Monoclonal Antibody-based Enzyme Immunoassay Research Articles

Monoclonal antibody-based enzyme immunoassay for detection of ricin.

A sensitive and specific enzyme-linked immunoadsorbent assay (ELISA) was developed to detect ricin in biological fluids. The assay is based on the sandwich format using monoclonal antibodies (MAbs) of two distinct specificities. An affinity-purified anti-ricin B chain MAb (1G7) is utilized to adsorb ricin from solution and the second anti-ricin A chain MAb (5E11) conjugated with peroxidase is then used to form a sandwich, and peroxidase allows color development and measurement of optical density at 450 nm. Standard curves were linear over the range of 2.5-100 ng/mL ricin. The limit of detection was below 5 ng/mL in assay buffer as well as in a 1:10 dilution of urine or 1:50 dilution of human serum spiked with ricin.

Incidence of enteric adenovirus gastroenteritis in Iranian children.

Enteric adenoviruses, i.e. adenovirus 40 (Ad40) and adenovirus 41 (Ad41), have been shown to be a substantial cause of pediatric gastroenteritis in various parts of the world, but no data are available for Iran. The present study was performed to determine the incidence of enteric adenoviruses in children presenting to the Children's Medical Center with gastroenteritis in Iran. Stool specimens from 872 children less than 7 years of age attending the Children's Medical Center in Tehran, Iran, with gastroenteritis were tested for the presence of Ad40, Ad41, and adenovirus-genus by a monoclonal antibody-based enzyme immunoassay. 6.7% of stool specimens contained enteric adenoviruses (3.3% Ad40 and 3.4% Ad41) and 2.0% nonenteric adenoviruses. Mean ages of Ad40, Ad41 and NEAd-positive children were 21, 19 and 29 months, respectively. Among the adenovirus-positive patients, 53.9% were male and 46.1% female. Watery diarrhea was present in 86.4% of children infected by adenoviruses. In conclusion, for the first time, we demonstrated the presence of enteric and nonenteric adenoviruses in a considerable proportion of stool samples from Iranian children with gastroenteritis.

Relationship between lipoprotein(a) levels, oxidative stress, and blood pressure levels in patients with essential hypertension.

High plasma levels of lipoprotein(a) [Lp(a)] are considered a risk factor for the development of coronary artery disease. In vitro experiments have shown that oxidized Lp(a) is able to impair the arterial endothelium-dependent dilation, thus suggesting a possible role of Lp(a) in the genesis of essential hypertension. The aim of our work was to investigate the correlation of blood pressure levels with plasma Lp(a) concentration, apo(a) isoform size, and peroxidative stress in patients with essential hypertension. The study was performed in 54 untreated hypertensive patients whose blood pressure was monitored for 24 h by ambulatory blood pressure monitoring. Lp(a) concentration was measured by a double monoclonal antibody-based enzyme immunoassay demonstrated to be insensitive to apo(a) size heterogeneity. Apo(a) isoforms were determined by a high-resolution SDS-agarose gel electrophoresis followed by immunoblotting. A significant correlation was found between Lp(a) levels and the night-time systolic and diastolic pressures (r=0.32, P<0.05 and r=0.30, P<0.05, respectively), as well as with the mean night-time fall in systolic and diastolic blood pressures (r=-0.28, P<0.05 and r=-0.29, P<0.05, respectively). These relationships were further potentiated when peroxidative stress data were taken into consideration (r=0.37 and r=0.40, P<0.01 for the night-time systolic and diastolic pressures, respectively and r=-0.34 and r=-0.38, P<0.01 for the night-time fall in systolic and diastolic blood pressures, respectively). Apo(a) isoform size did not affect these relationships. Our data suggest that Lp(a) and peroxidative stress may be involved as cofactors in essential hypertension, with a mechanism that remains to be elucidated.

