Development of a monoclonal antibody to 6 beta-hydroxycortisol and its application in an enzyme-linked immunosorbent assay (ELISA) for 6 beta-hydroxycortisol in urine

A novel monoclonal antibody to 6 beta-hydroxycortisol (6 beta-OHC) was generated and incorporated into an antigen-coated indirect enzyme-linked immunosorbent assay (ELISA) using 6 beta-OHC-protein conjugate as the steroid-coating antigen. The monoclonal antibody is specific to 6 beta-OHC and 6 beta-OHC-3-carboxymethyloxime. Cross-reactivity with other structurally related steroids such as cortisol, cortisone, and 6 beta-hydroxycortisone was less than 10%. Two different clones (clone 5C1 and 19F) of the monoclonal anti-6 beta-OHC antibody have been developed, each with slightly different sensitivity and specificity. The sensitivity of the MAb clones was not significantly improved when compared to the rabbit polyclonal antibodies in this study, but still within the accepted detection limit for 6 beta-OHC in both human and laboratory animals. The assay had a detection limit of 200 ng/ml, an intraassay variation of 6.4% and an interassay variation of 7.3%. The application of the anti-6 beta-OHC-MAb-based-ELISA was tested by measuring the urinary output of 6 beta-OHC in human before and after enzyme induction by rifampicin treatment. The mean 24-h urine output of 6 beta-OHC in human subjects was 485 +/- 100 micrograms and 1478 +/- 281 micrograms before and after rifampicin administration, respectively. In conclusion, the monoclonal anti-6 beta-OHC antibody developed in this study has the required specificity and sensitivity as an alternative method for measuring urinary 6 beta-OHC in the detection of enzyme induction or enzyme inhibition of CYP3A in humans and laboratory animals.     

A rapid and sensitive enzyme linked immunofilter assay (ELIFA) for whole bacterial cells

An improved method is described for the detection of Escherichia coli by an enzyme linked immunofilter assay (ELIFA) using nitrocellulose membrane sandwiched between two 96-well plates. The incorporation of a pumping system permits a continuous flow of reagents and/or wash fluids through the membrane and provides an assay procedure capable of detecting 10(3) bacteria per well within 40 min. Quantitative bacterial detection was based on precipitated chromogen determined by scanning densitometry. The procedure represents a significant improvement in assay time and/or sensitivity over previously described ELIFA and ELISA methods for whole bacterial cells.     

Seroepidemiology of rinderpest in bovids in Sri Lanka using the enzyme linked immunosorbent assay (ELISA) technique

Approximately 0.2% (n = 4397) of the bovids (cattle and buffalo) in Sri Lanka were sampled, from June 1992 using a multi-stage sampling procedure. Serum antibodies for the rinderpest virus were detected using the competitive enzyme-linked immunosorbent assay. The age, the agroclimatic zone, the management system practiced in the farms, and the vaccination history of the sampled bovids were studied as potential risk factors for being seropositive. The prevalence of rinderpest antibodies in non-vaccinated bovids was 3.5% (n = 4101). The prevalence was higher in the dry zone (9%; where the outbreak emerged in 1987), compared to bovids in the other zones (1%). Seropositive bovids over three years of age were approximately at fourfold higher chances of being seropositive compared to those that were < or = 3 years old. The higher prevalence in older animals is probably due to exposure to the virus during the 1987 epidemic. Bovids from the dry zone (annual rainfall 20 to 35 inches) were at higher odds of being seropositive even after controlling for the possible effects of age, agroclimatic zone, management system and vaccination. The fact that 62% of bovids from the dry zone in this study were reared under extensive management system (free grazing) which allow unrestricted contact between animals, may be the reason for the above finding. A relatively poor response to vaccination observed in vaccinated bovids (seroprevalence = 12%; n = 296) could be attributed to difficulties in maintaining the vaccine at recommended temperatures in the field. This is the first island-wide study on seroprevalence of rinderpest in Sri Lanka.     

