Detection of live and heat‐treated Salmonella enteritidis by a D1‐serospecific anti‐lipopolysaccharide O‐9 monoclonal antibody

A murine monoclonal antibody 2F11 (IgG_2a) against Salmonella enteritidis was produced by a fusion of P3X63 Ag8.653 myeloma cells with splenocytes of a mouse immunized with heat‐attenuated (80°C, 20 min) S. enteritidis cells. The specificity of this antibody was tested in an indirect ELISA and sodium dodecyl sulphate polyacrylamide gel electrophoresis followed by immunoblotting. The monoclonal antibody was specific to D₁‐serogroup Salmonella, exhibiting the highest reactivity with all tested phage types of S. enteritidis (1, 4, 8, 13, and 13a). The monoclonal antibody was reactive with heat‐attenuated, as well as live S. enteritidis cells. In addition, this antibody exhibited high and equal avidity to lipopolysaccharides isolated from S. enteritidis, regardless of phage types. The monoclonal antibody 2F11 proved to be specific to lipopolysaccharide O‐9 present in D₁‐serogroup Salmonella. Immunoblotting and ELISA results demonstrated that the epitope recognized by this antibody was partially composed of tyvelose and mannose. It was also determined by the nature of glycosidic bonds between monosaccharides in the polysaccharide backbone region. Employing poly‐L‐lysine precoated microplates, this antibody exhibited the detection limit of 10⁴ S. enteritidis cells ml^‐1 of buffer, as assessed by ELISA.     

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.     

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.     

Comparison of immunofluorescence method with histochemical and ELISA methods focusing on wheat protein detection in meat products

Gliadin is a major allergen causing allergies occurring also in meat products. Since wheat protein is used as a meat substitute to reduce cost of meat products. Sensitive consumers of these products are really threatened by food allergies in different allergic reaction. The objective of the study was to compare the histochemical, immunochemical (ALERT gliadin screening test) and immunofluorescence methods for the detection of wheat protein in model meat samples and meat products. The limit of detection for the ALERT gliadin screening test was 10?×?10⁴?mg?kg^?1 of addition, while the histochemical method demonstrated concentration of wheat protein already from 10?×?10³?mg?kg^?1, and the immunofluorescence method from the concentration of 20?mg?kg^?1. Comparison of the methods using McNemar’s test shows a statistically highly significant difference (p?=?.01) between the immunofluorescence method and ELISA and a statistically highly significant difference (p?=?.01) between the immunofluorescence and histochemical methods.     

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.     

Strip ELISA for detection of staphylococcal enterotoxins in culture supernatants and foods

For the easy and rapid detection of staphylococcal enterotoxins (SE) A, B, C₁, D and E, a sandwich ELISA (enzyme‐linked immunosorbent assay) was developed using an activated nylon membrane with covalently bound antibodies as a solid phase. Strips coated with antibodies against one SE type were incubated with culture supernatants of strains of Staphylococcus aureus as well as with the extracts of food previously contaminated with SE. The assay of bound toxin was performed using horseradish peroxidase labelled antibodies which were partly purified by affinity chromatography. After addition of 3,3’,5,5'‐tetramethylbenzidine, a distinct blue dot developed in the case of positive samples. The detection limit for individual SE is in the range of 0.5 ng ml^‐1 to 1 ng ml^‐1. A semi‐quantitative evaluation is possible without any special reading equipment. The test can be done within 2 h, sample preparation excluded. While milk needed no extraction, in the case of minced meat or noodles, homogenization with 1.5 or 2 volumes of phosphatebuffered saline followed by centrifugation for 20 min at 10 000 × g and 4° C proved to be adequate. Interferences by constituents of the food extracts were not observed. The results showed good agreement with those of the microtiter plate ELISA and also with those of a commercially available SE ELISA kit.     

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.     

New developments in rapid microbiology using immunoassays

Rapid microbial screening tests are slowly being accepted by food microbiologists. The ability to obtain test results while still maintaining the test's reliability would be an important benefit to food companies. However, even rapid tests require enrichment of the target bacteria to the level of the assay's detection limit. The bacteria may be sublethally injured by heating, and they may also be outnumbered by competitive bacteria. The complete enrichment procedure therefore comprises a number of steps and takes several days. Because this procedure is crucial for the success of any microbial screening method, alterations in the protocol should only be made with the utmost caution. In this study, selective enrichment is replaced by fast immunomagnetic separation (IMS) of Salmonella and its competitors. Detection of Salmonella in the enriched culture is subsequently conducted using a rapid enzyme immunoassay (EIA). If the Salmonella concentration is higher than 5000 bacteria ml^?1 after pre‐enrichment, the combination of IMS and EIA can detect the presence of Salmonella in 24 h.     

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.     