Use of a New Monoclonal Antibody-Based Enzyme Immunoassay for Soluble Fibrin to Exclude Pulmonary Embolism

We prospectively evaluated the diagnostic performance of a new soluble fibrin assay in 303 consecutive patients with suspected pulmonary embolism and examined potentially useful cut-off levels at which this disease can be safely excluded. In addition, the diagnostic accuracy was calculated in the subgroups of in- and outpatients. The ROC curve of the assay in the total study cohort had an area under the curve of 0.69. The cut-off level associated with a sensitivity and negative predictive value of 100% was 20 ng/ml, but the specificity was only 4%. The cut-off level with a sensitivity of 90% was 30 ng/ml, which corresponded with a specificity and negative predictive value of 27% and 86%. respectively. The diagnostic performance was comparable in the subgroups of in- and outpatients. We conclude that the soluble fibrin assay has a low diagnostic accuracy and seems unsuitable as a screening test for the exclusion of pulmonary embolism.

Clinical Evaluation of a Monoclonal Antibody-based Enzyme Immunoassay for Fibrin Degradation Products in Patients with Clinically Suspected Pulmonary Embolism

We prospectively evaluated the diagnostic accuracy of the Fibrinostika FbDP assay in 304 consecutive patients with suspected pulmonary embolism and examined potentially useful cut-off points at which the disease can be excluded. The prevalence of pulmonary embolism was 31%. The assay generated an area under the Receiver Operating Characteristic curve of 0.79 (95% CI 0.73-0.84). A cut-off point of 0.05 microg/ml yielded a sensitivity, specificity, negative predictive value and an exclusion efficiency of 100% (95% CI 96-100), 5% (95% CI 2-9), 100% (95% CI 69-100) and 3% (95% CI 2-6), respectively. A clinically useful cut-off point seems to be 0.11 microg/ml which corresponded with a sensitivity, specificity, negative predictive value and an exclusion efficiency of 96% (95% CI 90-99), 27% (95% CI 24-28), 93% (95% CI 84-98) and 20% (95% CI 16-25), respectively. We conclude that the assay has potential clinical utility for the exclusion of pulmonary embolism, but it cannot be used as a sole test.

Evidence of high-frequency genomic reassortment of group A rotavirus strains in Bangladesh: emergence of type G9 in 1995.

We characterized 1,534 rotavirus (RV) strains collected in Bangladesh from 1992 to 1997 to assess temporal changes in G type and to study the most common G and P types using reverse transcription-PCR, oligonucleotide probe hybridization, and monoclonal antibody-based enzyme immunoassay. Results from this study combined with our previous findings from 1987 to 1991 (F. Bingnan et al., J. Clin. Microbiol. 29:862-868, 1991, and L. E. Unicomb et al., Arch. Virol. 132:201-208, 1993) (n = 2,515 fecal specimens) demonstrated that the distribution of the four major G types varied from year to year, types G1 to G4 constituted 51% of all strains tested (n = 1,364), and type G4 was the most prevalent type (22%), followed by type G2 (17%). Of 351 strains tested for both G and P types, three globally common types, type P[8], G1, type P[4], G2, and type P[8], G4, comprised 45% (n = 159) of the strains, although eight other strains were circulating during the study period. Mixed G and/or P types were found in 23% (n = 79) of the samples tested. Type G9 RVs that were genotype P[6] and P[8] with both long and short electrophoretic patterns emerged in 1995. The finding of five different genotypes among G9 strains, of which three were frequently detected, suggests that they may have an unusual propensity for reassortment that exceeds that found among the common G types. We also detected antigenic changes in serotypes G2 and G4 over time, as indicated by the loss of reactivity with standard typing monoclonal antibodies. Our data suggest that a vaccine must provide protection against type G9 RVs as well as against the four major G types because G9 strains constituted 16% (n = 56) of the typeable RV strains and have predominated since 1996.

High prevalence of sensitization to cat allergen among Japanese children with asthma, living without cats.