Development of a sensitive ELISA for human leptin, using monoclonal antibodies

A new, sensitive ELISA for human leptin in plasma and cerebrospinal fluid (CSF) was developed, using monoclonal antibodies. The lower limit of detection of this ELISA was 0.78 pg/assay. Both intra- and interassay imprecision values were <7%. The dilution curves of plasma and CSF showed good linearity, and the recovery was 83.2-95.6%. There was good correlation between plasma leptin concentrations by the ELISA and a commercially available RIA (r = 0.99). Our ELISA is advantageous because it does not require radioisotopes, it produces results in hours rather than days, and more importantly, it improves on the detection limit and plasma interference of the RIA kit. The new ELISA enables measurement of low concentrations of leptin, as are seen in CSF and in plasma of patients with anorexia nervosa.     

Evaluation of an enzyme linked immunosorbent assay (ELISA) for determination of insulin in dogs

Between 1970 and 1983, 345 patients with ovarian cancer clinical stage I, II, and III were irradiated postoperatively. Five-year NED survival was achieved in 41.7% of patients. The most important prognostic factors were histological grade and clinical stage of cancer. Postoperative external beam radiotherapy appeared to be highly efficient for the patients with microscopic residual disease, giving 70% 5-year survival, and moderately efficient for patients with small, i.e. < or = 3 cm in diameter residual disease, giving 40% 5-year survival. The optimal technique of irradiation appeared to be the irradiation given to the entire abdominal cavity with additional irradiation coned down to the pelvis. External beam radiotherapy was ineffective in patients with gross residual disease, i.e. > 3 cm in diameter, and useless as palliative treatment given to patients with inoperable cancer of the ovary.     

An enzyme linked immunosorbent assay (ELISA) for detection of seminal fluid using a monoclonal antibody to prostatic acid phosphatase

A microtitre plate format enzyme linked immunosorbent assay (ELISA), employing commercially available PASE/4LJ mouse monoclonal hybridoma antibody is described. The technique is a solid phase indirect ELISA for prostatic acid phosphatase, applicable to specific detection of semen. Maximal detectability was found to be one hundred thousand fold dilution of pooled seminal plasma. No cross reactivities with human vaginal fluid, blood, saliva, female urine, nasal discharge, earwax, sweat or faeces have been found.     

A novel ELISA format for the rapid and sensitive detection of staphylococcal enterotoxin A

Staphylococcal food poisoning is one of the leading causes of bacterial food poisoning each year. Detection kits for staphylococcal enterotoxins are commercially available and the assays can require from one and a half to twenty-four hours to complete with detection limits ranging from 0.5 to 2 ng enterotoxin per gram of food. We have successfully demonstrated a microsphere-packed capillary (MPC) ELISA for the detection of staphylococcal enterotoxin A (SEA) and have compared it to two commercially available kits. The MPC assay detected a lower amount of SEA in ham, chicken, cheese, and bean sprouts than either of the two commercially available kits. In addition, the novel MPC assay was completed in less than ten minutes, as compared to three and twenty-four hours for the two commercially available kits. This research also demonstrated that the MPC ELISA can contain integrated positive and negative controls and has the potential to simultaneously detect and identify multiple enterotoxins.     

Comparison of a radioallergosorbent (RAST) inhibition method and a monoclonal enzyme linked immunosorbent assay (ELISA) for aeroallergen measurement

BACKGROUND: Mouse and rat urinary proteins are potent occupational allergens for exposed personnel. Methods of measuring airborne allergens differ greatly, and reported levels of allergens vary considerably between laboratories. OBJECTIVES: To compare the values obtained using two different methods of allergen detection. METHODS: Air samples were collected in rat rooms in Sweden and the United Kingdom at 2 L/min on to polytetrafluoroethylene (PTFE) filters and extracted in buffer containing 0.5% v/v Tween 20. Airborne rat urinary allergen (RUA) was measured in all samples by both RAST inhibition using a polyclonal human serum pool (UK) and a two monoclonal antibody sandwich ELISA employing antibodies specific for Rat n 1.02 (alpha2u-globulin) (Sweden). RESULTS: The two methods gave values which were correlated (r2 log values = 0.72, P<0.0001), but differed by several orders of magnitude (median [range] ratio of RAST inhibition/ELISA = 316 [7-26(80)]. There was a systematic bias: as the absolute values increased, the difference in the measurements increased. The rat urine standards used were antigenically similar. CONCLUSIONS: A large contrast in RUA values obtained from the two assays was observed in this study. This may be primarily due to methodological differences, but variations in antibody specificities or composition of allergenic epitopes in the air samples may contribute. The results demonstrate that standardization of methods and antibodies is necessary before interlaboratory comparisons can be made.     