How should a district general hospital immunology service screen for anti-nuclear antibodies? An ‘in-the-field’ audit

Anti‐nuclear antibody (ANA) testing assists in the diagnosis of several immune‐mediated disorders. The gold standard method for detection of these antibodies is by indirect immunofluorescence testing on human epidermoid laryngeal carcinoma (HEp‐2) cells. However, many laboratories test for these antibodies using solid‐phase assays such as enzyme‐linked immunosorbent assay (ELISA), which allows for higher throughput testing at reduced cost. In this study, we have audited the performance of a previously established ELISA assay to screen for ANA, making comparison with the gold standard HEp‐2 immunofluorescence test. A prospective and unselected sample of 89 consecutive ANA test requests by consultant rheumatologists were evaluated in parallel over a period of 10 months using both tests. ELISA and HEp‐2 screening assays yielded 40 (45%) and 72 (81%) positive test results, respectively, demonstrating lack of concordance between test methods. Using standard and clinical samples, it was demonstrated that the ELISA method did not detect several ANA with nucleolar, homogeneous and speckled immunofluorescence patterns. None of these ELISA^NEG HEp‐2^POS ANA were reactive with a panel of six extractable nuclear antigens or with double‐stranded DNA. Nonetheless, 13 of these samples (15%) originated from patients with recognized ANA‐associated disease (n = 7) or Raynaud's phenomenon (n = 6). We conclude that ELISA screening may fail to detect clinically relevant ANA that lack defined specificity for antigen.     

Production of antibodies to pseudomonas solanacearum the causative agent of bacterial wilt

Both polyclonal and monoclonal antibodies have been developed to Pseudomonas solanacearum, the causative agent of bacterial wilt. When used in an indirect enzyme‐linked immunosorbent assay, the polyclonal antibodies detected as few as 1 X 10⁴ bacterial cells ml^‐1 in infected plant or soil samples, but they could not distinguish between P. solanacearum, P. celebensis, P. syzygii and P. picketti. The monoclonal antibodies developed had differing specificities, but, using selective immunization schedules, several were obtained which no longer cross‐reacted with closely related bacteria. These monoclonal antibodies were not as sensitive as the polyclonal antibodies and were only able to detect down to 1 X 10⁶ cells ml^‐1.     

A NEW MODEL ELISA,BASED ON TWO MONOCLONAL ANTIBODIES,FOR QUANTIFICATION OF FATTY ACID SYNTHASE

ABSTRACT

A new model ELISA, based on two monoclonal antibodies, was developed for the quantification of fatty acid synthase (FAS). In this sandwich assay, a monoclonal antibody M6 was used as a capture on Nunc MaxiSorp ELISA/EIA Modules and another monoclonal antibody M3, labeled with biotin, was used as a detection antibody. More than 10 molecules of biotin were labeled on the anti-FAS monoclonal antibody using modified biotinylation conditions. The within- and between-run CVs were less than 10%, and the detection limit was 3.22 ng/mL. Recoveries were 98.54–121.95%, averaging 106.05%. The average FAS concentration obtained from the total 55 healthy volunteers blood was 4.07 ± 1.81 ng/mL, 4.25 ± 2.14 ng/mL in women (n = 37) and 3.70 ± 0.74 ng/mL in men (n = 18). When compared with the previously developed polyclonal–monoclonal ELISA, a different pattern of FAS levels was observed in the supernatant of two cultured breast cancer cell lines in a time course study and there was no linear correlation between the two assays using 215 human blood samples. Thus, this new model FAS–ELISA could be used as an independent assay in measuring clinical samples. In summary, this monoclonal–monoclonal FAS–ELISA is sensitive, accurate, and precise in quantification of fatty acid synthase and has potential as a complementary tool in testing clinical samples.     

Monoclonal antibody for the development of specific immunoassays to detect Enrofloxacin in foods of animal origin

The carbodiimide active ester method was employed to synthesise the antigen of 1-cyclopropyl-7-(4-ethylpiperazin-1-yl)-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid [Enrofloxacin (ENFX)], and male Balb/c mice were used to produce anti-ENFX monoclonal antibody. Based on the checkerboard titration, an indirect competitive enzyme-linked immunosorbent assay standard curve was established. This assay was sensitive and highly specific to ENFX with the half maximal inhibitory concentration and limit of detection values of 0.15?ng?mL^?1 and 0.028ng?mL^?1, respectively. Similarly, the antibody had a high affinity as seen in A_max values. Spiked cow milk beef liver and fish samples analysed using this kit had recoveries in the range of 90.2–110.2%, 70.7–81.5% and 89.1–101%, respectively, showing satisfactory results. The results suggest that this ELISA kit could be applied as a screening method to detect and control the illegal content of ENFX in food products.     

