Development of an Immunoassay for Detection of Staphylococcal Enterotoxin-Like J, A Non-Characterized Toxin

Staphylococcal enterotoxins (SEs) are the cause of staphylococcal food poisoning (SFP) outbreaks. Recently, many new types of SEs and SE-like toxins have been reported, but it has not been proved whether these new toxins cause food poisoning. To develop an immunoassay for detection of SE-like J (SElJ), a non-characterized toxin in SFP, a mutant SElJ with C-terminus deletion (SElJ∆C) was expressed and purified in an E. coli expression system. Anti-SElJ antibody was produced in rabbits immunized with the SElJ∆C. Western blotting and sandwich enzyme-linked immunosorbent assay (ELISA) detection systems were established and showed that the antibody specifically recognizes SElJ without cross reaction to other SEs tested. The limit of detection for the sandwich ELISA was 0.078 ng/mL, showing high sensitivity. SElJ production in S. aureus was detected by using the sandwich ELISA and showed that selj-horboring isolates produced a large amount of SElJ in the culture supernatants, especially in that of the strain isolated from a food poisoning outbreak in Japan. These results demonstrate that the immunoassay for detection of SElJ is specific and sensitive and is useful for determining the native SElJ production in S. aureus isolated from food poisoning cases.

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Development of an Indirect Competitive Enzyme-Linked Immunosorbent Assay Based on the Multiepitope Peptide for the Synchronous Detection of Staphylococcal Enterotoxin A and G Proteins in Milk

Journal of Food Protection ◽

10.4315/0362-028x.jfp-14-323 ◽

2015 ◽

Vol 78 (2) ◽

pp. 362-369 ◽

Cited By ~ 9

Author(s):

MINGYAN LIANG ◽

TINGTING ZHANG ◽

XUELAN LIU ◽

YANAN FAN ◽

SHENGLIN XIA ◽

...

Keyword(s):

Immunosorbent Assay ◽

Limit Of Detection ◽

Signal To Noise Ratio ◽

Food Poisoning ◽

High Sensitivity ◽

Cross Reactivity ◽

Enzyme Linked Immunosorbent Assay ◽

Synchronous Detection ◽

Standard Curve ◽

Stability And Accuracy

Staphylococcal food poisoning (SFP), one of the most common foodborne diseases, results from ingestion of staphylococcal enterotoxins (SEs) in foods. In our previous studies, we found that SEA and SEG were two predominant SE proteins produced by milk-acquired S. aureus isolates. Here, a tandemly arranged multiepitope peptide (named SEAGepis) was designed with six linear B-cell epitopes derived from SEA or SEG and was heterologously expressed. The SEAGepis-specific antibody was prepared by immunizing rabbit with rSEAGepis. Then, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) based on rSEAGepis and the corresponding antibody was developed to simultaneously detect SEA and SEG. Under the optimized conditions, the ic-ELISA standard curve for rSEAGepis was constructed in the concentration range of 0.5 to 512 ng/ml, and the average coefficients of variation of intra-and interassay were 4.28 and 5.61% during six standard concentrations. The average half-maximal inhibitory concentration was 5.07 ng/ml, and the limit of detection at a signal-to-noise ratio of 3 was 0.52 ng/ml. The anti-rSEAGepis antibody displayed over 90% cross-reactivity with SEA and SEG but less than 0.5% cross-reactivity with other enterotoxins. Artificially contaminated milk with different concentrations of rSEAGepis, SEA, and SEG was detected by the established ic-ELISA; the recoveries of rSEAGepis, SEA, and SEG were 91.1 to 157.5%, 90.3 to 134.5%, and 89.1 to 117.5%, respectively, with a coefficient of variation below 12%. These results demonstrated that the newly established ic-ELISA possessed high sensitivity, specificity, stability, and accuracy and could potentially be a useful analytical method for synchronous detection of SEA and SEG in milk.