Detection of antibody IgG to HIV-1 in urine by ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) using recombinant p24 as antigen for diagnosis of HIV-1 infection

Anti-HIV-1 IgG in urine was detected by an ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) using recombinant p24 gag protein (p24) of HIV-1 as antigen and beta-D-galactosidase from Escherichia coli as label. Anti-HIV-1 IgG in urine was reacted simultaneously with 2,4-dinitrophenyl-bovine serum albumin-recombinant p24 conjugate and recombinant p24-beta-D-galactosidase conjugate. The complex formed, consisting of the three components, was trapped onto polystyrene balls coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG, eluted with epsilon N-2,4-dinitrophenyl-L-lysine, and transferred to polystyrene balls coated with affinity-purified (anti-human IgG gamma-chain) IgG. Bound beta-D-galactosidase activity was assayed by fluorometry. This assay was at least 3,000-fold more sensitive than conventional methods. The lowest signal among 49 asymptomatic carriers was 3.1-fold higher than the highest nonspecific signal among 100 seronegative subjects. The sensitivity and specificity were both 100%. The positivity could be confirmed by preincubation of urine samples with excess of the antigen. Thus, this assay would be a powerful tool for detecting IgG antibody to HIV-1 in urine.     

The ubiquitin-conjugating enzyme Rad6 (Ubc2) is required for silencing in Saccharomyces cerevisiae

It has been previously shown that genes transcribed by RNA polymerase II (RNAP II) are subject to position effect variegation when located near yeast telomeres. This telomere position effect requires a number of gene products that are also required for silencing at the HML and HMR loci. Here, we show that a null mutation of the DNA repair gene RAD6 reduces silencing of the HM loci and lowers the mating efficiency of MATa strains. Likewise, rad6-delta reduces silencing of the telomere-located RNAP II-transcribed genes URA3 and ADE2. We also show that the RNAP III-transcribed tyrosyl tRNA gene, SUP4-o, is subject to position effect variegation when located near a telomere and that this silencing requires the RAD6 and SIR genes. Neither of the two known Rad6 binding factors, Rad18 and Ubr1, is required for telomeric silencing. Since Ubrl is the recognition component of the N-end rule-dependent protein degradation pathway, this suggests that N-end rule-dependent protein degradation is not involved in telomeric silencing. Telomeric silencing requires the amino terminus of Rad6. Two rad6 point mutations, rad6(C88A) and rad6(C88S), which are defective in ubiquitin-conjugating activity fail to complement the silencing defect, indicating that the ubiquitin-conjugating activity of RAD6 is essential for full telomeric silencing.     

Detection of thioredoxin in human serum and biological samples using a sensitive sandwich ELISA with digoxigenin-labeled antibody

Currently, recombinational cloning procedures based upon methods developed for yeast, Saccharomyces cerevisiae, are being exploited for targeted cloning and in-vivo modification of genomic clones. In this review, we will discuss the development of large-insert vectors, homologous recombination-based techniques for cloning and modification, and their application towards functional analysis of genes using transgenic mouse model systems.     