A comparison of time-resolved fluoroimmunoassay and ELISA in the detection of casein in foodstuffs

With the aim of lowering the detection limit for casein in foods, three competitive assays are described: direct time-resolved fluoroimmunoassay (TR-FIA), using europium-conjugated antibody, indirect TR-FIA, using biotinylated antibody with europium-conjugated streptavidin and ELISA, using a HRP-conjugated secondary antibody. Food samples (instant potato, flour mix, packet soup, spice-mix) were analysed. Standard curve sensitivities in direct and indirect TR-FIAs did not differ significantly (p=0.097), but both TR-FIAs were considerably less sensitive (both p<0.0001) than ELISA (LOQs 1.3, 1.5 and?<?1.0 mg kg^?1, respectively). The precision and working analyte range was similar in all three methods. Casein content measured in food products was comparable, using the three assays and rocket immunoelectrophoresis. The TR-FIA approach provided no improvement over the ELISA. All three assays allowed quantification of casein in foodstuffs in the order of 1–1.5 mg kg^?1, providing a basis for more rigorous validation and collaborative testing.     

High Prevalence of Human Anti‐bovine IgG Antibodies as the Major Cause of False Positive Reactions in Two‐Site Immunoassays Based on Monoclonal Antibodies

Abstract

A sandwich ELISA for quantification of the endometrial protein PP14 revealed false positive reactions in 81% of male sera (n?=?54). The PP14 ELISA was based on two monoclonal antibodies (Mabs) with different epitope specificities—a catcher and a biotinylated indicator. The monoclonal antibodies were purified by protein G affinity chromatography from culture supernatant containing 10% (v/v) fetal calf serum (FCS). Human anti‐animal IgG (bovine, mouse, horse, and swine) antibodies and human anti‐bovine serum albumin antibodies were measured using an ELISA design, with direct bridging of the solid phase and biotinylated antigens. The false positive reactions were abolished by addition of 1% (v/v) bovine serum to the dilution buffer (DB). Human anti‐bovine IgG antibodies (HABIA) were detected in 99 out of 104 sera from blood donors (50 females; 54 males). HABIA levels in male sera (n?=?54) were positively correlated to the false positive signals in the PP14 ELISA (r?=?0.923; p?<?0.0001). Antibodies to IgG from other mammalian species (mouse, horse, and swine) were also detected in the donor sera, but levels and frequencies were lower compared to that of HABIA. Furthermore, HABIA were positively correlated to human anti‐bovine serum albumin antibodies in the donor sera (r?=?0.639; p?<?0.0001; n?=?103). HABIA (prevalence 95%) cause false positive reactions due to crossbinding of contaminating bovine IgG and/or crossreaction with mouse IgG in two‐site immunoassays. The apparent presence of human anti‐mouse IgG antibodies (HAMA), described to create false positive results, may be due to a crossreacting fraction of the polyclonal circulating antibodies against bovine IgG.     

Development of an ELISA for detection of Sudan I in food samples using monoclonal antibody

A highly sensitive and specific indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) has been developed using a new monoclonal antibody for detecting the food colourant Sudan I. The half-maximum inhibition concentrations (IC₅₀) and the limit of detection (calculated as IC₂₀) of ELISA for Sudan I were 2 and 0.01 ng mL^?1, respectively. The study showed little cross-activity with Sudan I structural analogues (below 0.01%). The average recoveries in intra- and interassays for Sudan I from fortified fresh tomato and chilli samples by ELISA were in ranges of 82–94% and 79–91%, respectively. The coefficients of variation of intra- and interassays were 6–8% and 6–10%, respectively. The IC₅₀ was approximately 2.0 ng mL^?1 after the Sudan I-Kit had been kept for 180 days.     

Immunophenotype of peripheral blood natural killer cells and IL‐10 serum levels in relation to Helicobacter pylori status

Recent findings suggest that NK (Natural Killer) cells may directly modulate the antimicrobial immune responses. In this study, we performed immunophenotypic analysis of peripheral blood NK cells with regard to CD56, CD16, Nkp46, and CD25 markers, as well as IL‐10 levels quantification in the sera samples of asymptomatic, H. pylori (Hp)‐infected or uninfected individuals, and combined these results with our previous findings on lymphocyte cytotoxic activity. Twenty healthy volunteers [10 Hp(?);10 Hp(+)] were included in the study. The percentages of classic lymphocytes (CD3⁺) and NK cells (CD3^?CD56⁺, CD3^?Nkp46⁺, CD3^?CD16⁺) with or without CD25 receptor were evaluated by fluorochrome‐conjugated monoclonal antibody staining and flow cytometry analysis. IL‐10 quantification was performed by enzyme‐linked immunosorbent assay‐ELISA. Our study showed elevated levels of IL‐10 and higher NK cell numbers of both CD3^?CD56⁺CD25⁺ and CD3^?Nkp46⁺CD25⁺ phenotypes, as well as CD3⁺CD25⁺ classic lymphocytes in Hp(+) compared with Hp(?) individuals. No differences between Hp(?) and Hp(+) individuals were found either in total number of classic lymphocytes or NK cell subtypes. Our data suggest that in Hp(+) donors, there is a domination of lymphocytes and NK cells co‐expressing CD25 marker, which might be influenced by the regulatory IL‐10. This phenomenon may be a result of H. pylori adaptation to a changing environment in vivo leading to a chronic infection and lack of severe gastric pathologies.     