Cat allergy is common among children with asthma. Many cat-allergic patients in Japan and elsewhere do not keep cats, but nonetheless become sensitized through environmental exposure to cat allergen. To assess the frequency of cat allergy and cat-specific immunoglobulin E (IgE) and immunoglobulin G (IgG) antibody responses in young Japanese patients with asthma in relation to self-reported cat exposure and Fel d 1 levels in dust samples. Cat dander-specific IgE antibody was measured in sera from asthma patients using the CAP system. IgE and IgG antibody to Fel d 1 was measured by antigen binding radioimmunoassay and by chimeric enzyme immunoassay. Fel d 1 levels in dust samples from a subset of patients' homes were measured by monoclonal antibody-based enzyme immunoassay. Cat-specific IgE (CAP class>/=2) was found in sera from 70% of 44 patients who kept cats and 34% of 394 patients who had never kept cats. The prevalence of sensitization increased progressively to age 6 years (40%: positive), and then increased gradually to age 16 years (approximately 60%: positive) in patients who had never kept cats. There was an excellent correlation between cat CAP values and IgE levels to Fel d 1. The absolute amount of IgE antibody to Fel d 1 ranged from 0.01 to 15.6% of total IgE. Most patients who did not keep cats were exposed to Fel d 1 levels ranging from 0.07-8 microg/g dust. Sensitization to cat allergen is common among young asthmatic patients in Japan, even among patients who do not keep cats. Use of CAP and the chimeric enzyme-linked immunosorbent assay allows accurate diagnosis of cat allergy and quantification of specific IgE antibody levels.

A monoclonal antibody-based enzyme immunoassay for the determination of glycated high density lipoproteins

A monoclonal antibody-based enzyme immunoassay for the determination of glycated high density lipoproteins

Development of a simple enzyme immunoassay for blood haptoglobin concentration in cattle and its application in improving food safety

Abstract Objective To verify the role of haptoglobin, a major acute-phase reactant protein in cattle, as a marker to identify health/disease status in cattle and further assess its potential in improving food safety. Sample Population Serum samples from various cattle groups: clinically normal cattle comprising steers (n = 157) and culled dairy cows (n = 92) before death (antemortem [AM]); retained carcasses (n = 57) railed off the line during postmortem (PM) inspection; and apparently AM normal culled dairy cows (n = 57). Procedure -Efficacy of the simplified monoclonal antibody-based enzyme immunoassay was established by comparing results of haptoglobin tests performed independently on aliquots of serum samples by 3 laboratories. Results Haptoglobin concentration was significantly (P ≤ 0.0001) different between the PM retained carcass group (n = 57) and the AM steer (n = 157) and culled dairy cow (n = 92) groups. In addition, haptoglobin concentration in AM steers (n = 157) and culled dairy cows (n = 92) was significantly (P &lt; 0.0012) different, possibly reflecting a higher percentage of underlying pathologic or inflammatory conditions in animals of the latter group. Evaluation in 3 laboratories of sera from a group of culled dairy cows (n = 57), each laboratory performing a different test procedure, indicated that correlation of haptoglobin concentrations was good between the reported test procedure and the unmodified test and the classical hemoglobin-binding assay that measures peroxidase activity. Conclusion Haptoglobin determination is effective in identifying diseased and healthy cattle. It may be a potentially important tool for application at the farm and slaughterhouse as an aid in improving food safety. (Am J Vet Res 1998;59:1101-1107)

An immunoenzymatic procedure for human apo B-containing particles quantification using monoclonal antibodies.

The present report describes a new monoclonal antibody-based enzyme immunoassay (ELISA) for the quantification of apolipoprotein (apo) B-containing particles in plasma. Native low-density lipoprotein (LDL) and a reference serum were utilized to prepare the standard curve. Three different antibodies to apo B-100--4A6E3, 6A10B10, and 2D9--all produced in our laboratory, were examined. The apparent affinity constants of the monoclonal antibodies (MAbs) 4A6E3, 6A10B10, and 2D9 were 2.9 x 10(9), 1.74 x 10(9), and 0.63 x 10(8), respectively. The standard curve was generated for an apo B-LDL range of 0.1 to 4.0 microg/ml. Evaluating the concentration of apo B-containing particles in plasma may allow for a more accurate assessment of the risk of coronary artery disease.

A monoclonal antibody-based enzyme immunoassay for the measurement of native and glycated apolipoprotein B-containing particles.