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Rationally Designed Synthetic Haptens to Generate Anti-Ciguatoxin Monoclonal Antibodies, and Development of a Practical Sandwich ELISA to Detect Ciguatoxins

Toxins ◽

10.3390/toxins11090533 ◽

2019 ◽

Vol 11 (9) ◽

pp. 533 ◽

Cited By ~ 4

Author(s):

Takeshi Tsumuraya ◽

Masahiro Hirama

Keyword(s):

Monoclonal Antibodies ◽

Limit Of Detection ◽

Sandwich Elisa ◽

Food Poisoning ◽

Enzyme Linked Immunosorbent Assay ◽

Fda Guidance ◽

Fish Poisoning ◽

Highly Sensitive ◽

Taste And Smell ◽

Reliable Methods

“Ciguatera” fish poisoning (CFP) is one of the well-known food poisoning caused by the ingestion of fish that have accumulated trace amounts of ciguatoxins (CTXs). CFP affects more than 50,000 individuals annually. The difficulty in preventing CFP comes from the lack of reliable methods for analysis of CTXs in contaminated fish, together with the normal appearance, taste, and smell of CTX-contaminated fish. Thus, a sensitive, accurate, routine, and portable analytical method to detect CTXs is urgently required. Monoclonal antibodies (mAbs) specific against either wing of major CTX congeners (CTX1B, 54-deoxyCTX1B, CTX3C, and 51-hydroxyCTX3C) were generated by immunizing mice with rationally designed synthetic haptens-KLH conjugates instead of the CTXs. Haptenic groups with a surface area greater than 400 Å2 are required to produce mAbs that can strongly bind to CTXs. Furthermore, a highly sensitive fluorescence-based sandwich enzyme-linked immunosorbent assay (ELISA) was developed. This protocol can detect and quantify four major CTX congeners (CTX1B, 54-deoxyCTX1B, CTX3C, and 51-hydroxyCTX3C) with a limit of detection (LOD) of less than 1 pg/mL. The LOD determined for this sandwich ELISA is sufficient to detect CTX1B-contaminated fish at the FDA guidance level of 0.01 ppb.

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Application of Monoclonal Antibodies to Dulcitol 1-Phosphate Dehydrogenase for Rapid Detection of Salmonella

Journal of Food Protection ◽

10.4315/0362-028x-58.8.847 ◽

1995 ◽

Vol 58 (8) ◽

pp. 847-852 ◽

Cited By ~ 2

Author(s):

TAKAHISA MIYAMOTO ◽

HUAIZE TIAN ◽

KIYOSHI MATSUNO ◽

RYOJI TAKATA ◽

SHOJI HATANO

Keyword(s):

Monoclonal Antibodies ◽

Salmonella Typhimurium ◽

Magnesium Chloride ◽

Rapid Detection ◽

Limit Of Detection ◽

Sandwich Elisa ◽

Enzyme Linked Immunosorbent Assay ◽

The Novel ◽

Inoculum Level ◽

E Coli

Monoclonal antibodies raised against dulcitol 1-phosphate dehydrogenase of Salmonella typhimurium IFO 12529 were screened against 20 serotypes of Salmonella and 13 non-Salmonella bacteria. A sandwich-capture, enzyme-linked immunosorbent assay (sandwich ELISA) was developed for detection of Salmonella in food. The assay utilizes two monoclonal antibodies (DUI2 and DU28) which show no cross-reactions with non-Salmonella bacteria. The limit of detection of the sandwich ELISA was about 1 × 107 CPU/ml. After cultivation in a medium containing dulcitol at 37°C for 18 h followed by the sandwich ELISA. 1 CPU of Salmonella was detected. Although a high inoculum level of E. coli interfered with the detection of Salmonella, the interference was minimized by using a selective dulcitol-magnesium chloride-pyridinesulfonic acid medium for enrichment. The novel ELISA procedure detected Salmonella in chicken filtrates inoculated with 1.4 CPU/50 m1 and 1.3 × 107 CPU/50 ml of E. coli within 25 h.

