Immunoaffinity chromatography and a biotin-streptavidin amplified enzymeimmunoassay for sensitive and specific estimation of estradiol-17β

A sensitive test system has been developed for estimation ofestradiol-17β (E₂) in bovine plasma. Plasma extracts are first purified by a selective immunoaffinity chromatography (IAC) using an antibody raised against estradiol-6-carboxymethyloxime-bovine serum albumin and immobilized to Sepharose. The eluate was analysed by a competitive enzyme immunoassay (EIA) on microtitration plates. For the assay the wells of microtitration plates were coated with affinity purified sheep IgG (antirabbit IgG) that binds the hormone specific antibody raised in rabbits against estradiol-17-hemisuccinate-bovine serum albumin. E₂ is estimated by displacement of biocytinyl-E₂, that was produced by ligation of estradiol-17β, d-glucuronic acid and biocytin. Bound biocytinyl-E₂ is detected after binding of streptavidin-peroxidase and colour production by the enzyme. A very high amplification was possible with this technique and the absolute detection limit amounted to ≈120fg/well at 94% relative binding. By combination of IAC and EIA the following levels of E₂ were found in bovine plasma: male or female calves <2.7pg/ml, cycling cow 0.5–7 pg/ml, cow during last month of pregnancy 9–310 pg/ml, mature bull 5–30 pg/ml. However, up to 1110 pg E₂/ml were found in plasma of a calf after treatment with an illicit hormone preparation used for growth promotion; after 21 days levels declined to 6 pg/ml which is hardly different from controls. In conclusion, the IAC/EIA can be used for sentitive estimation ofestradiol-17β in plasma from all type of cattle and for control of improper use of E₂ after commitment of a threshhold level.     

Ochratoxin A in human blood serum – retrospective long-term data

In a long-term study (1990–1997) on ochratoxin A (OTA) in human blood serum, 102 serum samples from 36 persons of the Munich Institute for Hygiene and Technology of Food of Animal Origin were analysed by enzyme immunoassay (EIA), and by high performance liquid chromatography (HPLC) for control. Detection limits were at 50 pg/ml (EIA) and 50–70 pg/ml (HPLC), recoveries were 80–120% (EIA) and 30–60% (LC). OTA was detected in 98% (EIA, 368 ± 217 pg/ml) and 93% (HPLC, 271 ± 170 pg/ml) of samples (maximum 1,290 pg/ml). Using published conversion factors for serum/intake estimates (1.34 or 1.97), the mean daily OTA intake of these 36 persons was 493–725 pg/kg bw. Long-term individual mean OTA levels of nine persons ranged from 162 ± 80 pg/ml to 549 ± 172 pg/ml. Our data were compared with published OTA serum levels (1985–2008) for apparently healthy persons from a total of 30 countries. On a worldwide basis, the mean of means for OTA in human serum was estimated to be 700 pg/ml, corresponding to a mean daily OTA intake of 940–1380 pg/kg bw. This level, which was relatively stable over the last decades, is well below published tolerable daily intake values (14,000–18,000 pg/kg bw).     

An enzyme immunoassay for secretin

A sensitive and specific enzyme immunoassay for secretin was developed with the use of enzyme-labeled antigens. Synthetic porcine secretin and its carboxy-terminal fragments (residues 11-27 and 18-27) were conjugated with beta-D-galactosidase for use in the immunoassay, and the assay method with the latter fragment (residues 18-27) linked to beta-D-galactosidase was found to be the most sensitive. The minimum amount of secretin detectable by this method was 1-2.5 pg/assay. Serum levels of secretin after intravenous injection of the peptide in rats were determined by both the enzyme immunoassay and a commercial radioimmunoassay kit. The correlation coefficient between the levels measured by the two methods was 0.984. The enzyme immunoassay could detect immunoreactive secretin levels in normal human sera, giving a value of 16.9 +/- 2.2 pg/ml (mean +/- SE of six human subjects).     

