Development of an ELISA specific for Listeria monocytogenes using a polyclonal antibody raised against a cell extract containing internalin B

We have developed a new enzyme-linked immunosorbent assay (ELISA) that is specific to the foodborne pathogenic micro-organism Listeria monocytogenes. It is based on an antibody raised against an L. monocytogenes cell preparation optimized for extraction of internalin B. Only in a sandwich ELISA format was the protein A-purified antibody specific to L. monocytogenes. In a competitive ELISA format, the antibody recognizes other Listeria species. The sandwich ELISA shows no recognition of L. innocua, L. ivanovii, L. welshimeri, L. seeligeri, or L. grayii. It has a minimum detectable level for L. monocytogenes of log₁₀ 6.37 cfu ml^-1 in pure culture, is reproducible, and is unaffected by the presence of high numbers (approximately log₁₀ 8.0 cfu ml^-1) of the other Listeria species. Possible reasons for the format-dependent specificity are discussed. When the ELISA was applied to milk samples inoculated with L. monocytogenes reference material (5 cfu ml^-1), there was a strong response to the enrichment cultures. The new assay may prove useful in detection of L. monocytogenes in enrichment cultures of food samples.     

Characterization and application of a Listeria monocytogenes reactive monoclonal antibody C11E9 in a resonant mirror biosensor

Typical detection of Listeria monocytogenes involves selective enrichment, isolation and biochemical testing. Development of antibodies to Listeria species has improved detection; however, most antibodies detect all species of Listeria. A previously developed monoclonal antibody (MAb)-C11E9 was examined for its reaction to 13 L. innocua and 40 L. monocytogenes strains representing all 13 serotypes by ELISA. Absorbance values for L. monocytogenes strains were 0.44-3.58 and for L. innocua 0.22-1.44. ELISA reactions were divided into three arbitrary groups of high (Abs 1.0 or higher), intermediate (0.6-0.99) and low (0.18-0.59). Most L. monocytogenes strains (32/41, 78%) were in the high group while only 23% (3/13) of L. innocua were in the same group. In the Western blot assay, antibody reacted with phosphate-buffered saline (PBS) extracted protein preparations of 52, 66 and 97 kDa. Ribopattern of all strains was analyzed and no clear relationship was observed for antibody reaction and ribotype of a given strain. MAb C11E9 was used in a resonant mirror biosensor (IAsys sensor), but failed to detect any viable intact L. monocytogenes cells at levels as high as 10(8) cells/ml; however, it showed binding (85-150 arc/s) with the surface protein preparations containing the 97-, 66- and 52-kDa proteins at 208 mug/ml. Binding kinetics of L. monocytogenes and L. innocua surface protein extracts showed significantly (p<0.05) higher responses than the three other Listeria species (L. ivanovii, L. welshimeri and L. grayi), which could be detected in 10-20 min. These data corroborate with ELISA results. In summary, this study suggest that MAb-C11E9 is suitable for detection of all serotypes of L. monocytogenes despite cross-reaction with L. innocua and could be used for detection of soluble protein extracts in the resonant mirror (IAsys) biosensor.     

Combined Immunomagnetic Separation and Detection of Salmonella enteritidis in Food Samples

A model system for immunochemical detection of Salmonella enteritidis has been developed, using immunomagnetic separation (IMS) with both commercially‐available and laboratory‐prepared antibodies, followed by an enrichment stage and end‐point detection by ELISA. IMS alone gave an average of 77% recovery of cells artificially inoculated into the food, and a range of 20–110% recovery, depending on food type. The combined model system (IMS‐ELISA) enables detection of either < 10 cells m^?1 whole egg extract (using overnight enrichment after IMS), or > 10 cells ml^?1 (3 h enrichment after IMS). Cross‐reactivity of antibodies with Citrobacter was decreased by using two different immunochemical steps: IMS with monoclonal antibody‐coated beads, and a ‘sandwich’ ELISA with polyclonal capture antibody and monoclonal detector antibody. Discussion is presented on the best food preparation method, optimal substrate type for the ELISA and on the potential of IMS.     