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Dramatically Enhancing the Sensitivity of Immunoassay for Ochratoxin A Detection by Cascade-Amplifying Enzyme Loading

Toxins ◽

10.3390/toxins13110781 ◽

2021 ◽

Vol 13 (11) ◽

pp. 781

Author(s):

Zhuolin Song ◽

Lin Feng ◽

Yuankui Leng ◽

Mingzhu Huang ◽

Hao Fang ◽

...

Keyword(s):

Ochratoxin A ◽

Limit Of Detection ◽

High Sensitivity ◽

Cross Reaction ◽

Molar Ratio ◽

Quantitative Detection ◽

Enzyme Linked Immunosorbent Assay ◽

Enzyme Loading ◽

M13 Bacteriophage ◽

Mimic Peptide

Enzyme-linked immunosorbent assay (ELISA) is widely used in the routine screening of mycotoxin contamination in various agricultural and food products. Herein, a cascade-amplifying system was introduced to dramatically promote the sensitivity of an immunoassay for ochratoxin A (OTA) detection. Specifically, a biotinylated M13 bacteriophage was introduced as a biofunctional competing antigen, in which a seven-peptide OTA mimotope fused on the p3 protein of M13 was used to specifically recognize an anti-OTA monoclonal antibody, and the biotin molecules modified on capsid p8 proteins were used in loading numerous streptavidin-labeled polymeric horseradish peroxidases (HRPs). Owing to the abundance of biotinylated p8 proteins in M13 and the high molar ratio between HRP and streptavidin in streptavidin-polyHRP, the loading amount of HRP enzymes on the M13 bacteriophage were greatly boosted. Hence, the proposed method exhibited high sensitivity, with a limit of detection of 2.0 pg/mL for OTA detection, which was 250-fold lower than that of conventional ELISA. In addition, the proposed method showed a slight cross-reaction of 2.3% to OTB, a negligible cross-reaction for other common mycotoxins, and an acceptable accuracy for OTA quantitative detection in real corn samples. The practicability of the method was further confirmed with a traditional HRP-based ELISA method. In conclusion, the biotinylated bacteriophage and polyHRP structure showed potential as a cascade-amplifying enzyme loading system for ultra-trace OTA detemination, and its application can be extended to the detection of other analytes by altering specific mimic peptide sequences.

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Evaluation of an alternative S100b assay for use in cardiac surgery: relationship with microemboli and neuropsychological outcome

Perfusion ◽

10.1177/0267659107083243 ◽

2007 ◽

Vol 22 (4) ◽

pp. 267-272 ◽

Cited By ~ 10

Author(s):

D.C. Whitaker ◽

A.J.E. Green ◽

J. Stygall ◽

M.J.G. Harrison ◽

S.P. Newman

Keyword(s):

