Monoclonal Antibody-based Enzyme Immunoassay Research Articles

Cloning of cervine interferon-gamma cDNA by polymerase chain reaction

As the first step in the development of a cervine IFN-gamma assay for the diagnosis of tuberculosis in deer, cervine IFN-gamma, cDNA was amplified by polymerase chain reaction using primers based on the bovine IFN-y sequence. A high level of amino acid homology was found between the cervine and the ovine and bovine sequences (94% and 91% respectively). There was less identity with the porcine, human, mouse and rat sequences (78%, 62%, 37% and 39%, respectively). The amino terminus of the mature IFN-gamma protein, which is critical for interaction with its receptor and for triggering biological activity, is highly conserved between the cervine, bovine and ovine proteins. A monoclonal antibody-based sandwich enzyme immunoassay (EIA) specific for bovine IFN-gamma also detects ovine but not cervine IFN-gamma. This suggests that the antibodies recognise epitopes common to the bovine and ovine protein but not cervine IFN-gamma. Seven amino acid residues that were common to the bovine and ovine sequence differed in the cervine sequence, suggesting that the specificity of the monoclonal antibodies may be dependent on one or more of these residues. The possibility of the development of an EIA for cervine IFN-gamma as a commercial in vitro diagnostic assay for tuberculosis in deer is discussed.

Optimization of a monoclonal antibody-based enzyme immunoassay for the detection of terbuthylazine

The application of immunoassays in pesticide residue analysis is of increasing interest due to the sensitivity, simple handling and fast throughout of samples. For a wide application of these assays, a sufficient supply of standardized antibodies over a long period of time is necessary. The monoclonal antibody technology is solving this problem with an increasing number of cell lines which produce antibodies against different pesticides. Hybridomas were produced by cell fusion of spleen cells from mice immunized with dichloroatrazine conjugated to bovine serum albumin and mouse myeloma cells (PAI-B3 Ag8I). After screening with a competitive enzyme immunoassay, a monoclonal antibody that was specific for terbuthylazine and was produced by a permanent hybridoma cell line was selected for immunoassay development and optimization. For this purpose, an antigen- and antibody-immobilized ELISA technique was improved by varying the test parameters. Comparing both methods, the latter turned out to be superior (50% binding = 0.8 μg/l, detection limit = 0.14 μg/l).

Adenovirus infection and childhood intussusception.

To investigate the possible relationship between enteric adenovirus types 40 and 41 and intestinal intussusception in children. Prospective, case-control patient study. Sixty-three consecutive children suspected clinically of having intestinal intussusception were enrolled in this study. Of these, 25 children (mean age, 1.4 years; range, 3 months to 5 years) had barium enema examination-proved intussusception. Age-matched normal controls (24) and controls with diarrhea (21) were obtained within 1 month of the index case. Stools were tested for the presence of nonenteric adenovirus and enteric adenovirus using a monoclonal antibody-based enzyme immunoassay. Five (20%) of 25 children with intussusception had nonenteric adenovirus in their stools compared with one (4%) of 24 normal controls, none (0%) of 21 of the controls with diarrhea, and none (0%) of 37 patients suspected of having intussusception who had negative results on barium enema examination. However, no stool samples were positive for enteric adenovirus. Nonenteric adenovirus infection and intestinal intussusception may be associated. However, because enteric adenovirus was not found in any of the groups studied, no conclusions can be made regarding their possible influence on the risk for developing intussusception.

A monoclonal antibody-based enzyme immunoassay for human GMP-140/P-selectin

Two hybridoma cell lines producing monoclonal antibodies WGA-1 and PL7-6, reactive only with thrombin-stimulated human platelet have been established. Both these antibodies were investigated for their specific reactivity against GMP-140, based on the amino acid composition analysis of immunopurified antigen and N terminal amino acid sequencing of its protease fragments. A two-site enzyme immunoassay for quantification of human GMP-140 was developed using WGA-1 monoclonal antibody immobilized on 96-well microplates and horseradish peroxidase-labeled PL7-6 monoclonal antibody as detector. The assay was able to measure GMP-140 in serum and plasma with a sensitivity of about 5 ng/ml and a precision better than 10%. This assay will be useful for the detection of GMP-140 derived from platelets or endothelium in biological fluids and tissue extracts.

