Double-antibody Enzyme-linked Immunosorbent Assay Research Articles

Immunodiagnosis of sleeping sickness due to Trypanosoma brucei gambiense by detection of antiprocyclic antibodies and trypanosome antigens in patients' sera

Documented sera from 39 Trypanosoma brucei gambiense sleeping sickness patients from Ivory Coast (Côte d'Ivoire) were tested using the Procyclic Agglutination Trypanosomiasis Test (PATT) for the presence of anti-trypanosome antibodies and using an antigen-capture double antibody enzyme-linked immunosorbent assay (ELISA) for the presence of trypanosomal antigens. All 39 sera contained antiprocyclic antibodies and trypanosome antigens whereas 5 control sera did not. The results show that the PATT (for antibody detection) and the double antibody ELISA (for antigen detection) are useful for immunodiagnosis of African sleeping sickness due to T.b. gambiense and that these assays should be simplified for further testing and evaluation in the field.

Quantification in Enzyme‐Linked Immunosorbent Assay of a C3 Neoepitope Expressed on Activated Human Complement Factor C3

A sensitive double antibody enzyme-linked immunosorbent assay (ELISA) for quantification of C3 activation products in human plasma, synovial fluid, and cerebrospinal fluid is described. The monoclonal antibody MoAb bH6, which is specific for a C3 neoepitope expressed on C3b, iC3b, and C3c, was used as capture antibody. Detection antibody was a polyclonal rabbit anti-human C3c followed by development with a peroxidase-conjugated anti-rabbit Ig antiserum. The activity in normal human EDTA plasma was 1.5% of that in zymosan-activated serum (ZAS). The interassay and intra-assay coefficients of variation were 15% and 3%, respectively. The lower detection limit was 0.0005% of the ZAS standard. Reference range (1.1-2.1% of ZAS) was obtained by measuring EDTA plasma from 40 healthy blood donors. A positive correlation rs = +0.92; P less than 0.0002) was found between the present assay and an already established C3'g' activation ELISA, when samples from 16 patients were examined in both assays simultaneously. The present assay and an assay detecting the terminal complement complex showed virtually identical activation patterns in consecutively drawn samples from a patient undergoing extracorporal circulation.

Detection of lectins in nodulated peanut and soybean plants

The direct double-antibody enzymelinked immunosorbent assay system was used in the detection and measurement of seed lectins from peanut (Arachis hypogaea L.) and soybean (Glycine max L.) plants (PSL and SBL, respectively) that had been inoculated with their respective rhizobia. Concentrations of PSL dropped to undetectable levels in peanut roots at 9 d and stems and leaves at 27 d after planting; SBL could no longer be detected in soybean roots at 9 d and in stems and leaves at 12 d. A lectin antigenically similar to PSL was first detected in root nodules of peanuts at 21 d reaching a maximum of 8 μg/g at 29 d then decreasing to 2.5 μg/g at 60 d. There was no evidence of a corresponding lectin in soybean nodules.Sugar haemagglutination inhibition tests with neuraminidase-treated human blood cells established that PSL and the peanut nodule lectin were both galactose/lactose-specific. Further tests with rabbit blood cells demonstrated a second mannosespecific lectin in peanut nodule extracts that was not detected in root extracts of four-week-old inoculated plants or six-week-old uninoculated plants, although six-week-old root extracts from inoculated plants showed weak lectin activity. The root extracts from both nodulated and uninoculated plants contained another peanut lectin that agglutinated rabbit but not human blood cells. Haemagglutination by this lectin was, however, not inhibited by simple sugars but a glycoprotein, asialothyroglobulin, was effective in this respect.

Kinetics study of immunological response to Clostridium botulinum toxin.

A double-antibody enzyme-linked immunosorbent assay (ELISA) for detection of humoral antibody to type A botulinal toxin was developed. This assay was used to study the kinetics of antibody response of a volunteer to botulinal toxoid. The circulating type A antitoxin was first detected by the ELISA 2 weeks after the first booster injection of the toxoid. The antibody titer stayed level until the second booster at 12 weeks. The titer then continued to rise throughout the remaining study period. The neutralizing antibody to type A toxin was detected by mouse assay 15 weeks after detection of antitoxin by the ELISA.

