins. In 58 of 58 samples that contained HSV-2-specific IgG, antibodies were present that reacted strongly with a gB-2 amino-proximal segment between amino acids (aa) 18 and 75. Three of 33 serum samples that contained HSV-1- and not HSV-2-specific IgG (as defined by the HSV lysate Western blot assay) reacted with this segment. Both HSV-2 antibodies and HSV-1 antibodies reacted strongly with a carboxy-terminal gB-2 segment between aa 819 and 904; a second minor cross-reactive region was mapped to a gB-2 segment between aa 564 and 626. The gB-2 segment from aa 18 to 75 may constitute a useful reagent for the virus type-specific serodiagnosis of HSV-2 infections. Further studies will be required to determine the relative sensitivities and specificities of the assay for gB-2 aa 18 to 75, HSV gG assays, and HSV lysate Western blot assays for detecting virus type-specific antibody responses in acute and chronic HSV-2 infections.     

Production of monoclonal antibodies to estriol and their application in the development of a sensitive nonisotopic immunoassay

The development of highly specific monoclonal antibodies to estriol and a nonisotopic immunoassay (EIA) for unconjugated estriol based on the use of these monoclonal antibodies have been described. The monoclonal antibodies show little cross reactivity with other steroids and steroid conjugates and can be used directly in immunoassays without any purification. The EIA described here can be performed in 96-well microtiter plates or polystyrene tubes that have been coated with estriol-bovine serum albumin conjugate. In this assay, estriol in the standard or clinical samples (serum or saliva) competes with the immobilized steroid on the plate or the tube for binding with the antibody. The assay shows good agreement with radioimmunoassay (RIA) and is highly sensitive and reliable. Since no prior processing or extraction of the clinical samples is necessary, the method is potentially applicable for routine use in fetal monitoring as well as in a steroid laboratory.     

Assay of ethynyloestradiol in human serum and its binding to plasma proteins

A radioimmunoassay for estimating both unconjugated and conjugated ethinyl estradiol (EE) in serum is described, along with a report of levels of EE attained after its administration and the binding of EE in plasma, as determined by this radioimmunoassay. Mean values for concentration of free EE were 38-87 pg/ml, 1-4 hours after administration, and by 24 hours levels in most subjects were below the sensitivity level of this assay, which is less than 25 pg/ml. Conjugated EE concentration in serum was considerably higher, with mean values of 370-770 pg/ml 1-4 hours after ingestion, decreasing slowly to mean values of 285 pg/ml at 8 hours and 100 pg/ml at 24 hours postadministration. Total EE (conjugated plus unconjugated) had a mean half-life of 3.8 hours during the period 2-8 hours after administration and 10.7 hours for the period 8-24 hours postadministration. EE in plasma was shown, by equilibrium dialysis, gel filtration on Sephadex G200, and polyacrylamide gel electrophoresis, to bind only to albumin, and no specific binding of EE occurred. The apparent association constant for the binding of EE to human serum albumin was 1.7 times 10 5 liter per mol.     

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.     

Antibody to platelet activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; PAF)

Specific antibodies to platelet activating factor (PAF) were prepared by immunizing rabbits with a hapten-bovine serum albumin (BSA) conjugate. As the hapten we used the synthetic PAF derivative which is resistant against enzymatic inactivation by plasma or tissues and which can bind to BSA through covalent bonding. Antibody activity was determined by an enzyme-linked immunosorbent assay (ELISA). Anti-PAF IgG reacted strongly with PAF. By means of the ELISA inhibition assay, we found that the antibody did not cross-react with phosphocholine, glycerophosphocholine, dilaurylglycerophosphocholine or PAF analogues which have ethanolamine-type polar head groups instead of choline group.     

Development and optimization of an enzyme-linked immunosorbent assay employing two murine monoclonal antibodies for absolute quantitation of human beta-glucuronidase.

We have developed and optimized an enzyme-linked immunosorbent assay (ELISA) for absolute quantitation of human beta-glucuronidase. This is a double antibody sandwich system employing two murine monoclonal antibodies specific for human beta-glucuronidase developed in our laboratories. The method involves (a) coating of the high binding polystyrene microtitration plate with the first antibody (7B6 IgG), (b) blocking of remaining active sites with 3% bovine serum albumin in phosphate-buffered saline, (c) application of samples, (d) addition of the biotinylated second antibody (6D2 IgG), (e) addition of streptavidin-horseradish peroxidase, and (f) development of color with o-phenylenediamine dihydrochloride-H2O2 and reading in a microplate reader at a wavelength of 490 nm. The method is highly sensitive with an optimal range of 10 to 100 ng/ml of the enzyme and is reproducible with intraday and interday precisions of 3.2 and 4.1%, respectively. The enzyme contents of 20 urine and 20 bile samples quantitated by this ELISA method were, respectively, 148 +/- 101 and 6380 +/- 3780 ng/ml (means +/- SD) which correlated well with their enzyme activities. Such a method for absolute quantitation of human beta-glucuronidase is essential for studying its pathophysiologic roles in cholelithiasis and carcinogenesis and can also be used clinically as an indicator for tissue damage or malignancy.