Determination of triasulfuron in soil by monoclonal antibody-based enzyme immunoassay

Determination of triasulfuron in soil by monoclonal antibody-based enzyme immunoassay

Outbreaks of human enteric adenovirus types 40 and 41 in Houston day care centers

Human enteric adenovirus (EAd) types 40 and 41 cause diarrhea in young children, but little is known about their association with outbreaks of diarrhea in the child care setting. This study evaluated EAd as a cause of outbreaks of diarrhea among infants and toddlers in day care centers. Stool specimens were collected weekly regardless of symptoms during four periods from January 1986 to April 1991, from children 6 to 24 months of age enrolled in prospective studies of diarrhea in day care centers. All diarrhea stool specimens were tested for bacterial enteropathogens, rotavirus, and Giardia lamblia. A total of 131 outbreaks occurred during the study. No etiologic agent was identified in 77 outbreaks. Stool specimens from 75 of these 77 outbreaks and from another 21 outbreaks of diarrhea with a known cause were evaluated for EAd with a monoclonal antibody-based enzyme immunoassay. A total of 4402 stool specimens from 613 children from these 96 outbreaks was tested for EAd. The virus was detected in specimens collected during 10 outbreaks, 3 of which occurred in 1986, 3 in 1988, 1 in 1989, 1 in 1990, and 2 in 1991. Of 249 children, 94 (38%) in these 10 EAd outbreaks were infected with EAd. In 51 children (54%) the infection was symptomatic and in 43 (46%) it was asymptomatic. Outbreaks lasted 7 to 44 days (mean 24.5 days). Duration of EAd excretion ranged from 1 to 14 days (mean 3.9 days), with excretion occurring from 7 days (mean 2.6) before diarrhea began to 11 days (mean 5.3 days) after diarrhea stopped. Enteric adenovirus types 40 and 41 are an important cause of outbreaks of diarrhea among children attending day care centers, often involve children in more than one room, and frequently produce asymptomatic infection.

Germ cell alkaline phosphatase in human seminal plasma following vasectomy.

Germ cell alkaline phosphatase (also termed placental-like alkaline phosphatase or Nagao isoenzyme) was assayed in seminal plasma using monoclonal antibody-based enzyme immunoassays in samples obtained both before and after vasectomy. In all cases, germ cell alkaline phosphatase fell to undetectable levels within 14 days of operation. A fall in seminal plasma germ cell alkaline phosphatase may thus be an early indication of successful vasectomy.

Monoclonal Antibody-Based Enzyme Immunoassay for the Quantitative Determination of the Tropane Alkaloid, Scopolamine

A monoclonal antibody (SP1-4-A2) against scopolamine was produced, characterized, and used to develop a sensitive and selective, competitive enzyme immunoassay for the quantitation of the alkaloid. The assay uses nor-scopolamine-N-beta-propionic acid coupled to alkaline phosphatase as tracer and is linear from 10 pg to 10 ng of scopolamine. As little as 10 pg of scopolamine can be quantitated in an unprocessed plant extract or in human serum after suitable dilution, corresponding to detection limits of 0.1 ng scopolamine/ml of plant extract or 0.5 ng/ml of serum. The assay is more selective for scopolamine (percent cross-reactions for hyoscyamine = 0.21%, 6-hydroxy-hyoscyamine = 0.17%) than previously reported immunoassays. The assay format was designed to minimize intra- and inter-plate variabilities which are, on an average, all below 3% (coefficients of variation). The assay reported here has been validated against an HPLC-based technique using plant samples and was shown to correlate closely (y = 0.959 x + 0.14, r = 0.982).