Cardiac Surgery ◽

Limit Of Detection ◽

Artery Bypass ◽

Sandwich Elisa ◽

High Sensitivity ◽

Cross Reactivity ◽

Cell Protein ◽

Skin Closure ◽

Neuropsychological Outcome ◽

The Right

Introduction. The aim of the study was to investigate the relationship between S100b release, neuropsychological outcome and cerebral microemboli. Peri-operative assay of the astroglial cell protein S100b has been used as a marker of cerebral damage after cardiac surgery but potential assay cross-reactivity has limited its specificity. The present study uses an alternative enzyme-linked immunoabsorbant assay (ELISA) for serum S100b that has documented sensitivity and specificity data in patients undergoing coronary artery bypass grafting (CABG). Methods. Fifty-five consecutive patients undergoing routine CABG surgery received serial venous S100b sampling at five time points: i) Pre-operative, ii) At the end of cardiopulmonary bypass (CPB), iii) 6 hrs, iv) 24 hrs and v) 48 hrs post skin closure. A previously described sandwich ELISA with monoclonal anti- S100b was used. This assay has a lower limit of detection of 0.04 μ g/L and < 0.006% reactivity with S100a at a concentration of 100 μg/L S100a. Cerebral microemboli during surgery were recorded by transcranial Doppler monitor over the right middle cerebral artery. Evidence of cerebral impairment was obtained by comparing patients' performance in a neuropsychological battery of 9 tests administered 6—8 weeks post-operatively with their pre-operative scores. Results. There was a significant increase in S100b only at the end of bypass (mean 0.30 μg/L, SD ± 0.33 and range .00 to 1.57). S100b levels at the end of bypass did not correlate with neuropsychological outcome or microemboli counts. Conclusions. The low levels of S100b detected using the present assay, despite its high sensitivity and despite the routine use of cardiotomy suction, suggest that the assay may have higher specificity for cerebral S100b than previously used assays. There was no evidence that this assay is related to neuropsychological change or cerebral microemboli in cardiac surgery. Perfusion (2007) 22, 267—272.

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Multiplex Detection of Salmonella spp., E. coli O157 and L. monocytogenes by qPCR Melt Curve Analysis in Spiked Infant Formula

Microorganisms ◽

10.3390/microorganisms8091359 ◽

2020 ◽

Vol 8 (9) ◽

pp. 1359

Author(s):

Sarah Azinheiro ◽

Joana Carvalho ◽

Marta Prado ◽

Alejandro Garrido-Maestu

Keyword(s):

Food Industry ◽

High Resolution Melting ◽

Limit Of Detection ◽

Food Poisoning ◽

Relative Sensitivity ◽

Multiplex Detection ◽

Salmonella Spp ◽

Melt Curve Analysis ◽

E Coli ◽

Reaction Inhibition

Food poisoning continue to be a threat in the food industry showing a need to improve the detection of the pathogen responsible for the hospitalization cases and death. DNA-based techniques represent a real advantage and allow the detection of several targets at the same time, reducing cost and time of analysis. The development of new methodology using SYBR Green qPCR for the detection of L. monocytogenes, Salmonella spp. and E. coli O157 simultaneously was developed and a non-competitive internal amplification control (NC-IAC) was implemented to detect reaction inhibition. The formulation and supplementation of the enrichment medium was also optimized to allow the growth of all pathogens. The limit of detection (LoD) 95% obtained was <1 CFU/25 g for E. coli O157, and 2 CFU/25 g for Salmonella spp. and L. monocytogenes and regarding the multiplex detection a LoD 95% of 1.7 CFU/25 g was observed. The specificity, relative sensitivity and accuracy of full methodology were 100% and the use of the NC-IAC allowed the reliability of the results without interfering with the sensitivity of the methodology. The described study proved to obtain results comparable to those of probe-based qPCR, and more economically than classical high resolution melting qPCR, being both important aspects for its implementation in the food industry.

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The Use of Chicken Igy in a Double Antibody Sandwich Elisa for the Quantification of Melittin in Bee Venom and Bee Venom Melittin Content in Cosmetics

Journal of Apicultural Science ◽

10.1515/jas-2015-0011 ◽

2015 ◽

Vol 59 (1) ◽

pp. 97-107

Author(s):

Lindsey Y. K. Suh ◽

Tayabaa Kartoon ◽

Naiyana Gujral ◽

Youngmee Yoon ◽

Joo Won Suh ◽

...