We describe the development of sandwich enzyme-linked immunosorbent assays designed to measure native and glycated apolipoprotein B containing particles in plasma. The assays utilize monoclonal antibodies anti native or glycated apo B-LDL for coating and a polyclonal anti apoB-LDL-peroxidase conjugate as the detecting antibody. The method is specific, sensitive and precise. The intra assay coefficient of variation for the plasma native and glycated apolipoprotein B-containing particles was determine to be 7.8% and 7.5%, respectively. The method described can provide specific and reproducible determinations of apoB and glycated-apoB containing particles in plasma; it will be of great interest in the evaluation of atherosclerotic risk in dyslipoproteinemic states in diabetic and non-diabetic subjects.

Monoclonal antibody based enzyme immunoassay for marihuana (cannabinoid) compounds.

MAb against delta 1-THCA was produced by fusing hybridoma with splenocytes immunized with delta 1-THCA-BSA conjugate and hypoxanthine, aminopterine, thymidine-sensitive mouse myeloma cell line, P3-X63-Ag8-653. The cross-reaction of anti-delta 1-THCA antibody against other cannabinoids was very wide, thus many cannabinoids and a spiro-compound were reactive suggesting that 2'-hydroxyl, 6'-hydroxyl or 6'-O-alkyl, 4'-alkylbenzene ring moiety is necessary for its reactivity. It became evident that this ELISA was able to be applied to the biotransformation experiments of cannabinoids in plant tissue culture system. The metabolites of delta 6-THC such as two major metabolites, 7-oxo-delta 6-THC and 7-hydroxyl-delta 6-THC were also detectable by this ELISA.

Detection of enteric adenoviruses 40 and 41 in stool specimens by monoclonal antibody-based enzyme immunoassays

To examine the role of enteric adenoviruses (Ad40 and Ad41) in children with acute gastroenteritis, we evaluated 273 children with diarrhoea and 137 without enteric symptoms in Palermo, Italy, during an 8-month period. Stools were tested by two home-made monoclonal-based ELISAs to detected genus-specific adenovirus antigen and to type Ad40 and Ad41. Twenty-five samples (6.1%) were found to contain adenovirus, 18 of which were grown in Graham 293 and in HEp-2 cells. Ad40 and Ad41 were detected in 2.6% of children with diarrhoea and in none in the control group, while non-enteric adenoviruses were obtained from both patients (3.2%) and controls (6.5%). Samples containing Ad40 and Ad41 were positive by the virus isolation procedure in Graham and in HEp-2 cells, showing no distinct growth pattern in these cell lines. The evaluation of a latex agglutination test (Adenolex) and of a commercial ELISA (Adenoclone), respectively available for the detection of genus adenovirus antigen and for the typing of Ad40 and Ad41 suggests that both tests enable the identification of enteric adenoviruses in stool specimens, giving results comparable to our ELISAs.

Testosterone-induced suppression of lipoprotein(a) in normal men; relation to basal lipoprotein(a) level

The concentration of lipoprotein(a) [Lp(a)] in human plasma is largely genetically determined and is inversely correlated to the size of apolipoprotein(a) [apo(a)]. Additionally, Lp(a) values are relatively stable within individuals and are only marginally susceptible to therapeutic treatment. The aim of our study was to evaluate the effect of exogenous testosterone on plasma Lp(a) concentration. The study was carried out on 19 healthy men who were receiving weekly intramuscular injections of 200 mg testosterone enanthate. Lp(a) values were determined at multiple time-points by a double monoclonal antibody-based enzyme immunoassay. This method is not sensitive to variation in Lp(a) size and the values are expressed in nmol/l. Apo(a) size isoforms were determined by agarose gel electrophoresis followed by immunoblotting. No correlation was found between the baseline Lp(a) values and the baseline values of testosterone or estradiol. The Lp(a) response to testosterone treatment varied widely among subjects and was dependent upon the pretreatment Lp(a) concentration. For 10 subjects with low Lp(a) values (< 25 nmol/l), no significant decrease in Lp(a) was observed while, for the nine individuals with Lp(a) values > 25 nmol/l, there was a significant and consistent reduction in Lp(a) ranging from 25 to 59%. Lp(a) levels returned to baseline values following cessation of testosterone administration. Apo(a) size polymorphism did not appear to play a role in the determination of Lp(a) response to testosterone.