Enzyme immunoassays for picloram detection: A comparison of assay formats

Two direct enzyme immunoassays for picloram (4‐amino‐3,5,6‐trichloro‐2‐pyridinecarboxylic acid) detection were developed. The assay using IgG isolated from a rabbit antipicloram antiserum had a working range from 5 to 5000 ng ml^?1 with a mean I₅₀ value of 180 ng mh^?1 and a limit of quantification of 10 ng ml^?1. The assay using a monoclonal anti‐picloram antibody had a working range from 1 to 200 ng mh^?1 with a mean I₅₀ value of 18 ng mh^?1 and a limit of quantification of 5 ng ml^?1. The direct assays were compared to an existing monoclonal antibody‐based indirect enzymes immunoassay for accuracy and precision of picloram determinations in fortified ground and surface waters. In both matrices, the monoclonal antibody‐based indirect enzyme immunoassay was shown to be the superior format in terms of greater precision and equal or greater accuracy.     

Use of monoclonal antibodies that recognize p60 for identification of Listeria monocytogenes

Listeria monocytogenes causes major food-borne outbreaks of disease worldwide. Specific identification of this microorganism is of utmost importance to public health and industry. Listeria species are known to secrete a 60-kDa protein collectively termed p60, which is encoded by the iap (invasion-associated protein) gene and secreted in large quantities into the growth media. p60 is a highly immunogenic murein hydrolase that is essential for cell division. Due to these properties, p60 is an ideal diagnostic target for the development of immunological detection systems for L. monocytogenes. We report here two independent lines of monoclonal antibody (MAb): p6007, which specifically recognizes L. monocytogenes p60, and p6017, which reacts with a wide range of Listeria p60 proteins. By combining these antibodies with a polyclonal antibody, we developed efficient sandwich enzyme-linked immunosorbent assay (ELISA) systems which can specifically identify L. monocytogenes or generally detect Listeria species. Since an excess amount of the peptide corresponding to PepA or PepD did not interfere with the ELISA, and direct ELISAs were unable to detect both peptides, we concluded that the epitope presumed to be recognized by p6007 or p6017 could be distinguished from PepA and PepD as described by Bubert et al. (Appl. Environ. Microbiol. 60:3120-3127, 1997). To our best knowledge, this is the first example of an immunological identification system that uses p60-recognizing MAbs.     

Development of a monoclonal antibody-based ELISA to detect Escherichia coli O157:H7

A pair of monoclonal antibodies (mAb) from 10 murine hybridomas secreting Escherichia coli O157:H7 (E. coli O157:H7)-specific mAbs were selected for the development of the sandwich ELISA to detect E. coli O157:H7. On the basis of pairwise interaction analysis, mAb-1 was selected as a capture antibody while mAb-6 was used as a detection antibody. The buffer system which provided the greatest difference between the specific E. coli O157:H7-positive antigen and the negative control was chosen. This sandwich ELISA showed good linearity when the concentration of E. coli O157:H7 was in the range of 10⁵–10⁸ cfu/mL, and the sensitivity was 1×10⁴ cfu/mL. With 8-h enrichment of bacteria, this ELISA was found to detect 0.4 cfu/g E. coli O157:H7 in artificially contaminated green tea samples.     

Serotyping of Listeria monocytogenes by enzyme-linked immunosorbent assay and identification of mixed-serotype cultures by colony immunoblotting

Routine analysis of Listeria monocytogenes by serotyping using traditional agglutination methods is limited in use because of the expense and limited availability of commercially prepared antisera and intra- and interlaboratory discrepancies arising from differences in antiserum preparation and visual determination of agglutination. We have adapted a commercially available set of L. monocytogenes antisera to an enzyme-linked immunosorbent assay (ELISA) format for high-throughput, low-cost serotype determination. Rather than subjective visualization of agglutination, positive antigen and antiserum reactions were scored by a quantitative, colorimetric reaction. ELISA serotyping of 89 of 101 L. monocytogenes isolates agreed with slide agglutination serotyping data, and 100 previously uncharacterized isolates were serotyped unambiguously by the ELISA method. In addition, mixed-serotype cultures of L. monocytogenes were identified by a colony immunoblot procedure, in which serogroup 1/2 and serogroup 4 colonies were discriminated by differential staining.     