The Complement—Fixing Activity of Immune Complexes Containing IgG Antibodies of Different Functional Affinities: Effects on Superoxide Production by Rabbit Neutrophils

When neutrophil phagocytes are stimulated by IgG containing immune complexes (IgG‐IC), with or without the participation of the complement system, they show a sharp increase in oxygen uptake and begin to release large quantities of superoxide anions (O₂^?) and hydrogen peroxide (H₂O₂) into the surrounding medium. The aim of the present investigation was to provide insights into the production and release of O₂^? by rabbit neutrophils activated with immune complexes (IC) containing IgG antibodies of different functional affinity, opsonized and not opsonized by complement system components. For this purpose, two populations of polyclonal anti‐ovalbumin (OVA) IgG antibodies with different functional affinity, 5 × 10⁸ M^{? 1} and 2 × 10⁷ M^{? 1}, were prepared. The production of O₂^? was measured spectrophotometrically by a method using the superoxide dismutase‐inhibited reduction of ferricytochrome C to the ferrous form. The activation of complement by different IgG‐IC was determined by estimating the total residual haemolytic activity of the alternative and classical pathways in sera treated with different concentrations of anti‐OVA IgG/OVA immune complexes formed at equivalence. The results showed that: 1) antibody functional affinity influenced O₂^? production and the complement‐fixing activity induced by the IC. In general, the higher functional affinity antibodies were more efficient in stimulating the respiratory burst of neutrophils and in activating complement by the classical and alternative pathways than the lower functional affinity antibodies at all IC concentrations tested; 2) complement components incorporated into the immune complex lattice caused an increase in the stimulatory activity of both IgG antibodies to produce O₂^? (? 15% for the IC of IgG with K_a = 5 × 10⁸ M^{? 1} and ? 7% for the IC of IgG with K_a = 2 × 10⁷ M^{? 1}). This effect was dependent on antibody affinity and concentration; 3) there was a direct relationship between the overall level of complement activation, antibody affinity and superoxide production by neutrophils. Thus, we conclude that antibody affinity influences immune complex lattice formation, modulating its three‐dimensional structure and the disposition of Fc fragments interfering with the antibody's biological properties. These results can help understand the precise role of antibody functional affinity in antigen‐antibody complex diseases and define the immunochemical characteristics of pathogenic complexes.     

Rapid monitoring of dicyclohexyl phthalate in foods using the direct competitive ELISA

A direct competitive enzyme-linked immunosorbent assay (dc-ELISA) has been developed for the quantitative detection of the dicyclohexyl phthalate (DCHP) in some liquid food. Specific polyclonal antisera to DCHP was raised, using the hapten-bovine serum albumin conjugates as the immunogen. The conjugate of horseradish peroxidase (HRP) with antibody was used as the detectable probe to provide the direct measurement of the antigen and analyte, which was synthesised by a modified glutaraldehyde method. Under the optimised assay, the quantitative working range was from 0.1 to 100 ng mL^?1 (R ²=0.9989), with a limit of detection (LOD) of 0.03 ng mL^?1, and a recovery of 96.6–112.4%. The specificity and accuracy of developed method were evaluated. The cross-reactivities of antibody with structurally related phthalate esters were less than 10%. Results obtained indicated that the dc-ELISA was a sensitive, low expense method to improve the routine monitoring of trace constituents in some liquid food.     

Production of a monoclonal antibody with specificity for deoxynivalenol, 3‐acetyldeoxynivalenol and 15‐acetyldeoxynivalenol

Monoclonal antibodies reactive with deoxynivalenol were generated following the immunization of mice with a deoxynivalenol‐mouse serum albumin conjugate. One of the anti‐deoxynivalenol monoclonal antibodies, designated C6–1, exhibited cross‐reactivity with 3‐acetyldeoxynivalenol and 15‐acetyldeoxynivalenol but not with nivalenol, T‐2 tetraol or scirpentriol. An indirect competitive ELISA based on this monoclonal antibody gave 50% inhibition values of 0–6 μg ml^‐1 for deoxynivalenol, 0–2 μg ml^‐1 for 15‐acetyldeoxynivalenol and 10 μg ml^‐1 for 3‐acetyldeoxynivalenol.