A monoclonal antibody-based enzyme immunoassay for quantitation of human tumor necrosis factor binding protein I, a soluble fragment of the 60 kDa TNF receptor, in biological fluids

Three hybridoma cell lines secreting monoclonal IgG antibodies specific for human tumor necrosis factor-binding protein I (TNF-BP I), the extracellular domain of the 60 kDa TNF receptor, were developed by fusion of spleen cells from mice immunized with TNF-BP I purified from urine. The antibodies recognize three different epitopes on TNF-BP I. Two of the antibodies were used to develop a two-site (‘sandwich’) enzyme immunoassay with horseradish peroxidase as the marker enzyme. The assay was able to measure TNF-BP I in serum, urine and cell culture supernatants with a sensitivity of about 200 ng/1 and a precision better than 10%. TNF-BP I was detected in the serum of healthy individuals at a mean concentration of 2.1±1.0 μg/l(mean± standard deviation; range, 0.52–5.4 μg/l, n=42); no significant difference was seen in patients with chronic polyarthritis (2.3±0.79 μg/l; n=15). Serum TNF-BP I was significantly elevated in patients with burns (6.5±1.7 μg/l; n=10) and markedly increased in patients with renal failure (49±17 μg/l; n=6). TNF-BP I was also detectable in urine from normal individuals (2.2±1.2 μg/l; range 0.78−4.3 μl; n=16). Culture supernatants of several human tumor cell lines also contained TNF-BP I. The assay will be a useful tool to detect activation of the TNF receptor by the physiological ligands, TNF-α and TNF-β, as well as transmodulation by other mediators in various pathological conditions.

Astroviruses as a cause of gastroenteritis in children.

Infection with astroviruses has been associated with gastroenteritis in children, and serologic surveys indicate that this infection may be frequent. The importance of astroviruses as agents of gastroenteritis has not been shown in a controlled study, however. We used monoclonal antibody-based enzyme immunoassays to detect astroviruses, enteric adenoviruses, and rotaviruses in stool samples obtained from age-matched children with and children without gastroenteritis. The samples were obtained in two studies, three years apart, among patients attending an outpatient clinic in Bangkok, Thailand. In the first study, astroviruses were detected in 8.6 percent (96 of 1111) of the children with gastroenteritis and in 2.0 percent (19 of 947) of the children without gastroenteritis. In the second study the rates were 8.6 percent (50 of 580) and 2.1 percent (11 of 512), respectively. For both studies combined, enteric adenoviruses were detected in 2.6 percent of those with gastroenteritis and in 0.5 percent of the controls, whereas rotaviruses were detected in 19 percent of those with gastroenteritis and in 1.0 percent of the controls. The clinical findings associated with astrovirus infection were similar to those associated with rotavirus infection, except for a trend toward greater dehydration in the children infected with rotaviruses. These two controlled studies involving a total of 3150 Thai children provide evidence that astroviruses are a common cause of viral gastroenteritis. Astroviruses were found in association with gastroenteritis more frequently than were enteric adenoviruses, and with nearly half the frequency of rotaviruses.

Sensitivity of an Antigen Detection Enzyme Immunoassay for Diagnosis of Trypanosoma congolense Infections in Goats and Cattle

The sensitivity of a monoclonal antibody-based antigen-detection enzyme immunoassay (antigen-ELISA) for the diagnosis of Trypanosoma congolense was evaluated using sera from experimentally infected goats and cattle. Ten goats (Galla x East African Masai) and 7 steers (Bos indicus) were infected with different clones of T. congolense and left to run a chronic course for 46 and 24 mo, respectively. During this period, monthly blood samples were collected and analyzed for the presence of trypanosomes and antigens in peripheral blood. Of 383 caprine blood samples, 361 (94.3%) were positive for circulating antigens whereas only 42 (10.9%) had demonstrable trypanosomes as revealed by the microhematocrit centrifugation technique. In cattle, 570 (82.5%) of 691 blood samples were antigen-ELISA positive compared to 136 (19.7%) samples with detectable trypanosomes. In an analysis of serum samples from goats in an area known to be endemic for trypanosomiasis, 106 (80.9%) of 131 were positive for T. congolense antigens whereas none of the corresponding blood samples had detectable trypanosomes. Control sera from 24 goats in a trypanosomiasis-free region were all antigen-ELISA negative. Hence, the antigen-ELISA was at least 4 times more sensitive than the microhematocrit centrifugation technique in monitoring T. congolense infections in goats and cattle.