The radioimmunological estimation of 13, 14-dihydro-15-ketoprostaglandin E2 in plasma and cervical mucus

Highly specific antibodies to 13,14-dihydro-15-ketoprostaglandin E2 (PGEM) were raised in rabbits. The animals were immunized with PGEM-bovine serum albumin (BSA)-conjugates. The metabolites were extracted with dichloromethane followed by column chromatography. The final antisera dilution was 1:15000 and the cross-reactivity towards prostaglandin A2, F2 alpha, I2, 13,14-dihydro-15-ketoprostaglandin F2 alpha was less than 0.1%. The limit of detection was 7.8 +/- 4.7 pg/ml plasma at the standard range of 3.9 to 500 pg/ml. The intra- and inter-assay variations were 5 and 12%, respectively. PGEM was measured throughout the menstrual cycle in female volunteers. In normal ovulatory women (n = 6) plasma concentrations of PGEM varied between 0.94 to 2.19 ng/ml. A significant increase of plasma PGEM was detected in the preovulatory phase of the cycle (P less than 0.01) over basal levels. In three of these volunteers cervical mucus was analyzed on PGEM and PGFM concentrations showing a fluctuation from 2 pg to 109 pg for PGEM and 0.05 pg to 2.4 pg for PGFM per ml of cervical mucus. The lowest concentrations have been found at the time of ovulation. The application of the radioimmunological method to the measurement of PGEM in addition to the measurement of prostaglandin E2 may be useful for estimating the turnover rates of this fatty acid.     

Enzyme immunoassay for serum 18-hydroxycorticosterone and its clinical application

An enzyme immunoassay for serum 18-hydroxycorticosterone was established using alkaline phosphatase as a label. The antiserum for 18-hydroxycorticosterone was produced by immunization of rabbits with 18-hydroxycorticosterone 3-(O-carboxymethyl)oxime conjugated to bovine serum albumin. Sephadex LH-20 column chromatography was used to separate 18-hydroxycorticosterone from other steroids in serum samples. The minimal detectable amount of 18-hydroxycorticosterone was 50 pg/tube and the measurable range was from 5 to 1000 ng/dl when a 1.0 ml serum sample was used. Intra- and inter-assay coefficients of variance were 5.0% (n=6) and 5.8% (n=6), respectively. Four of 5 patients with aldosterone-producing adenoma had above-normal serum 18-hydroxycorticosterone levels.     

Development of a sensitive, direct luminescent enzyme immunoassay for plasma estradiol-17 beta

A rapid, sensitive, precise, chemiluminescent enzyme immunoassay for estradiol-17 beta has been developed and validated. Antibodies were produced in rabbits using estradiol-17 beta-6-(O-carboxymethyl)oxime coupled to bovine serum albumin, purified and immobilized on polystyrene beads (6.4 mm diameter). The same derivative was used to prepare the enzymatic tracer by coupling with horseradish peroxidase. The assay, direct on the serum sample, featured a 4-h binding step at 4 degrees C followed by the chemiluminescent detection using luminol/H2O2. The detection limit was 0.15 pg/tube and the assay was carried out on 20-100 microliter of sample, allowing measurement of estradiol-17 beta in plasma concentrations from 1.5 to 500 pg/ml. The method fulfills all the standard requisites of precision and accuracy and the results agree well with a radioimmunoassay procedure on extracted serum.     

Enzyme-Linked Immunosorbent Assay for Detection of Ochratoxin A

A simple microtest plate enzyme-linked immunosorbent assay was developed for the detection of ochratoxin A at levels as low as 25 pg per assay. The relative cross-reactivities of the antibody in this system with ochratoxin A (OA), OB, OC, and Oα were found to be 1.0, 0.14, 0.44, and 0.01, respectively.     