Sensitive and highly specific detection of Cronobacter sakazakii based on monoclonal sandwich ELISA

A sensitive and specific monoclonal antibody-based sandwich enzyme-linked immunosorbent assay (ELISA) was established to detect the Cronobacter sakazakii. A pair of monoclonal antibodies (mAbs) selected from mAbs produced by 11 murine hybridomas was selected for the sandwich ELISA procedure. Targets of two mAbs were 100 kDa and 42 kDa protein extracted from the bacteria, respectively, which were proved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting analysis. The limit of detection of this method was established as 1 × 10⁴ cfu/mL, and the linear range from 1 × 10⁵ to 1 × 10⁸ cfu/mL. In the real sample test, 1 cfu/g C. sakazakii was detected in artificially contaminated powdered infant formula with 4 h enrichment.     

Development of high‐sensitive enzyme immunoassays for gliadin quantification using the streptavidin‐biotin amplification system

Optimization of three enzyme immunoassays of very high sensitivity using three antiprolamin monoclonal antibodies (MAbs) (13B4, 11C4 and 12A1) is presented here. These MAbs are specific for those prolamins toxic for coeliac patients, as determined by immunoblotting analysis. Biotinylated MAbs were used in two of the assays. In a competitive ELISA, the binding of each biotinylated MAb to a gliadin‐coated solid phase was inhibited by gliadin in the fluid phase. The best result was obtained using the biotinylated MAbl3B4 (detection limit: 20 ng ml^?1). With regard to capture ELISA, we tested the performance of the three MAbs. In this sandwich ELISA, the MAb used for antigenic capture was the same as that used as secondary biotinylated antibody. The MAbl2Al had the best performance (detection limit: 1 ng ml^?1). The use of biotin‐labelled gliadin in a quantitative immunoassay with a detection limit of 5 ng ml^?1 is also reported. This assay involves an antigenic capture using the MAbl2Al followed by a competition between biotinylated and non‐biotinylated gliadin. We have found the use of the streptavidin‐biotin interaction as signal amplification system to be very useful. This technique, as far as we know, has not been previously reported for gliadin quantification.     

Development and application of immunochromatographic tests for the detection of staphylococcal enterotoxin E

Two rapid immunochromatographic assay formats for the detection of Staphylococcus aureus enterotoxin serotype E (SEE) were developed. For both tests, polyclonal antibodies against SEE were immobilized on a membrane strip representing the test zone, while second anti‐SEE antibodies conjugated to dyed latex particles were employed as markers in sandwich immunoassay. In a two‐step test format, sample and labelled antibodies were preincubated prior to immunochromatography, whereas in a one‐step test the labelled antibodies were integrated in the test pad and activated by addition of the sample solution. In the presence of SEE in a sample solution, colour development at the antibody‐coated zone occured within 20 min. The visual detection limits for SEE in buffer solution were at 5 ng ml^?1 (two‐step test) and at 10 ng ml^?1 (one‐step test), respectively. Both assays were specific for SEE and showed no cross‐reaction with serotype A, B, C, and D enterotoxins. When the two‐step test format was used for the identification of SEE in S. aureus culture broth, the detection limit was found to be 10 ng ml^?1. Culture supernatants of 10 enterotoxigenic strains of S. aureus were analysed with the immunochromatographic two‐step test and with a microtitre plate enzyme immunoassay (EIA) test kit for enterotoxins A‐E. Results obtained by the immunochromatographic test (presence or absence of SEE) were in excellent agreement with those of the microtitre EIA.     

Development of Immunochromatographic Assay for Identification of Organophosphate Pesticides in Environmental Samples

Microtiter plate enzyme linked immunoassay (ELISA) experiments in competitive format were performed utilizing polyclonal antibody for establishing a detection system for organophosphate pesticides. IC₅₀ value of and limit of detection (LOD) value was determined by standard inhibition curve and value obtained were 0.05 μgmL^?1 and 0.001 μgmL^?1, respectively. Specificity of antibody was investigated with different organophosphate pesticides. Immunochromatographic assay (ICA) experiments were also designed in competitive format by making use of immunochromatographic strip which was assembly of three main components: conjugate pad, membrane and adsorbent pad. Membrane was coated with hapten-OVA conjugate (test line) and antirabbit IgG (control line). ICA experiments were performed by employing gold-labeled antibody as a detector reagent which was applied over conjugate pad. Visual detection limit obtained from ICA was 0.5 μgmL^?1. Major advantage of strip assay was rapid result, i.e., less than 10 min. which makes it suitable for onsite applications.     