Enzyme Immunoassay for Determination of Gluten in Foods: Collaborative Study

A collaborative study was performed in 15 laboratories to validate a monoclonal antibody-based enzyme immunoassay (EIA) for determination of gluten in foods. The study included 13 samples: maize starch, "gluten-free" baking mixes, wheat flours, cookies, cooked meats, and a soup. Gluten was present in these samples at either zero or 0.02 to 10% by weight, i.e., over almost 3 orders of magnitude. The mean assay values for the foods varied from 88 to 105% of the actual amounts. The assay was quantitative for cereal products and the soup with repeatability (RDS-r, relative standard deviation) and reproducibility (RSD-R) of 16-22% and 24-33%, respectively. The assay was semiquantitative for the processed meat products (RSD-r 14 and 26% and RSD-R 46 and 56%), probably because gluten was unevenly distributed in the small (1 g) samples that were analyzed. The ELISA method produced no false positive results, and false negatives obtained with tannin-containing foods could be avoided by use of a modified sample extractant. None of the collaborators reported problems in following the protocol. The method has been adopted official first action by AOAC for determination of wheat gluten in foods.

Environmental exposure to cockroach allergens: Analysis with monoclonal antibody-based enzyme immunoassays

Quantitative two-site monoclonal antibody (MAb)-based enzyme-linked immunoassays for two cockroach (CR) allergens, Bla g I and Bla g II, have been developed and used to measure allergen levels in house-dust samples. Dust collected from the CR-infested homes of two patients with asthma from Charlottesville, Va., demonstrated wide variation in the levels of Bla g I, depending on the location of dust collection. Dust from kitchen floors and cabinets contained 50-fold more allergen (mean, 10,755 U/gm of dust) than dust from bedrooms and upholstered furniture (mean, 204 U/gm). One hundred forty-five dust samples were collected from the bedrooms and living rooms of 22 children with asthma and 16 control subjects without asthma living in Atlanta, Ga. Twenty-seven of the 38 homes ( 17 22 children with asthma; 10 16 control subjects) had detectable Bla g I (4 to 1340 U/gm of dust). Bla g II levels were assayed in 40 kitchen, bedroom, and living room samples from homes in Wilmington, Del. Highest levels of Bla g II were detected in kitchen-floor dust (300 U/gm of dust). Additionally, ~20% of homes with no visual evidence of CR infestation had significant levels of Bla g II in at least one dust sample (>4 U/gm of dust). Our results demonstrate that CR may be an occult allergen in homes. The kitchen appears to be the primary site of CR-allergen accumulation, but significant CR-allergen levels can also be found at other sites in the home. The MAb-based assays can be used for quantitation of environmental exposure to CR allergens. MAbs should be very useful in epidemiologic studies to establish levels of CR-allergen exposure that should be considered as a risk factor for the development of allergic diseases, particularly asthma.

Rapid culture confirmation of herpes simplex virus by a monoclonal antibody-based enzyme immunoassay

Rapid confirmation of herpes simplex virus (HSV) is essential in many clinical settings. Viral isolation in cell culture is the standard method for diagnosing HSV infection, with confirmation by specific immunological staining. The performance of the Rapid Absorbent Matrix Pad (RAMP) HSV culture confirmation test was evaluated with specimens obtained from 71 patients with suspected HSV infection and inoculated into African green monkey kidney cell lines. Forty-one culture-positive specimens were confirmed to be HSV by both the RAMP HSV test and the Bartels HSV immunoperoxidase test. Thirty immunoperoxidase-negative specimens were also negative in the RAMP HSV test. The sensitivity and specificity of the RAMP HSV test were 100%. Twelve specimens positive for cytomegalovirus, adenovirus, or enterovirus tested negative by the RAMP HSV test. Thus, the RAMP HSV test was faster than, easier to perform than, as sensitive as, and as specific as other well-documented confirmation methods.

