Evaluation of the use of chromogenic and fluorogenic substrates in solid-phase enzyme linked immunosorbent assays (ELISA)

Fluorogenic and chromogenic substrates were used in direct and trapping enzyme-linked immunosorbent assays (ELISA) for the detection of mouse IgG and foot-and-mouth disease virus (FMDV). The detection limits for both antigens were compared using different combinations of enzymes and substrates. Various times and concentrations of chemicals were used to obtain maximum sensitivity for both systems. Similar sensitivities were found using fluorogenic and chromogenic substrates. Tetramethyl benzidine substrate for horse-radish peroxidase enzyme conjugates was found to attain the highest sensitivity levels for chromogenic assays (0.12 ng IgG/ml and 1.0 ng/ml FMDV respectively), after 10 min incubation. Of the two fluorogenic enzyme/substrates studied, B-galactosidase was the most sensitive but required extended incubation times (2-3 h) as compared with chromogenic systems. Special microplates for fluoro-immunoassay (FIA) were compared with conventional microplates and no advantage was found to justify their use. An alkaline phosphatase anti-guinea-pig conjugate was used to confirm the equivalence of fluorogenic and chromogenic substrates in terms of sensitivity. A comparison of the amount of signal generated using various concentrations of enzyme in the absence of antigen was made for two different alkaline phosphatase conjugates to obtain theoretical sensitivity limits. One possible advantage of fluorogenic substrates is that high binding ratio can improve the confidence in discrimination of positive results.     

Comparison of enzyme-linked immunosorbent assay, electron microscopy, and reversed passive haemagglutination for detection of human rotavirus in stool specimens

An enzyme-linked immunosorbent assay (ELISA) using microplates as solid phase, rabbit antiserum against human rotavirus Wa strain as catching antibody, and the same reagent labeled with beta-D-galactosidase as conjugate, has been developed for detection of human rotavirus antigen(s) in stool specimens from patients with acute gastroenteritis. The limit of detection of purified human rotavirus by ELISA was 15.6 ng/ml (1.56 ng/well) of viral protein. The sensitivities of ELISA, electron microscopy, and the reversed passive haemagglutination method (ROTA-CELL) were compared. ELISA was more sensitive than electron microscopy and the reversed passive haemagglutination method. The ELISA blocking assay was useful for detection of an antibody response to human rotavirus in paired sera from children in two institutions during outbreaks of rotavirus gastroenteritis.     

High sensitivity immunoassays using particulate fluorescent labels.

The use of polystyrene fluorescent microspheres as sensitive labels in direct-detection (not enzymatically amplified) heterogeneous equilibrium "sandwich" immunoassays in 96-well plates is described. With mouse IgG as a model antigen, a fluorescent particulate label is more sensitive than a corresponding soluble reporter. The limit of detection of mouse IgG in the multiparametrically optimized assay was 0.2 ng/ml (7.6 x 10(8) antigens/ml) for the particulate reporter and 50 ng/ml (1.9 x 10(11) antigens/ml) for the soluble reporter. The sensitivities of assays using the particulate label were dependent on the surface densities of the capture and reporter antibodies and the concentration of reporter beads. Sensitivity was improved by adding the preformed reporter antibody/fluorescent microsphere complex to trapped antigen on the well surfaces instead of sequentially adding the reporter antibody and then the fluorescent microspheres. Maximal (equilibrium) binding of the particulate reporter to captured antigen occurred after 20 h with a concentration of 1.4 x 10(9) reporter beads/ml. Thus, particulate fluorescent labels provide high sensitivity in direct-detection immunoassays.     

Highly sensitive enzyme-linked assay based on monoclonal antibodies for detection of Brucella antigens

Mice monoclonal antibodies against lypopolysaccharides (LPS) of Brucella abortus has been obtained and characterized. The antibodies detected LPS of B. abortus, B. melitensis and B. suis with high sensivity and specificity and did not react with LPS of Yersinia enterocolitica O:3, Y. enterocolitica O:9, Salmonella typhimurium, and Francisella tularensis. It has been shown that interaction of monoclonal antibodies and LPS of Brucella species can be critically dependent from buffer system. Obtained monoclonal antibodies allowed to develop highly sensitive assay which was able to detect antigens of Brucella species in concentrations 0.05 - 0.1 ng/ml. The assay can be used for detection and identification of Brucella species.     