Development of a polyclonal antibody-based indirect competitive enzyme-linked immunosorbent assay to detect dufulin residue in water,soil and agricultural samples

Dufulin (DFL) is a potent, new-generation agrochemical that protects crops, such as tobacco, from viruses. Herein, for the first time, we developed a sensitive and specific polyclonal antibody-based indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) to detect DFL residues in samples. The assay showed high sensitivity and specificity to DFL, with a half maximal inhibitory concentration (IC₅₀) of 48.5?ng?ml^?1 and limit of detection (IC₂₀) of 2.5?ng?ml^?1. The average recovery of DFL in different spiked samples, such as tobacco, rice, tomato, cucumber, soil and water, was in the range of 70.7–138.8%. A statistically significant correlation was observed between our developed ELISA and previously established methodology of high-performance liquid chromatography with a diode array detector. These results indicate our assay is a potentially useful analytical tool for rapid detection of DFL residue in actual samples.     

Polyclonal antibody responses against Listeria monocytogenes 4b surface proteins in asymptomatic human carriers

Listeria monocytogenes 4b surface protein extract was used in an immunoblot assay to analyze the antibody profile in sera from 40 healthy urban workers (U group), 40 healthy slaughterhouse workers (W group) and four healthy carriers with positive L. monocytogenes 4b feces culture (positive controls). In addition, pooled rabbit sera, before and after immunization with L. monocytogenes 4b whole‐cell suspension, were analyzed against L. monocytogenes 4b surface protein extract in order to determine the specific L. monocytogenes 4b antibody pattern. The degree of similarity (S) between such a pattern and each of those obtained with serum samples from the three subject groups was assessed. For U and W group sera, mean S values were 24.3 ± 13.5 and 32.8 ± 14.3, respectively. An S value greater than 65, corresponding to mean S_U value ± 3 standard deviation, was considered as an indicator of a healthy carrier. Thus, the estimated healthy carrier percentages found in U and W groups were 2.5 and 5%, respectively. The proposed immunoblot assay may prove a useful tool for epidemiological surveys to determine whether a healthy person is a L. monocytogenes 4b carrier.     

Occurrence of potentially pathogenic vibrios in final effluents of a wastewater treatment facility in a rural community of the Eastern Cape Province of South Africa

We assessed the occurrence of Vibrio pathogens in the final effluents of a rural wastewater treatment facility in the Eastern Cape Province of South Africa as free or plankton-associated (180 μm, 60 μm and 20 μm plankton sizes) entities using standard culture-based and molecular techniques. The free-living Vibrio densities varied from 0 to 3.45 × 10¹ cfu ml^?1, while the plankton-associated Vibrio densities vary with plankton sizes as follows: 180 μm (0–4.50 × 10³ cfu ml^?1); 60 μm (0–4.86 × 10³ cfu ml^?1); 20 μm (0–1.9 × 10⁵ cfu ml^?1). The seasonal variations in the Vibrio densities in the 180 and 60 μm plankton size samples were significant (p < 0.05), while the 20 μm plankton size and free-living Vibrio densities were not. Molecular confirmation of the presumptive vibrios isolates revealed fluvialis (36.5%), as the predominant species, followed by Vibrio vulnificus (34.6%), and Vibrio parahaemolyticus (23.1%); only API 20NE was employed to detect Vibrio metschnikovii (5.8%), suggesting a high incidence of pathogenic Vibrio species in the final effluent of the rural wastewater facility. Analysis suggested that the concentration of Vibrio species correlated negatively with salinity and temperature (p < 0.001 and p < 0.002 respectively) as well as with pH and turbidity (p < 0.001) in the final effluent. We conclude that rural wastewater treatment facilities in the Eastern Cape Province of South Africa are potential sources of Vibrio pathogens in the aquatic environment of the communities.     