An evaluation of an enzyme immunoassay method for immunoreactive trypsin in dried blood spots

A novel monoclonal antibody based enzyme immunoassay (EIA) method for the measurement of the human cationic trypsinogen (NeoScreen, AGEN Biomedical Ltd., Acacia Ridge, Australia) in dried blood spots for the neonatal screening of cystic fibrosis was evaluated. The calibration standards provided as dried blood spots by AGEN are highly unstable and must be replaced with user prepared materials. Reference values from control individuals were obtained by parametric methods. A preliminary comparison with a polyclonal antibody based RIA method (Trypsik, SORIN Biomedica, Saluggia, Italy) was performed. Regression analysis between the RIA and the EIA methods gave a coefficient of correlation of 0.58 for RIA values less than 40 micrograms/L and of 0.77 for RIA values greater than or equal to 40 micrograms/L. Average CV of the within-run imprecision for the EIA method was 19.6% and for the RIA method 28.8%. CVs of the between-run imprecision at low, intermediate and high values for the EIA method were 23.7%, 15.8%, 15.6% and for the RIA method 20.6%, 14.4%, 11.2%. The diagnostic accuracy analyzed by a Receiver Operating Characteristics (ROC) curve of the RIA method gave a maximum accuracy of 190.9 while that of a simulated ROC curve for the EIA method was 193.0. We found that the precision and the diagnostic accuracy of the EIA method (AGEN) are equal to or better than those of one of the RIA methods.

Immunodetection of human T-cell lymphotropic virus type I core protein in biological samples by using a monoclonal antibody immunoassay

A monoclonal antibody-based enzyme immunoassay (EIA) has been developed for detection of human T-cell lymphotropic virus type I (HTLV-I) core protein. The monoclonal antibody (clone 6.11) specifically recognizes the p19 gag gene-encoded protein of the virus. The EIA was over 100 times more sensitive than reverse transcriptase measurement and was capable of responding to less than 500 pg of whole-virus lysate. The assay exhibited type specificity in that HTLV-II antigens failed to produce a positive signal. In addition, a panel of other viruses demonstrated no antigenic cross-reactivity. These included herpesviruses, measles virus, human immunodeficiency viruses, and others. Viral p19 was followed during the course of density gradient ultracentrifugation in the presence of detergent, where it was noted to associate with viral membrane proteins. In comparison, reverse transcriptase activity localized in fractions of higher density containing envelope-free cores. Of clinical interest, the EIA was used to detect HTLV-I antigen in the viral cultures of patients with HTLV-I-associated myelopathies and from symptom-free individuals with proviral integration.

Use of Parasite Antigen Detection to Monitor Macrofilaricidal Therapy in Brugia malayi-Infected Jirds

Improved methods are needed to evaluate new treatments for filarial infections. We have recently developed a monoclonal antibody-based enzyme immunoassay to detect circulating parasite antigen in sera from Brugia malayi-infected jirds. In the present study, parasite antigen levels were compared to parasitological parameters after treatment of B. malayi-infected jirds with CGP 20376 that has been reported to be active against both microfilariae and adult worms of this parasite. Microfilariae were cleared promptly and permanently after CGP 20376 treatment, and no adult worm was recovered in jirds at necropsy 20 wk after treatment. In contrast, untreated animals had sustained microfilaremia throughout the course of the study, and adult worms were recovered in all control animals (mean worm recovery; 24.3 +/- 7.8 SE). Parasite antigen was present in sera from all infected animals before treatment. Parasite antigen titers in sera were unchanged 5 wk after treatment but fell to undetectable levels in 4 of 6 animals by 20 wk after treatment. Low-level antigenemia was detected in 2 of 6 animals at 20 wk, perhaps suggesting incomplete killing of parasites or incomplete clearance of antigen. Parasite antigen levels were stable throughout the study in control animals. These preliminary results suggest that parasite antigen detection is useful as a means of noninvasively monitoring the efficacy of anti-filarial drug therapy.