Characterization of a rapid and sensitive enzyme immunoassay (EIA) for progesterone applied to conditioned cell culture media

Progesterone accumulation in conditioned media is a frequently employed endpoint for in vitro cell culture of steroidogenic cells. Although radioimmunoassay (RIA) has been the predominant method for measurement of progesterone, a number of nonradiometric immunoassays have been described but they have not been applied to conditioned media. Here, we report the characterization of a microtitre plate enzyme immunoassay (EIA) for determination of progesterone in conditioned media. The EIA has a sensitivity of 0.3 pg per well with intraassay and interassay coefficients of variation of 7.3 and 10.2%, respectively. The specificity of the EIA is no different than that of a comparable RIA showing crossreactivities of less than 0.1% for other steroids except 5 alpha-pregnan-3,20-dione (47%) and 11 alpha-hydroxyprogesterone (18%). Progesterone levels from conditioned cell culture media of either rat or human granulosa cell cultures measured by both EIA and RIA were in close agreement (r = 0.96) and serial dilutions of culture samples in the EIA were parallel to those of the standards. Also, extraction of culture media prior to EIA was found not to be necessary. Thus, this EIA is a highly sensitive and specific assay that provides a rapid, simple, inexpensive, and non-radiometric alternative to radioimmunoassay for measurement of progesterone in conditioned cell culture media.     

Measurement of oxidation in plasma Lp(a) in CAPD patients using a novel ELISA

BACKGROUND: LGE2 is produced by the cyclooxygenase- or free radical-mediated modification of arachidonate and is formed during the oxidation of low density lipoprotein (LDL) with subsequent adduction to lysine residues in apo B. We have developed a sensitive enzyme-linked sandwich immunosorbent assay (ELISA) for detection and measurement of LGE2-protein adducts as an estimate of oxidation of plasma LDL and Lp(a). METHODS: The assay employs rabbit polyclonal antibodies directed against LGE2-protein adducts that form pyrroles, and alkaline phosphatase-conjugated polyclonal antibodies specific for apo B or apo (a). It demonstrates a high degree of specificity, sensitivity and validity. RESULTS: Epitopes characteristic for LGE2-pyrroles were quantified in patients with end-stage renal disease (ESRD) that had undergone continuous ambulatory peritoneal dialysis (CAPD) and in a gender- and age-matched control population. In addition to finding that both LDL and Lp(a) levels were elevated in CAPD patients, we also found that plasma Lp(a) but not LDL was more oxidized in CAPD patients when compared to corresponding lipoproteins from healthy subjects. Using density gradient ultra-centrifugation of plasma samples, we found that modified Lp(a) floats at the same density as total Lp(a). CONCLUSIONS: The results of this study demonstrate that oxidation of plasma Lp(a) is a characteristic of ESRD patients undergoing CAPD. This ELISA may be useful for further investigations on oxidation of lipoproteins in the circulation of specific patient populations.     

Validation of an inhibition ELISA using a monoclonal antibody for foot-and-mouth disease (FMD) primary diagnosis

An inhibition ELISA (IH-ELISA) test for foot-and-mouth disease virus (FMDV) was validated using 106 epithelial samples from suspected cases of FMD in Argentina submitted to the Argentine National Diagnostics Laboratory (GELAB) over a period of 12 months and examined in parallel with the complement fixation test (CFT). IH-ELISA was found to be more sensitive, detecting 25% (26 samples) more FMDV positives than the CFT in original suspensions of field samples. The effect of storage conditions on 12S stability was examined. Plates stored at 4 degrees C blocked with 1% ovalbumin and plates stored at -20 degrees C with or without blocking buffer could be used for at least 90 days. When various brands of polystyrene plates were compared for 12ps adsorption it was found that those microplates of higher binding capacity were more efficient.     

Sensitive ELISA dipstick test for the detection of chloroquine in urine under field conditions

OBJECTIVE: To evaluate a new enzyme-linked immunosorbent assay (ELISA) dipstick test for detecting chloroquine (CQ) in urine in a malaria-endemic region of north-western Namibia. METHOD: Urine samples from 92 patients attending the outpatient department of Kamhaku Hospital with suspected malaria infection were tested for CQ with both the Dill-Glazko test and the ELISA dipstick test. Results were compared to the history of CQ intake as documented in the patients' health passes. RESULTS: The dipstick test proved an easy-to-handle and very sensitive tool for the detection of CQ with a lower limit of detection at 120 nmol/l. It showed high agreement with the history of CQ intake within the last 6 months. The specificity in a negative control group was 100%. The Dill-Glazko test was far less sensitive and specific with a lower detection limit of 150 micromol/l. CONCLUSION: The dipstick test can be used in pharmacological studies to evaluate the use of CQ, and as an inclusion criterion for in vivo and in vitro sensitivity tests, whereas the Dill-Glazko test is appropriate to test compliance during and a few days after CQ intake.     