A sandwich ELISA for the detection of Wnt5a

Wnt5a is a noncanonical member of the Wnt family of signaling molecules that has been linked to various physiological and pathological processes including cell differentiation, cell migration, cell growth, vascular remodeling, cancer and chronic inflammation. To understand the role of Wnt5a in these processes, it is necessary to determine the function and expression level of Wnt5a. In this study we developed a sensitive and specific sandwich enzyme-linked immunosorbent assay (ELISA) for detecting Wnt5a. We found that a rabbit anti-human Wnt5a is a suitable capture antibody for establishing a sandwich ELISA. We used two systems to detect Wnt5a: (1) goat anti-mouse Wnt5a and horseradish peroxidase (HRP) conjugated F(ab′)₂ donkey anti-goat IgG as detection and enzyme-linked antibodies respectively, or (2) biotinylated goat anti-mouse Wnt5a and HRP–streptavidin as detection antibody and enzyme-linked avidin respectively. A sandwich ELISA using either of these systems failed to detect recombinant mouse (rm)-Wnt5a diluted in Hank's balanced salt solution supplemented with Ca²⁺ and Mg²⁺ and 1% bovine serum albumin (HBBS+, 1% BSA). Addition of polyethylene glycol (PEG) to the HBBS+, buffer during the binding stage of rm-Wnt5a, afforded the detection of rm-Wnt5a. The use of PEG during both the binding of rm-Wnt5a and detection antibody stages of the assay yielded the maximum signal for rm-Wnt5a. The relationship between the ELISA signal and concentration of Wnt5a was linear with an R² of 0.9934. In summary, we have developed a specific and sensitive sandwich ELISA that detects rm-Wnt5a.     

Identification of a Novel Protein Adjuvant Isolated from Abrus precatorius

Agglutinin and toxin was isolated and purified from Abrus precatorius seeds using conventional biochemical techniques such as Sepharose 4B affinity chromatography, Sephadex G-100 gel filtration and analytical HPLC chromatography. The yield of purified agglutinin was 272 mg per 50 g of seeds which was composed of 8.0% agglutinin and 6.1% toxin. SDS gel electrophoresis of purified protein under reducing and non-reducing conditions of native and denatured protein revealed four bands with molecular weights ranging from 31 to 66 kDa by protein and glycoprotein staining procedures. The adjuvant property of abrus agglutinin was evaluated using rat as an animal model, in comparison with Freund's complete adjuvant. Ovalbumin was used as a test antigen and was injected along with Freund's complete adjuvant (Group I experimental animals) and along with denatured abrus protein (Group II experimental animals). The humoral response was compared between two groups of animals (Group I and II) for total (by sandwich ELISA) and specific response (by antibody capture assay). Group II animals which received denatured abrus protein along with test antigen showed a significantly higher IgG response of 23.0 ± 2.37 mg ml^?1 (p < 0.001) when compared to Group I animals (14.0 ± 2.50 mg ml^?1). However, there was no statistically significant difference between Group I and Group II, with respect to specific IgG response to test antigen. Thus, the present investigation suggests that the denatured abrus agglutinin can be used as an effective alternative protein adjuvant.     

Single-chain Fv antibody with specificity for Listeria monocytogenes

Single chain antibodies (scFv) exhibiting specific binding to Listeria monocytogenes strains were isolated from a pool of random scFvs expressed on the surface of filamentous bacteriophages. Positive selection (panning) using L. monocytogenes was used to enrich for phage clones with the desired binding affinity, and negative selection using L. innocua and L. ivanovii was used to remove phages expressing cross-reactive antibody fragments. A single phage clone, P4:A8, was selected using two independent panning schemes. A rapid assay was devised to determine phage antibody binding specificity and was used to develop a selectivity profile for individual phage clones. The P4:A8 clone was screened against a panel of bacteria consisting of eight strains of L. monocytogenes, one each of the other six species of Listeria and nine other relevant bacterial species. A collection of individual clones from the penultimate panning was also screened against a subset of the panel of bacteria. The selectivity profiles indicate that multiple clones, including P4:A8, exhibit binding to one or more strains of L. monocytogenes without cross-reactivity toward any other species in the panel. This is the first report of a species-specific antibody for viable cells of L. monocytogenes (i.e., the ability to bind to L. monocytogenes without cross-reactivity toward any other species of Listeria).     