Amplification of microsphere-based microarrays using catalyzed reporter deposition

Assay sensitivities using three fluorescent signal generation schemes were evaluated on the Luminex flow cytometer. Following microsphere capture of antigen by immobilized antibodies, bound targets were quantified by use of (1) Cy3-labeled "tracer" antibodies (30min total time), (2) biotinylated tracers followed by streptavidin-R-phycoerythrin (60min total time), or (3) biotinylated tracers followed by avidin-peroxidase conjugates and tyramide signal amplification (TSA; 90min total time). Use of TSA for signal generation in three individual toxin assays improved performance up to 100-fold over Cy3-antibody-based detection, and while streptavidin-R-phycoerythrin provided equivalent sensitivities, TSA produced dramatic increases at low concentrations simplifying positive sample identification. Detection limits for TSA-interrogated assays for ricin, cholera toxin, and staphylococcal enterotoxin B were 64pg/ml, 4pg/ml, and 0.1ng/ml, respectively, using optimized conjugates; analogous detection limits for Cy3-antibody-interrogated assays were 8ng/ml, 1ng/ml, and 1ng/ml, respectively. No improvement was observed in botulinum toxoid A assays when TSA amplification was used. As unique preferences for specific avidin-peroxidase conjugates were observed in the individual assays, improvements in multiplexed assays utilizing a single conjugate were significantly lower (3-10-fold improvements). Furthermore, increases in variability resulted in poorer performance of TSA-interrogated assays for botulinum toxoid, indicating that assay-specific optimization should be performed, especially prior to multiplexing.     

Properties of monoclonal antibodies to the genus-specific antigen of Chlamydia and their use for antigen detection by reverse passive haemagglutination

The chlamydial genus-specific antigen was extracted with phenol/chloroform/petroleum ether (PCP) from preparations of Chlamydia trachomatis and C. psittaci, and quantities measured using an assay for lipopolysaccharide (LPS). The LPS from C. trachomatis contained 2.2% (w/w) of ketodeoxyoctanoic acid. Five IgG monoclonal antibodies reacted in an ELISA with LPS from both species, the antigen being periodate-sensitive and heat-resistant, confirming that all antibodies were against the genus-specific antigen. All the antibodies bound to the PCP extract of C. trachomatis on an immunoblot, at a position corresponding to the periodate-Schiff-stained bands of both C. trachomatis extract and Salmonella Re-LPS. When linked to trypsin-treated sheep erthrocytes and used in reverse passive haemagglutination tests, all antibodies gave indicator cells capable of detecting chlamydial LPS or crude preparations of chlamydiae grown in McCoy cells, the sensitivity varying with the antibody used. The antibodies varied in IgG subclass (either IgG2a or IgG3), and in ability to precipitate in immunodiffusion tests. Two antibodies cross-reacted with one strain of Acinetobacter in ELISA and with Salmonella Re-LPS in both ELISA and immunodiffusion tests. The other three did not react in ELISA with Acinetobacter strains or Salmonella Re-LPS, and none of the five reacted with LPS of E. coli or Pseudomonas morsprunorum.     

A competitive enzyme linked immunosorbent assay for recombinant Bm86 produced in Pichia pastoris

A competitive enzyme linked immonosorbent assay (ELISA) was developed for quantification of recombinant Bm86 protein (rBm86), antigen of the vaccine GavacTM against the cattle tick Boophilus microplus. Monoclonal antibody (Mab) SSBm1 showed identical recognition to folded and denatured antigen and was used in the competition assay. This ELISA was sensitive enough to detect 60 ng/ml of rBm86. Within-assay coefficient of variation was 5.7 to 6.2 % and between-assay variation was 7.7 to 11.6 %. The level of expression of recombinant Bm86 in Pichia pastoris reached about 2.7 g per liter of culture after 80 hour of fermentation. © Rapid Science Ltd. 1998     

The determination of the antibodies and the somatic O-antigen of Salmonella group B by using the complement-bound lysis of liposomes]

A simple and sensitive method for the determination of group B Salmonella O-antigen and specific antibodies to group B Salmonella by means of the complement-dependent lysis of liposomes sensitized with S. typhimurium lipopolysaccharide (LPS) is proposed. The factors affecting the sensitivity of the method during the determination of antibodies and free antigen have been studied. The method permits the determination of soluble LPS antigen in concentrations of 0.5-200 micrograms/ml.     