Increased Soluble Form of P-Selectin in Patients With Unstable Angina

P-selectin in platelets and endothelial cells mediates adhesive interaction with leukocytes to form thrombi. The purpose of the present study was to investigate the plasma levels of P-selectin in patients with unstable angina and in those with stable effort angina of different pathophysiologies. Plasma P-selectin levels were determined by a monoclonal antibody-based enzyme immunoassay on plasma samples taken from 12 patients with unstable angina, 11 patients with stable effort angina, and 15 healthy volunteers. Patients with unstable angina had angina at rest associated with ECG changes. In patients with unstable angina, plasma P-selectin levels within 1 hour (361 +/- 90 ng/mL) and at 3 hours (282 +/- 56 ng/mL) after angina were significantly (P < .05) higher than those in volunteers (177 +/- 31 ng/mL). Plasma P-selectin levels at 5 hours after attack (242 +/- 46 ng/mL) did not differ from those in volunteers. Although patients with stable effort angina developed angina with ST-segment depressions by treadmill exercise, their plasma P-selectin levels did not change (before, 178 +/- 45; immediately after, 186 +/- 36; and 1 hour after the exercise, 179 +/- 34 ng/mL). Plasma P-selectin levels after angina increased significantly in patients with unstable angina but did not in patients with stable effort angina. These findings may contribute to understanding of the pathophysiology of the acute coronary syndrome of unstable angina.

Identification ofShigella and enteroinvasiveEscherichia coli strains by a virulence-specific, monoclonal antibody-based enzyme immunoassay

BALB/c mice were immunised with water extracts made from an Escherichia coli K-12 strain harbouring the shigella invasion plasmid, and hybridomas secreting antibodies specific to invasion plasmid-coded antigens were selected. On Western blots, antibodies produced by one of these clones (MAIC-1) recognised a protein of 43 kDa, which is the molecular mass of invasion plasmid coded antigen C (IpaC). When used in enzyme immunoassay against whole bacterial cells or against proteins secreted by actively growing bacteria, MAIC-1 clearly differentiated between invasive and non-invasive strains. Testing 123 enteroinvasive and 139 non-enteroinvasive strains the MAIC-1 based assay proved to be highly specific and sensitive in recognising enteroinvasive isolates. This test could be an inexpensive and rapid alternative to cumbersome virulence assays and a helpful technique in identifying Shigella or enteroinvasive Escherichia coli isolates.

Monoclonal antibody-based enzyme immunoassay for simultaneous quantification of 2- and 16α-hydroxyesterone in urine

Alterations in the metabolism of estrogen have been implicated as an important factor in the etiology of diseases such as gynecological cancers and lupus erythematosus. The major metabolites of estradiol are hydroxylated at the C-2 or C-16α position yielding products with estrogen antagonist and agonist activities, respectively. A sensitive and specific immunodiagnostic assay to determine the balance between these competing pathways might serve as a routine biomarker for management of estrogen-related diseases. We describe here the generation of high affinity, specific murine monoclonal antibodies to 2-hydroxyesterone and 16α-hydroxyesterone by high efficiency fusion protocols. With these antibodies, we have developed a rapid and simple anzyme immunoassay (EIA) kit for the simultaneous quantitation of 2- and 16α-hydroxyestrone in unextracted urine. Initial validation studies established that urinary metabolite 2- and 16α-hydroxyestrone concentrations found by the EIA correlate well with values found by gas chromatography-mass spectroscopy. Preliminary studies with the EIA kit found total recovery of metabolites from spiked urine samples. The EIA inter- and intra-assay coefficients of variation for 2-hydroxyestrone and 16α-hydroxyestrone and the ratio of 2-hydroxyestrone to 16α-hydroxyestrone with the current EIA kit were consistently less than 9%. This kit, designated ESTRAMET ™ 2/16 may provide an important new tool for research in estrogen-related diseases.