A rapid ELISA for measurement of anti-glomerular basement membrane antibodies using microwaves

The presence of anti-glomerular basement membrane antibodies is one of the features of Goodpasture's syndrome. Since the disease has a rapidly progressive course, an early diagnosis is essential. As was already demonstrated in other ELISA methods, 2.45-GHz microwave irradiation can accelerate all kinds of time consuming processes in several laboratory techniques. The application of microwaves in an ELISA for the measurement of anti-GBM antibodies in serum indicated that a considerable time reduction of 75% can be achieved, resulting in a rapid and reliable assay. In addition, microwaves can also have a positive effect on the resolution of that particular ELISA as shown in this study.     

Highly sensitive high-performance liquid chromatographic determination method for a new erythromycin derivative, EM523, and its major metabolites in human plasma and urine using post-column tris(2,2'-bipyridine) ruthenium(III) chemiluminescence detection

A method for the simultaneous determination of de(N-methyl)-N-ethyl-8,9 -anhydroerythromycin A 6,9-hemiacetal (EM523, I) and its three metabolites in human plasma and urine has been developed using high-performance liquid chromatography (HPLC) with chemiluminescence (CL) detection. Plasma and urine samples spiked with erythromycin as an internal standard were extracted with a mixture of dichloromethane and diethyl ether under alkaline conditions. The organic layer was evaporated under a stream of nitrogen gas. The reconstituted sample was injected into an HPLC apparatus and separated on an ODS column using a gradient elution method. The eluate was reacted on-line with a mixture of tris(2,2'-bipyridine) ruthenium(II) and peroxodisulfate, and the generated CL intensity was detected. Optimization of the CL reaction conditions resulted in a sensitive and stable CL intensity for the determination of I and its metabolites. The recovery of each compound from human plasma and urine, and the sensitivity, linearity, accuracy and precision of the method were satisfactory. The lower limits of quantitation for each compound using 0.2 ml of plasma and 0.1 ml of urine were 1 and 10 ng/ml, respectively. This method has been used for the determination of 1 in samples from clinical trials.     

Delta-Aminolevulinic acid in urine (screening method)

Various methods for determining urinary delta-aminolevulinic acid (ALA) have been devised by many investigators since 1956. This paper introduces a history of the methodology in the determination of urinary ALA and application of some methods. These methods can be divided into two groups; one group consists of colorimetric methods based on the color reaction of ALA-pyrrole with Ehrlich's reagent, and the other group consists of fluorometric methods, based on the fluorescence derivatization of ALA and its separation by HPLC. Colorimetric methods are convenient and inexpensive, while these are less specific. On the other hand, the fluorometric HPLC methods are highly sensitive and specific, while these are expensive because of the high cost of the instruments.     

Studies of the repressor (BlaI) of beta-lactamase synthesis in Staphylococcus aureus

Purified BlaI, the putative repressor of the beta-lactamase operon in Staphylococcus aureus, binds specifically to two regions of dyad symmetry (operators) located in the blaZ-blaR1 intergenic region. BlaI binds with similar affinity to the two regions and to the related sequence upstream of the mec gene found in methicillin-resistant strains of S. aureus, providing physical evidence for the cross-talk previously observed between these systems. A change from a lysine in the N-terminus of BlaI to an alanine or deletion of the C-terminal 23 amino acids severely reduces its DNA-binding ability, demonstrating the functional importance of both the N- and C-termini. An operator DNA-protein complex observed with crude cell lysates from repressed cells, indistinguishable from that observed with purified BlaI, was eliminated by induction of the beta-lactamase operon. Furthermore, BlaI is proteolytically cleaved in response to the addition of inducer in a blaR1-dependent manner, providing primary evidence for the molecular basis of induction. Thus, BlaI is shown to be the repressor of the beta-lactamase system.