Detection of aflatoxins,trichothecenes, ochratoxin a and zearalenone by test strip enzyme immunoassay: A rapid method for screening cereals for mycotoxins

The development of test strip enzyme immunoassays (EIA) for the rapid detection of a number of mycotoxins is described. Monoclonal or polyclonal antibodies against aflatoxin B₁ (AFB₁), aflatoxin M₁ (AFM₁), ochratoxin A (OA), T‐2 toxin, diacetoxyscirpenol (DAS), 3‐acetyldeoxynivalenol (3‐AcDON), roridin A (RA) and zearalenone (ZEA) were immobilized on a nylon membrane. Using the corresponding toxin‐horseradish peroxidase conjugate in a direct competitive assay, dot colour development of toxinpositive test strips was visually discernible from that of the negative control. The results of the visual evaluation were compared with that of instrumental reflectance measurements of the test strips. The visual detection limits of the test strip assays for mycotoxins were at 0.6 ng ml^?1 (AFB₁) 0.2 ng ml^?1 (AFM₁), 2.0 ng ml^?1 (OA), 1.0 ng ml^?1(T‐2 toxin), 0.2 ng ml^?1 (DAS), 10.0 ng ml^?1 (3‐AcDON), 15.0 ng ml^?1 (RA), and 5.0 ppb (ZEA) respectively. Utilizing a simple extraction procedure, AFB₁, OA, T‐2 toxin, and ZEA in spiked corn samples were detected by test strip EIA at levels of 15 ng g^?1, 100 ng g^?1, 20 ng g^?1 and 80 ng g^?1 respectively.     

Determination of DDT in model samples by using solid-phase extraction and indirect competitive enzyme immunoassay

Two-step indirect competitive enzyme immunoassay of DDT has been developed. The limit of detection and limit of quantification were 0.3 nmol l^?1 and 4.2 nmol l^?1, respectively. To shorten the analysis time, the one-step enzyme immunoassay has been developed by replacing anti-DDT antibody with the complex anti-DDT antibody-HRP. The anti-DDT antibody-HRP conjugate was prepared by periodate method. A concentration of the preparation was 1.9?mg·ml^?1 and molar ratio Px/IgG was 1.87. The detection limit of the one-step ELISA reached 0.3?nmol l^?1, but the sensitivity of assay was very poor. The two-step enzyme immunoassay of DDT has been adapted for the chemiluminescent detection of the complex antigen-antibody. The detectability of chemiluminescent ELISA was comparable with that of chromogenic ELISA. A limited number of the artificially contamined samples have been tested. All samples were prepared by solid-phase extraction by using the commercial Agilent Zorbax SPE C₁₈ columns. A background was observed in most tested samples. The recoveries obtained from the analysis of DDT spikes reached values between 89.0–161.0%, and 81.5–111.6% at standard addition 50 μg·kg^?1, and 200 μg·kg^?1, respectively. The overestimated amounts of DDT were determined in spinach and nectarines at standard addition 50 μg·kg^?1.     

A SPECIFIC AND ULTRASENSITIVE CHEMILUMINESCENT SANDWICH ELISA TEST FOR THE DETECTION AND QUANTITATION OF PNEUMOLYSIN

A chemiluminescent sandwich ELISA test has been developed for the detection and quantitation of pneumolysin. The test is based on a mouse monoclonal as the capture antibody and on rabbit polyclonal IgGs as detection antibodies, in combination with an anti-rabbit IgG alkaline phosphatase conjugate. The estimated detection limit of the purified recombinant toxin in phosphate-buffered saline with 0.05% Triton X-100 is around 5 pg ml^?1, with averaged intra- and inter-assay variation coefficients of 7% and 13.5%, respectively.

The assay has been applied to the quantitation of pneumolysin in pneumococcal isolates, providing, for the first time, a direct measurement of the amount of the toxin produced by different strains; a variation has been found in their pneumolysin content. The test is highly specific as no other purified toxins or human pneumonia- or meningitis-associated bacteria yielded false-positive results. This specific and highly sensitive method could help in the diagnosis of human infections.