Monoclonal antibodies of IgA isotype specific for lipopolysaccharide of Salmonella enteritidis: production, purification, characterization and application as serotyping reagents

Hybridomas secreting immunoglobulin A (IgA) monoclonal antibodies (MAbs) against Salmonella enteritidis lipopolysaccharide (LPS) were generated after mucosal immunization of BALB/c mice with heat killed bacteria. Antigen binding properties and specificity of the produced MAbs were studied in ELISA and immunoblotting with purified LPS. Two IgA MAbs agglutinated all Salmonella OD1 strains and all S. enteritidis clinical isolates. MAb 178H11 recognized O:9 antigen of subserogroup OD1 LPS. MAb 177E6/A9 reacted also with OD3 LPS antigen and agglutinated OD3 strains. These data suggest the existence of different O:9 antigen subspecificities, one presented in subgroup OD1 and the other common for OD1 and OD3. Thus the produced IgA MAbs prove to be useful reagents, which could differentiate OD1 and OD3 from OD2 strains.     

Generation of polyclonal antibodies against nisin: immunization strategies and immunoassay development.

Murine polyclonal antibodies reactive to the lantibiotic bacteriocin nisin A (nisA) have been produced by immunization with nisA-cholera toxin and nisA-keyhole limpet hemocyanin (nisA-KLH) conjugates. Mice immunized with nisA-cholera toxin developed nisA-specific antibodies with low relative affinities and poor sensitivities, while the immunization of mice with nisA-KLH conjugates resulted in the production of nisA-specific antibodies with high relative affinities and much-increased sensitivities. nisA antibodies could also be readily mass produced in less than 8 weeks in ascites fluid by using the nisA-KLH conjugate. A competitive direct enzyme-linked immunosorbent assay (ELISA) whereby nisA-horseradish peroxidase and free nisA competed for antibody binding was devised. The detection limit for nisA in the competitive direct ELISA with the nisA-KLH-generated antibodies was from 5 to 100 ng/ml, while the amount of free nisA required for 50% antibody binding inhibition ranged from 0.3 to 5 micrograms /ml. Both antisera and ascites polyclonal antibodies cross-reacted with nisZ either in the supernatant of a producer strain or with the pure lantibiotic but did not cross-react at all with non-lantibiotic-type bacteriocins. These polyclonal antibodies should find a wide usage from nisA ELISA analysis in foods and other matrices.     

A monoclonal antibody to T-2 toxin

A specific, high affinity monoclonal antibody to T-2 toxin of Fusarium has been produced. The monoclonal antibody was conjugated to horse-radish peroxidase and employed to develop a direct competitive enzyme-linked immunosorbent assay (ELISA) for the toxin. The sensitivity of the ELISA was 10 ng/ml with a working range up to 500 ng/ml. The antibody cross-reacted with HT-2 toxin (25%) but did not bind to any other trichothecene tested.     

Production and characterization of the monoclonal antibodies to <Emphasis Type="Italic">Bacillus anthracis</Emphasis> protective antigen

Anthrax, a cosmopolitan acute infectious disease affecting animals and humans, is a category A bioterrorism threat agent because of the high resistance of Bacillus anthracis spores to adverse environmental factors and their easy production. In this work, we have obtained a representative panel of 20 monoclonal antibodies to the anthrax protective antigen, a key component of the pathogenic exotoxin. A sandwich ELISA for quantification of the protective antigen using these antibodies was developed. Six pairs of monoclonal antibodies are able to detect the protective antigen in blood serum with a detection limit of 1 ng/ml.     