Keyword(s):

Sandwich Elisa ◽

Competitive Elisa ◽

Bee Venom ◽

Raw Material ◽

Enzyme Linked Immunosorbent Assay ◽

Western Blot Assay ◽

Detection Systems ◽

Indirect Competitive Elisa ◽

Double Antibody ◽

Das Elisa

Abstract Two enzyme-linked immunosorbent assay (ELISA) - based detection systems: indirect competitive ELISA and biotinylated double antibody sandwich ELISA (DAS-ELISA) were developed to determine the melittin concentration in honeybee (Apis mellifera) venom and the melittin concentration in cosmetics which contain bee venom. The indirect competitive ELISA employed chicken anti-melittin IgY. The biotinylated DAS-ELISA employed anti-melittin monoclonal antibody (MAb) and biotinylated anti-melittin IgY. To produce anti-melittin IgY; Sigma melittin was emulsified with Freund‘s incomplete adjuvant and immunised to Leghorn laying chickens intramuscularly at four different sites (50 μg/mL, 0.25 mL per site) of the breast muscles. After 5 to 8 weeks of the immunisation, anti-melittin IgY was extracted and analysed by ELISA. The anti-melittin IgY antibody produced was highly specific to melittin and did not cross-react with other bee venom proteins, as examined by ELISA and a western-blot assay. Indirect competitive ELISA demonstrated a higher range of melittin detection (2.5 to 80 μg/mL). Double antibody sandwich ELISA using MAb as the capture antibody and biotinylated polyclonal IgY as the detection antibody, provided a lower range of detection (2.5 - 40 ng/mL), which has a 1000 times higher sensitivity than that of indirect competitive ELISA. Therefore, indirect competitive ELISA is a useful tool to measure the concentration of melittin in bee venom as a raw material. Biotinylated DAS-ELISA, on the other hand, is more suitable for nanoscale quantification of melittin in commercial products.

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Expression and Purification of Botulinum Neurotoxin Type ‘A’ Toxin Domain in E. coli and its Application in Detection

South Asian Journal of Experimental Biology ◽

10.38150/sajeb.11(4).p449-457 ◽

2021 ◽

Vol 11 (4) ◽

pp. 449-457

Author(s):

Monika Singh Jadon ◽

Jyotsna Dhubkarya ◽

Shiv Kumar Yadav ◽

Abdhesh Kumar ◽

S. Ponmariappan

Keyword(s):

Neutralizing Antibodies ◽

Limit Of Detection ◽

Polyclonal Antibodies ◽

Sandwich Elisa ◽

Type A ◽

Simple Method ◽

Elisa System ◽

E Coli ◽

Biowarfare Agent ◽

Botulinum Toxins Type A

Botulism is a neuroparalytic disease caused by botulinum toxins type A-G. It has been listed as category ‘A’ biowarfare agent by CDC due to its extreme toxicity.. In the present study, we developed a simple method for the pro-duction and purification of recombinant toxin domain of BoNT type ‘A’ from Escherichia coli and established its application in BoNT detection. The BoNT/A LC gene was cloned in pET28b+ vector and expressed in E.coli. The recom-binant BoNT/A LC protein expression was achieved at 25°C with the induc-tion of 0.5mM IPTG at 16 h. The recombinant protein was purified under native conditions with more than 98% purity using simple affinity chroma-tography and confirmed by Mass spectrometry and Western blot analysis. Generated polyclonal antibodies and the toxin domain elicited a strong im-mune response in mice and rabbit. Optimized the Sandwich ELISA system resulted with the sensitivity of 7.5 ng/ml for the detection of BoNT/A. The limit of detection (LOD) in different food matrices were studied through spik-ing studies using rBoNT/A LC protein as antigen and achieved a detection limit ranging from7.5 ng/ml to 250 ng/ml. The developed method has the potential for the industrial production towards its application in detection, development of candidate vaccine molecule and production of neutralizing antibodies. Current study explored for detection of BoNT/A.