An enzyme immunoassay for plasma betamethasone

A sensitive enzyme immunoassay for plasma betamethasone was developed using betamethasone-3-(O-carboxymethyl)oxime-β-D-galactosidase conjugate as a labelled antigen and 4-methylumbelliferyl-β-D-galactoside as a fluorescence substrate. The performances of the enzyme immunoassay were compared with that of a radioimmunoassay using ³H-betamethasone and the same antiserum. The minimal detectable level for the enzyme immunoassay was 0.15 pg/tube or 0.15 ng/ml of plasma, which was remarkably more sensitive than the radioimmunoassay level of 10 pg/tube or 2 ng/ml of plasma. The specificity was sufficient, in particular, the cross reactivity of cortisol was 0.008%. However, the precision of the enzyme immunoassay was inferior to that of the radioimmunoassay.     

Ochratoxin A: study of grain contamination

Ochratoxin A was quantitatively monitored in grain extracts by indirect solid-phase enzyme immunoassay with the use of an immobilized conjugate of the toxin with gelatin and polyclonal rabbit antibodies raised against the ochratoxin A-BSA conjugate. This monitoring found that 1.7 to 18.5% of the samples were contaminated with the toxin at a concentration of 25.9-291.7 micrograms/kg. An analysis of forage grain found ochratoxin A at concentrations of 440-3250 micrograms/kg.     

A sensitive enzyme immunoassay for human basic fibroblast growth factor

A sensitive sandwich enzyme immunoassay for human basic fibroblast growth factor (HbFGF) was developed employing three monoclonal antibodies (MAb3H3, MAb98 and MAb52). The Fab' fragment of MAb3H3 which inhibits HbFGF biological activity was conjugated to horseradish peroxidase. A mixture of MAb52 and MAb98 was used in the solid phase. Neither human acidic fibroblast growth factor, hst-1/KS3 product nor acid denatured HbFGF was cross-reactive in this assay system. The detection limit of this assay system was 1 pg/well. Using this assay, some tumor cell lines were revealed to produce a higher level of bFGF than a normal one. Serum samples from normal volunteers were also assayed, and immuno-reactive HbFGF could be detected in 16 out of 57 samples at range 30 approximately 206 pg/ml.     

Ochratoxin A: Contamination of grain

Ochratoxin A was quantitatively monitored in grain extracts by indirect solid-phase enzyme immunoassay with the use of an immobilized conjugate of the toxin with gelatin and polyclonal rabbit antibodies raised against the ochratoxin A-BSA conjugate. This monitoring found that 1.7 to 18.5% of the samples were contaminated with the toxin at a concentration of 25.9–291.7 μg/kg. An analysis of forage grain found ochratoxin A at concentrations of 440-3250 μg/kg.     

Enzyme immunoassay for serum dexamethasone using 4-(carboxymethylthio)dexamethasone as a new hapten.

A sensitive and simple enzyme immunoassay for direct quantitation of serum dexamethasone was established. An antiserum with high specificity was produced by the immunization of rabbits with a newly synthesized 4-(carboxymethylthio)dexamethasone-bovine serum albumin conjugate. Alkaline phosphatase was used as a labeling enzyme. The minimum amount of dexamethasone detected was 2 pg per tube on the basis of B/Bo 100 - 2 SD (%) of standard curve. However, taking into account the cross-reaction with steroids such as cortisol in dexamethasone-free serum, the measurable range was from approximately 0.13 to 10 micrograms/dl. Intra- and interassay coefficients of variation were 1.5 - 5.4% and 0.6 - 6.5%, respectively. Serum levels of dexamethasone and cortisol in four normal subjects after an oral administration of 1 mg of dexamethasone are also reported.     