Monoclonal antibodies specific for 18-hydroxycortisol and their use in an enzyme immunoassay for human urinary 18-hydroxycortisol for diagnosis of primary aldosteronism

We report the development of a monoclonal antibody-based enzyme immunoassay (EIA) specific for human urinary 18-hydroxycortisol, a biological marker of primary aldosteronism. Hybrid cell lines (hybridomas) were isolated after fusion between myeloma cells and spleen cells prepared from mice immunized with 18-hydroxycortisol conjugate. A competitive EIA suitable for the measurement of urinary 18-hydroxycortisol was developed using the mouse monoclonal antibody, KTM-41, which showed no practical cross-reaction with related endogeneous steroids and synthetic steroids. This EIA meets all the requirements of routine clinical assay in terms of sensitivity (detection limit: 20 nmol/L), reproducibility (total CV: 8–15%), accuracy (recovery: 88–115%), implicity and rapidity (if > 3h). Urinary 18-hydroxycortisol measured by the present assay was 153 ± 119 nmol/L (mean ± SD, range, 28–485) and 1787 ± 1180 (range, 810–4264) in normal subjects ( n = 20) and in patients with primary aldosteronism ( n = 7), respectively. Clinical validation of the assay was confirmed by an appropriate decrease in urinary 18-hydroxycortisol level in patients with primary aldosteronism subsequent to adrenalectomy: 171 ± 141 mol/L (range, 41–466).

Monoclonal antibody-based enzyme immunoassay for pediocins of Pediococcus acidilactici

Monoclonal antibody (MAb) R2-AR against pediocin RS2 was developed. Mice were immunized for 12 weeks with pediocin RS2 conjugated to a polyacrylamide gel. Two hybridoma fusions yielded an MAb that in Western blots (immunoblots) reacted only with pediocins RS2 and AcH (3 kDa) from Pediococcus acidilactici RS2 and H, respectively, and did not react with any other bacteriocin, including sakacin A from Lactobacillus sake Lb 706, leuconocin LCM1 from Leuconostoc carnosum LM1, nisin from Lactococcus lactis ATCC 11454, and pediocin A from Pediococcus pentosaceus FBB61. Each of the bacteriocin bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels was confirmed to be biologically active by a gel overlay test performed with sensitive indicator organisms. In dot immunoblot assays, the MAb could detect a minimum of 32,000 arbitrary units of pediocin RS2 or AcH per ml. In colony immunoblot assays, the MAb was used successfully to differentiate bac+ and bac- variants of P. acidilactici RS2 strains.

Outbreaks of astrovirus gastroenteritis in day care centers

This study evaluated astrovirus as a cause of diarrhea outbreaks among infants and toddlers in day care centers. Stool specimens were collected weekly during four periods (from January 1986 through December 1991) from children 6 to 30 months of age who were enrolled in prospective studies of diarrhea in day care centers. All diarrheal stool specimens were tested for bacterial enteropathogens, rotavirus, enteric adenovirus, and Giardia lamblia. A total of 1365 stool specimens from 70 outbreaks in which no etiologic agent was identified and from another 11 outbreaks with a known cause were tested for astrovirus, by means of a monoclonal antibody-based enzyme immunoassay. Confirmatory testing was performed by reverse transcriptase-polymerase chain reaction with primers designed to produce an 89 base-pair product. Astrovirus was detected in 6 (7%) of the 81 outbreaks. Of 217 children tested, 73 (34%) were infected with astrovirus; infections in 35 (48%) were symptomatic and in 38 (52%) asymptomatic. The six outbreaks lasted 11 to 44 days (median 22 days). Astrovirus excretion was detected for a duration of 2 to 30 days, with excretion occurring from 1 to 8 days (median 2 days) before diarrhea began to 1 to 20 days (median 2 days) after diarrhea ceased. Younger children (< or = 12 months) were at greater risk than older children (p = 0.011) of becoming infected with astrovirus during an outbreak and were more likely (p = 0.015) to have symptoms when infected. Of 24 specimens with astrovirus by enzyme immunoassay, 20 (83%) were confirmed to have the virus by reverse transcriptase-polymerase chain reaction. Astrovirus was an important cause of outbreaks of diarrhea among children attending day care centers, more frequently infected younger children, and often produced asymptomatic infections.