Circulating IgA immune complexes in head and neck cancer, nasopharyngeal carcinoma, lung cancer, and colon cancer.

A double antibody enzyme-linked immunosorbent assay (ELISA) was developed to quantitate circulating immune complexed IgA (IgA IC) in human serum. The serum panel for this study consisted of normal blood donors, benign surgery (BS), head and neck cancer (HN), nasopharyngeal carcinoma (NPC), lung cancer (LC), and colon cancer (CC) patients. Immune complexes (IC) were isolated from these sera by precipitation with 3.5% polyethylene glycol (PEG), washed and then redissolved in 0.1 M phosphate-buffered saline pH 7.2. The amount of IgA IC present were then quantified using the double antibody IgA ELISA. This assay was found to be both sensitive (26.0 ng/ml) and reproducible (intra-assay coefficient of variation 4.0%). The mean IgA IC for each cancer group tested (HN = 11.38 +/- 12.54 micrograms/ml; NPC = 13.36 +/- 17.56 micrograms/ml; LC = 17.39 +/- 13.04 micrograms/ml; CC = 26.50 +/- 4.60 micrograms/ml) were significantly elevated (P = 0.001) over both the normals (5.12 +/- 4.09 micrograms/ml) and the benign surgery controls (5.92 +/- 5.04 micrograms/ml). In addition to providing a new tumor marker the presence of high levels of IgA IC in cancer patients could provide a source of tumor-specific antibody as well as antigen and provide reagents to study immune regulation in cancer patients.

Enzyme-linked Immunosorbent Assay for a Soluble Antigen of Renibacterium salmoninarum, the Causative Agent of Salmonid Bacterial Kidney Disease

A double-antibody enzyme-linked immunosorbent assay (ELISA) for detection of a soluble fraction of Renibacterium salmoninarum was developed from components extracted from the supernatant of an R. salmoninarum broth culture. The Costar® Serocluster™ EIA microplate gave the highest absorbance and signal-to-noise ratios among seven types tested. Including Tween 80 in the wash buffer resulted in higher absorbances than Tween 20 when antigen was present. Background absorbance did not increase when Tween 80 was added to the wash buffer, but did when Tween 80 replaced Tween 20 in antigen and conjugate diluents. Adsorption of coating antibody peaked within 4 h at 37 °C and 16 h at 4 °C. Antigen attachment to antibody-coated microplate wells depended more on incubation temperature than duration; we adopted a 3-h incubation at 25 °C. Conjugate incubation for longer than 1 h at 37 °C or 3 h at 25 °C resulted in unacceptable background levels. No cross-reactions resulted from heat-extracted antigens of 10 other species of bacteria. The optimized ELISA is a 6-h test that enables detection of levels of soluble antigen as low as 2–20 ng.

Evaluation of a Reversed Passive Latex Agglutination Test Kit for Clostridium perfringens Enterotoxin

A reversed passive latex agglutination (RPLA) test kit for Clostridium perfringens enterotoxin (CPE) marketed by the Denka-Seiken Co., Tokyo, Japan, was evaluated by using culture supernatant fluids and extracts from feces of food poisoning patients. Nanograms of CPE were detectable with the assay and the reaction was specific, as shown by parallel activity in a double antibody enzyme-linked immunosorbent assay (ELISA). Although less sensitive, the RPLA method is easier to perform than the ELISA and counterimmunoelectrophoresis, both of which require special test reagents and equipment not generally available.

Use of fluorodinitrobenzene to identify monoclonal antibodies which are suitable for conjugation to periodate-Oxidized horseradish peroxidase

During the recent development of a double antibody enzyme-linked immunosorbent assay for detecting malaria sporozoites in mosquitoes it was found that a large number of potentially useful monoclonal antibodies lost their capacity for binding antigen after conjugation to periodate-oxidized horseradish peroxidase (HPO). Since HPO reacts with primary amino groups, we used a simple chemical reaction with fluorodinitrobenzene (FDNB) to determine if the loss of antigen binding was due to the requirement for unmodified primary amino groups in the binding site. FDNB-treated antibodies which reacted with antigen in an indirect fluorescent antibody assay (IFA) also yielded successful HPO-antibody conjugates. Conversely, those antibodies which did not react with antigen after treatment with FDNB failed to produce useful HPO-antibody conjugates. These data suggest that conjugation of oxidized HPO to primary amines in or near the antigen-combining site yields conjugates in which the antibody is inactive. Use of FDNB to predict the suitability of antibodies for conjugation to periodate-oxidized HPO may save a considerable amount of time over randomly selecting antibodies for conjugation and testing.