Effects of particle characteristics on magnetic immunoassay in a thin channel

The effects of size and porosity of particles on magnetic immunoassay in a thin channel were studied. Experimental parameters were investigated and compared using a model immunoassay complex of carcinoembryonic antigen (CEA)/anti-CEA. The rate constant for the affinity reaction between functional particles increased as the size of magnetic nanoparticles (800-80 nm) decreased. The affinity reaction between functional particles had no significant effect on the sizes of microparticles (1.0-4.4 μm) at commonly used thin channel flow-rates of 0.001-0.025 ml/min. Competitive and sandwich reactions of CEA/anti-CEA were studied for CEA detection. Microparticles of different porosities produced similar linear ranges of detection and limits of detection. The limits of detection for CEA were 0.29 pg/ml and 0.21 pg/ml for competitive and sandwich reactions, respectively. The linear ranges of detection were from 0.49 pg/ml to 4.9 ng/ml for both competitive and sandwich reactions. The detection limits were lower, and the linear ranges were wider than those of literature. There was a 9% difference in CEA detection measurements between competitive and sandwich magnetic immunoassay. The measurements of two magnetic immunoassays differed by less than 13% from the ELISA reference measurements. The running time was less than 30 min. Magnetic immunoassay in a thin channel has great potential for biochemical analysis and immunoassay-related applications.     

A colorimetric technique for detecting trichothecenes and assessing relative potencies

We tested a novel colorimetric toxicity test, based on inhibition of beta-galactosidase activity in the yeast Kluyveromyces marxianus, for sensitivity to a range of mycotoxins. A variety of trichothecene mycotoxins could be detected. The order of toxicity established with this bioassay was verrucarin A > roridin A > T-2 toxin > diacetoxyscirpenol > HT-2 toxin > acetyl T-2 toxin > neosolaniol > fusarenon X > T-2 triol > scirpentriol > nivalenol > deoxynivalenol > T-2 tetraol. The sensitivity of detection was high, with the most potent trichothecene tested, verrucarin A, having a 50% effective concentration (concentration of toxin causing 50% inhibition) of 2 ng/ml. Other mycotoxins (cyclopiazonic acid, fumonisin B1, ochratoxin A, patulin, sterigmatocystin, tenuazonic acid, and zearalenone) could not be detected at up to 10 micrograms/ml, nor could aflatoxins B1 and M1 be detected at concentrations up to 25 micrograms/ml. This test should be useful for trichothecene detection and for studies of relevant interactions-both between trichothecenes themselves and between trichothecenes and other food constituents.     

A novel sensitive immunoassay method based on the Invader technique

A novel and sensitive immunoassay method has been developed in which the conventional sandwich immunoassay and the highly sensitive DNA detection method, the Invader method, are combined. The signal amplification function of the latter method has been successfully used to enhance the sensitivity of the sandwich immunoassay. The new assay method may be called the Immuno-Invader assay. The assay format involves three important steps: (1) a target antigen is captured and flagged with a biotin-conjugated detection antibody by the sandwich method, (2) streptavidin and a biotin-conjugated oligonucleotide are added to form a complex with the detection antibody, and (3) the oligonucleotide in the complex is detected using the Invader method. The method was applied to the assay of human tumor necrosis factor-α (hTNF-α). Detection limits obtained were 0.1 pg/ml hTNF-α when a luminescent europium chelate was used with a time-resolved measurement mode, and 0.8 pg/ml when fluorescein was used with a normal prompt fluorescence measurement mode. On the other hand, the detection limit of a commercially available hTNF-α enzyme-linked immunosorbent assay that uses horseradish peroxidase was 3.5 pg/ml. These results demonstrate the feasibility and potential of the new assay method for highly sensitive immunoassay.     

An indirect enzyme immunoassay for the mycotoxin citrinin.

An indirect competitive enzyme immunoassay using rabbit antisera could detect citrinin in buffer solutions at 1 to 13 ng/ml (0.05 to 0.65 ng per assay). Cross-reactivity with austdiol, alternariol, ochratoxin A, and deoxynivalenol was < 0.1% relative to citrinin. Recovery of citrinin added to wheat flour at 200 to 2,000 ng/g was 89 to 104%, with a coefficient of variation of 6.9 to 13%.     