Immunoenzyme method for detecting microbial producers of palytoxin

A competitive ELISA using the intact toxin as a coating antigen for detecting palytoxin was developed. This immunoassay allows palytoxin (PTX) to be determined in the range of 6-250 ng/ml. In sensitivity, this determination is comparable with RIA but is three times inferior to ELISA using monoclonal antibodies. Inhibition experiments using some toxins of marine invertebrates proved the serological specificity of the palytoxin binding to antibodies. Both the indirect and competitive ELISA were used to find PTX-producing bacteria among 420 isolates of sea bacteria. It was found that gram-negative bacteria Aeromonas sp. and Vibrio sp. associated with toxic samples of the soft coral Palythoa sp. produced compounds antigenically related to PTX.     

重组人睫状神经营养因子蛋白质定量检测方法的建立

采用ELISA双抗体夹心法,建立一种快速灵敏的定量检测rhCNTF成品蛋白含量的方法。结果显示,rhC-NTF抗原浓度在(0～25)ng范围内线性良好(r>0.99),灵敏度为0.3ng/ml,与其他重组细胞因子无交叉反应,样品的检测结果与理论含量相吻合,CV<15%,该方法检测速度快、重复性好、灵敏度高、特异性好。     

Development of immunosensor based on OWLS technique for determining Aflatoxin B1 and Ochratoxin A

Mycotoxins are toxic secondary metabolites produced by a number of different fungi, and can be present in a wide range of food and feed commodities including cereal grains, oil seeds, dried fruits, apple juice, wine and meat products from animals fed contaminated meal. Many mycotoxins are highly resistant, and survive food processing, and therefore enter the food chain and provide a threat to human health. The optical waveguide lightmode spectroscopy (OWLS) technique has been applied to the detection of Aflatoxin and Ochratoxin in both competitive and in direct immunoassays. After immobilizing the antibody or antigen conjugate for the direct or indirect measurement, respectively, the sensor chip was used in flow-injection analyser (FIA) system. When using non-competitive method, sensor responses were obtained first only at analyte concentrations of 5-10 ng ml(-1). In both cases, the responses were very unstable. For competitive sensor investigation with the sensitized chip first the optimal dilution rate of monoclonal antibodies was determined, for the measurement of Ochratoxin A and Aflatoxin B1 the monoclonal antibody stock solution was diluted to 1 microg ml(-1) and to a 1:400 dilution, respectively. During the competitive measurement standard solutions were mixed with monoclonal antibodies at the appropriate concentration, the mixture was incubated for 1 min and injected into the OWLS system. The sensitive detection range of the competitive detection method was between 0.5 and 10 ng ml(-1) in both cases. After the establishment of the indirect method, barley and wheat flour samples were measured, and the results were in good correlation by those measured by enzyme linked immuno-sorbent assay (ELISA). Regression coefficient between the two methods for Ochratoxin and Aflatoxin was determined as 0.96 and 0.89, respectively.     

Development and characterization of monoclonal antibodies against Rift Valley fever virus nucleocapsid protein generated by DNA immunization

This paper describes the generation of monoclonal antibodies directed to immunogenic nucleoprotein N epitopes of Rift Valley fever virus (RVFV), and their application in diagnostics, both for antibody detection in competitive ELISA and for antigen capture in a sandwich ELISA. Monoclonal antibodies (mAbs) were generated after DNA immunization of Balb/c mice and characterized by Western blot, ELISA and cell immunostaining assays. At least three different immunorelevant epitopes were defined by mAb competition assays. Interestingly, two of the mAbs generated were able to distinguish between RVFV strains from Egyptian or South African lineages. These monoclonal antibodies constitute useful tools for diagnosis, especially for the detection of serum anti-RVFV antibodies from a broad range of species by means of competitive ELISA.     

Sensitivity enhancement of surface plasmon resonance biosensing of small molecules

Surface plasmon resonance (SPR) biosensor formats using gold nanoparticle or protein signal amplification for the sensitive assay of small molecules were developed using progesterone as a model compound. Progesterone was immobilized to a dextran surface in the Biacore biosensor through in situ covalent immobilization using an oligoethylene glycol linker attached to the 4 position of the steroid. This surface produced stable antibody binding for in excess of 1100 assay cycles. Using this surface, assays were developed for progesterone using 10- and 20-nm gold-streptavidin labels attached to biotinylated monoclonal antibody in both label prebinding and sequential binding formats. Prelabeling formats gave no signal enhancement but produced assays with limits of detection of 143 pg/ml, compared with approximately 1 ng/ml in previous studies. Sequential binding formats gave signal enhancements of 2.2-fold over the monoclonal antibody and a limit of detection of 23.1 pg/ml. It was found that secondary antibody labeling gave 8.1-fold signal enhancements and a limit of detection of 20.1 pg/ml, whereas use of secondary antibody-25 nm gold complexes provided more signal enhancement (13-fold) and a further improvement in limit of detection of 8.6 pg/ml.     