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Use of Bispecific Antibodies in Molecular Velcro Assays Whose Specificity Approaches the Theoretical Limit of Immunodetection for Bordetella pertussis

Clinical and Diagnostic Laboratory Immunology ◽

10.1128/cdli.11.4.752-757.2004 ◽

2004 ◽

Vol 11 (4) ◽

pp. 752-757 ◽

Cited By ~ 12

Author(s):

X. L. Tang ◽

M. S. Peppler ◽

R. T. Irvin ◽

M. R. Suresh

Keyword(s):

Bordetella Pertussis ◽

Whooping Cough ◽

Limit Of Detection ◽

Solid Phase ◽

High Sensitivity ◽

Enzyme Linked Immunosorbent Assay ◽

Nasopharyngeal Swab ◽

Theoretical Limit ◽

Benzhydroxamic Acid ◽

New Generation

ABSTRACT A bispecific monoclonal antibody (bsMAb) that detects Bordetella pertussis, the causative agent of whooping cough, and horseradish peroxidase (HRPO) has been developed by use of the quadroma technology. A quadroma, P123, was produced by fusing two well-characterized hybridomas against the bacterium and the enzyme and was subcloned to obtain a stable bsMAb-secreting cell line. The quadroma was theoretically expected to produce up to 10 different molecular species of immunoglobulins, so secreted bispecific antibody was complexed with excess HRPO and the HRPO-bsMAb complex was purified in one step by benzhydroxamic acid-agarose affinity cochromatography. An ultrasensitive homosandwich molecular “velcro” enzyme-linked immunosorbent assay for the detection of B. pertussis whole bacteria with HRPO-bsMAb was established in both microplate and nasopharyngeal swab formats. This assay demonstrates a high sensitivity that approaches the theoretical limit of detection of one bacterium. This new nanoprobe can be used to develop a new generation of assays that are simple, inexpensive alternatives to quantitative PCR and that can be used by clinical laboratories. This strategy of homosandwich assays with solid-phase monospecific antibodies and solution-phase bsMAb with specificity for the same repeating surface determinants can be applied to generate ultrasensitive immunodiagnostic assays for viruses and bacteria.

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Gamma Irradiation Can Be Used To Inactivate Bacillus anthracis Spores without Compromising the Sensitivity of Diagnostic Assays

Applied and Environmental Microbiology ◽

10.1128/aem.00557-08 ◽

2008 ◽

Vol 74 (14) ◽

pp. 4427-4433 ◽

Cited By ~ 29

Author(s):

Leslie A. Dauphin ◽

Bruce R. Newton ◽

Max V. Rasmussen ◽

Richard F. Meyer ◽

Michael D. Bowen

Keyword(s):

Gamma Irradiation ◽

Bacillus Anthracis ◽

Real Time ◽

Real Time Pcr ◽

Limit Of Detection ◽

Sandwich Elisa ◽

Pcr Analysis ◽

Enzyme Linked Immunosorbent Assay ◽

Chromosomal Dna ◽

Unit Reduction

ABSTRACT The use of Bacillus anthracis as a biological weapon in 2001 heightened awareness of the need for validated methods for the inactivation of B. anthracis spores. This study determined the gamma irradiation dose for inactivating virulent B. anthracis spores in suspension and its effects on real-time PCR and antigen detection assays. Strains representing eight genetic groups of B. anthracis were exposed to gamma radiation, and it was found that subjecting spores at a concentration of 107 CFU/ml to a dose of 2.5 × 106 rads resulted in a 6-log-unit reduction of spore viability. TaqMan real-time PCR analysis of untreated versus irradiated Ames strain (K1694) spores showed that treatment significantly enhanced the detection of B. anthracis chromosomal DNA targets but had no significant effect on the ability to detect targets on the pXO1 and pXO2 plasmids of B. anthracis. When analyzed by an enzyme-linked immunosorbent assay (ELISA), irradiation affected the detection of B. anthracis spores in a direct ELISA but had no effect on the limit of detection in a sandwich ELISA. The results of this study showed that gamma irradiation-inactivated spores can be tested by real-time PCR or sandwich ELISA without decreasing the sensitivity of either type of assay. Furthermore, the results suggest that clinical and public health laboratories which test specimens for B. anthracis could potentially incorporate gamma irradiation into sample processing protocols without compromising the sensitivity of the B. anthracis assays.

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