Quantitation of plasma membrane fatty acid-binding protein by enzyme dilution and monoclonal antibody based immunoassay

A plasma membrane fatty acid-binding protein (h-FABPpm) has been isolated from rat hepatocytes. Analogous proteins have also been identified in adipocytes, jejunal enterocytes and cardiac myocytes, all cells with high transmembrane fluxes of fatty acids. These 43 kDa, highly basic (pI = 9.1) FABPpm's appear unrelated to the smaller, cytosolic FABP's (designated FABP's) identified previously in the same tissues. h-FABPpm appears closely related to the mitochondrial isoform of glutamic-oxaloacetic transaminase (mGOT), and both the purified protein and liver cell plasma membranes (LPM) possess GOT enzymatic activity. From their relative GOT specific activities it is estimated that h-FABPpm constitutes approximately 2% of LPM protein, or about 0.7 x 10(7) sites per cell. A monoclonal antibody-based competitive inhibition enzyme immunoassay (CIEIA) for h-FABPpm is described; it yields an estimate of 3.4 x 10(7) h-FABPpm sites per hepatocyte. Quantitated by either method, h-FABPpm appears to be a highly abundant protein constituent of LPM.

Monoclonal antibody–based immunoassays for serum myoglobin quantification in acute myocardial infarction

We have developed a monoclonal antibody-based enzyme immunoassay and a solid-phase radioimmunoassay for human myoglobin. Both assays are based on competition for the monoclonal antibody between the free myoglobin present in the standards or serum samples and the myoglobin coated to the wells of microtiter plates. Consequently, the absorbance at 630 nm and the radioactivity are inversely related to the concentrations of free myoglobin. The sensitivity of both assays was 10 micrograms/L with linearity up to 1,000 micrograms/L. There was no interference with other serum proteins, as judged from analysis of specimens with high concentrations of lactate dehydrogenase, creatine kinase, or hemoglobin. The average serum myoglobin concentration in 30 normal individuals was 67 micrograms/L. Five patients with cardiac arrhythmias had normal values (average, 63 micrograms/L) while four patients with myocardial infarction had abnormally high concentrations of myoglobin (300-1,000 + micrograms/L). In a typical case of myocardial infarction, serum myoglobin rose 21 hr earlier and peaked 12 hr earlier than creatine kinase and its cardiac isoenzyme. These rapid immunoassays appear to be useful for the early detection of increased serum myoglobin indicative of myocardial infarction.

Monoclonal antibody-based enzyme immunoassay for Giardia lamblia antigen in human stool.

A visually readable monoclonal antibody-based antigen-capture enzyme immunoassay for the detection of Giardia lamblia antigen in human stool specimens was developed and found to be 97% (30 of 31 stool specimens) sensitive for formalinized stools and 82% (49 of 60 stool specimens) sensitive for unfixed stool specimens by visual reading. The storage of specimens in 10% Formalin resulted in increased absorbance in 20 of 26 G. lamblia-positive specimens tested as both formalinized and unfixed specimens; the increase averaged 1,336%. The assay was specific for antigens of this organism and for antigens derived from the cyst, as opposed to the trophozoite, stage. The assay could detect the antigens of five cysts per well, but could not detect antigen in in vitro-cultured trophozoites. A mouse monoclonal antibody of the immunoglobulin G1 (IgG1) subclass, which was prepared against cysts of G. lamblia, was used as the solid-phase capture antibody. The antibody was reactive with the cyst wall, as determined by immunofluorescence. Polyclonal rabbit anti-cyst IgG was used as the secondary antibody, and peroxidase-labeled goat anti-rabbit IgG was used as the tertiary antibody in the assay format. Maximal capture of antigen from stool specimens occurred by 30 min. Optimal dilution of specimens was in the range of 1:60 to 1:600. Preliminary characterization of affinity-purified antigen recognized by the monoclonal antibody showed that it is heat stable (100 degrees C, 12 min) and resistant to sodium periodate treatment and that it may exist in multiple molecular weights from 45,000 to 110,000.