Production and characterization of KDO-specific monoclonal antibodies recognizing lipopolysaccharides from heptoseless mutants of Salmonella

Abstract Hybrid cell lines producing monoclonal antibodies with specificity for the lipopolysaccharide (LPS) from the deep rough mutant Salmonella minnesota R595 have been established. Spleen cells from BALB/c mice immunized with live R595 bacteria were fused with Sp 2/0 myeloma cells and three hybridomas producing antibodies specific for heptoseless LPS from Salmonella were selected. All three monoclonal antibodies were shown to bind only to heptoseless, but 3-deoxy- d -manno-octulosonic acid (KDO) containing LPS when tested in enzyme-linked immunosorbent assay (ELISA) against a set of structurally defined LPS and lipid A from Salmonella, Shigella and Escherichia coli . Synthetic KDO was an efficient inhibitor of the antibody-R595 LPS interaction defining that KDO is in an immunodeterminant position interacting with the monoclonal antibodies.     

An equilibrium ELISA for the dosage of Androctonus australis garzonii (Aag) and Buthus occitanus tunetanus (Bot) scorpion venoms: set up and calibration

Scorpion stings are very frequent in Tunisia; yet a method for evaluating envenoming severity and consequently victim treatment, has never been adequately established nor has its efficiency been properly evaluated. Indeed, a management of envenomed patients requires the optimization of envenoming antivenom immunotherapy. This task requires either, an accurate evaluation of toxicokinetic parameters of scorpion venoms in absence and in presence of antivenom, using animals as models, and the establishment of a quantitative relationship between human blood scorption venom levels, envenoming severities and clinical symptoms. A performant sandwich ELISA was set up and calibrated for measuring scorpion venom levels in human and rabbit sera. This assays was performed with polyclonal F(ab')2 specific to the two North African scorpion (Androctonus australis garzonii; Aag and Buthus occitanus tunetanus: Bot) venoms. It is simple, rapid, very sensitive (detection limit = 0.9 ng/ml) and shows a good linearity for venom concentrations in human sera comprised between 0.5 and 15 ng/ml. The ELISA is also reproducible: the coefficient of variation, determined at different venom concentrations (low: 4 ng/ml; medium: 8 ng/ml and high: 12 ng/ml) prepared in a pool of sera collected from several healthy donors, were lower than 10%. Such an ELISA has been successfully used, either in experimental toxinokinetic and immunotherapeutic studies carried out in rabbits or for the quantification of Aag and Bot venom levels in the serum of human victims stung by these scorpion.     

Determination of 5-chloro-7-iodo-8-quinolinol (clioquinol) in plasma and tissues of hamsters by high-performance liquid chromatography and electrochemical detection

This paper describes a method of determining clioquinol levels in hamster plasma and tissue by means of HPLC and electrochemical detection. Clioquinol was separated on a Nucleosil C18 300 mm x 3.9 mm i.d. 7 microm column at 1 ml/min using a phosphate/citrate buffer 0.1M (400 ml) with 600 ml of a methanol:acetonitrile (1:1, v/v) mobile phase. The retention times of clioquinol and the IS were, respectively, 11.6 and 8.1 min; the quantitation limit (CV>8%) was 5 ng/ml in plasma and 10 ng/ml in tissues. The intra- and inter-assay accuracies of the method were more than 95%, with coefficients of variation between 3.0 and 7.7%, and plasma and tissue recovery rates of 72-77%. There was a linear response to clioquinol 5-2000 ng/ml in plasma, and 10-1000 ng/g in tissues. The method is highly sensitive and selective, makes it possible to study the pharmacokinetics of plasma clioquinol after oral administration and the distribution of clioquinol in tissues, and could be used to monitor plasma clioquinol levels in humans.