Double-antibody ELISA for detection of trace amounts of pig meat in raw meat mixtures

A modification of double-antibody sandwich ELISA (enzyme-linked immunosorbent assay) has been applied successfully to the problem of the detection of very low amounts of pig meat (1–3%) in beef and manufactured beef products made with rusk and other additives. The assay uses a monospec fic antiserum bound to the solid plastic support to sequester the porcine serum albumin (PSA) from amongst the many proteins co-extracted from the complex sample extracts. Further immuno-recognition was made with a second antibody, also specific for PSA, which was detected by a conjugated enzyme anti-IgG that interacted only with the second anti-PSA antibodies. Subsequent enzymic conversion of substrate gave clear visual differences between beef and beef containing 0·5% of pig meat and beef products with 1% of the lean replaced by lean pork compared with reference samples.

Identification of Plasmodium Falciparum-Infected Mosquitoes by a Double Antibody Enzyme-Linked Immunosorbent Assay *

A double antibody micro enzyme-linked immunosorbent assay (ELISA) for identifying Plasmodium falciparum sporozoites in mosquitoes is described. Using monoclonal antibodies made against South American P. falciparum sporozoites, the ELISA was able to detect and identify sporozoite antigens of South American and Asian origins in extracts of dried infected mosquitoes.

Circulating antigens, antibodies and immune complexes in experimental Taenia pisiformis infections of rabbits.

Circulating antigen has been detected in the serum of rabbits experimentally infected with Taenia pisiformis. A rabbit antiserum against an antigen preparation derived from the in vitro maintenance of living metacestodes of T. pisiformis was used in a double-antibody enzyme-linked immunosorbent assay (ELISA). Antigen was detected in infected rabbit serum 48 h after infection and peak levels were recorded at 2 weeks post-infection or post-challenge. The pattern of circulating immune complex formation followed closely that of circulating antigen, and infected rabbit serum contained both anti-metacestode and antioncospheral antibodies. Serum antigen levels were just detectable (0.4-0.8 micrograms protein/ml) over the period (greater than 2 weeks post-infection) of peritoneal cyst development. The circulating antigen probably has antigenic determinants in common with the oncosphere, juvenile and mature metacestode stages. Possible reasons for the temporal variation in serum antigen levels are discussed.

Enzyme-Linked Immunosorbent Assay of Ochratoxin A in Barley

A noncompetitive, double antibody enzyme-linked immunosorbent assay for ochratoxin A using microtitration plates has been developed and applied to samples of barley. The anti-ochratoxin A antiserum, which is used at high dilution, does not cross-react significantly with ochratoxin B or ochratoxin a. Assay sensitivity for determination of the toxin in barley samples is 60 ng/kg. Minimal sample preparation is required before assay.

Enzyme-linked immunosorbent assay for detection and identification of coxsackieviruses A.

Coxsackieviruses A are known to cause a wide range of human disease processes. However, because many coxsackieviruses A present in clinical specimens do not produce a recognizable cytopathic effect in readily available tissue culture systems, infections with coxsackieviruses A are often difficult to diagnose. We have thus developed enzyme-linked immunosorbent assay (ELISA) systems for the detection and serotyping of coxsackievirus A antigens. The assays consist of a double-antibody ELISA which utilizes type-specific monkey and mouse coxsackievirus antisera. Although some cross-reactivity was noted, the ELISA systems correctly identified the serotypes of 22 to 23 coxsackievirus A complement fixation antigens available for testing. Testing of tissue culture fluids revealed that antigen could often be detected by ELISA before the appearance of a cytopathic effect. In addition, the infecting coxsackievirus A antigen could be unequivocally identified in 8 of 11 stool specimens obtained from patients with coxsackievirus A infections. The ELISA system might thus represent an important tool in the diagnosis and study of coxsackievirus A infections.