An enzyme immunoassay of estrone in swine serum

An enzyme immunoassay for estrone in swine serum was established. For this, beta-galactosidase from E. coli was conjugated through estrone-17 (O-carboxymethyl)oxime using a mixed anhydride reaction. The percentage of immunoreactive estrone-17 (O-carboxymethyl)oxime-beta-galactosidase conjugate was estimated to be about 70%. The recovery rate of estrone (25-500 pg) added to 0.05 ml of swine serum averaged 91.4%. The sensitivity of the present enzyme immunoassay was 5 pg/tube. The coefficients of variation (CV) were 5.9-8.2% (within assays) and 4.1-5.9% (between assays), respectively. Estrone values determined by the present enzyme immunoassay were highly correlated with those determined by radioimmunoassay (r = 0.99, P less than 0.005). This method of enzyme immunoassay was determined to be suitable for the routine assay of serum estrone.     

Sex-Dependent and Sex-Independent Distribution of the β-Subunit of Nerve Growth Factor in the Central Nervous and Peripheral Tissues of Mice

Levels of the beta-subunit of nerve growth factor (beta-NGF) were measured in the central nervous and peripheral tissues of mice using a highly sensitive, sandwich-type enzyme immunoassay system. Antiserum was raised in rabbits against the 7S form of NGF, which was purified from mouse submandibular glands. beta-NGF-specific antibody isolated on a column of Sepharose CL-4B coupled with purified beta-NGF reacted only with beta-NGF. The assay for beta-NGF was performed by incubation of F(ab')2 fragments of the antibody immobilized on a polystyrene ball with tissue extract and then with the same antibody Fab' fragments labeled with beta-D-galactosidase, followed by measurement of galactosidase activity. Our assay system was found to be highly sensitive (minimal detection limit, 0.3 pg/0.3 ml of assay mixture). Furthermore, the presence of gelatin hydrolysates and protease inhibitors during preparation of tissue extracts enabled us to determine the precise levels of beta-NGF in almost all organs of mice. The amount of beta-NGF in submandibular glands was extremely high, and its level increased rapidly until mice were 2 months of age; then, the level continued to increase slowly until mice were 1 year old (3-5 mg/g of tissue). In serum, some of the 2-month-old males, but none of the females, exhibited a fairly high level of beta-NGF (greater than 100 pg/ml).(ABSTRACT TRUNCATED AT 250 WORDS)     

A new monoclonal antibody detecting ochratoxin A at the picogram level

Á. GYÖNGYÖSI-HORVÁTH, I. BARNA-VETRÓ AND L. SOLTI. 1996. A monoclonal antibody against ochratoxin A was produced after immunization of Balb/c mice with ochratoxin A-BSA. This antibody was of the IgGl heavy chain subclass with a k type light chain. The 50% inhibition value was 0.45 ng ml^-1 in a direct competitive ELISA and the detection limit was 42 pg ml^-1. This antibody is very specific, cross-reacting only with ochratoxin B (9.3%).     

A highly sensitive enzyme immunoassay (EIA) system for mouse epidermal growth factor (mEGF)

A highly sensitive two-site enzyme immunoassay system for mouse epidermal growth factor (mEGF) was developed, based on the sandwiching of an antigen between anti-mouse EGF IgG antibody-coated on a polystyrene bead and anti-mouse EGF Fab' antibody-linked peroxidase (horseradish peroxidase, EC. 1.11.1.7). The procedure is simple and rapid compared to a bioassay. Also, the Fab' antibody-peroxidase complex is more stable than the 125I-labeled antibody. Purified mEGF is detectable at a concentration as low as 3 pg/ml. The detection range was 0.3 to 680 pg/sample with 0.1 ml samples. Levels of immunoreactive mEGF in extracts from adult male mice well agreed with those determined by a radioimmunoassay and a radioreceptor assay. The submaxillary gland contained an extremely high concentration of EGF, while other tissues had low levels of EGF.