Rapid Detection of Salmonella spp. in Foods by Combination of a New Selective Enrichment and a Sandwich ELISA Using Two Monoclonal Antibodies against Dulcitol 1-Phosphate Dehydrogenase

1996 ◽  
Vol 59 (11) ◽  
pp. 1158-1163 ◽  
Author(s):  
HUAIZE TIAN ◽  
TAKAHISA MIYAMOTO ◽  
TAKASHI OKABE ◽  
YOICHIRO KURAMITSU ◽  
KEN-ICHI HONJOH ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Rapid Detection ◽  
Enrichment Culture ◽  
Sandwich Elisa ◽  
False Negative ◽  
Detection Sensitivity ◽  
Elisa Method ◽  
Salmonella Spp ◽  
Selective Enrichment ◽  
Raw Foods

A rapid-detection method was developed for food-borne dulcitol-positive Salmonella spp. in foods that involves a new preenrichment and selective enrichment system and a sandwich ELISA using two monoclonal antibodies against dulcitol 1-phosphate dehydrogenase. Preenrichment and selective enrichment were in Enterobacteriaceae enrichment mannitol (EEM) broth at 42°C for 6 h and in a new dulcitol-magnesium chloride-pyridinesulfonic acid brilliant green-novobiocin (DMPBN) medium at 42°C for 27 h, respectively. The cells were collected from the selective enrichment culture and suspended in 0.1 ml of 1 N NaOH for 2 min. The solution was neutralized with 0.1 ml of 2 M Tris-HCl buffer (pH 7.5) and the mixture was used as a sample for ELISA. The detection sensitivity of the ELISA was 105 CFU of Salmonella spp. per ml of culture. Competing non-Salmonella organisms in raw food did not interfere with the detection of Salmonella cells even when present at 107: 1 (non-Salmonella: Salmonella ratio) in food. Nonmotile Salmonella gallinarum was detected by the ELISA. The minimum detectable number of initial inoculum of Salmonella typhimurium was 0.69 CFU/25 g of raw chicken after the preenrichment in EEM broth and the selective enrichment in DMPBN medium. The present ELISA method required a total analysis time of 36 h including the preenrichment and selective enrichment periods. The ELISA method was compared with a conventional cultural method for the detection of Salmonella cells in 130 samples of raw foods. Of the samples tested, 16 were Salmonella-positive and 114 samples were negative by both methods. False-positive and false-negative results were not encountered.

scholarly journals Contamination of Local Laying Hen’s Egg Shell with Salmonella Serotypes

2013 ◽  
Vol 37 (1) ◽  
pp. 13-16
Author(s):  
Maysoon R. Jaffer
Keyword(s):  
Salmonella Enteritidis ◽  
Enrichment Culture ◽  
Identification System ◽  
Salmonella Spp ◽  
Isolation And Identification ◽  
Selective Enrichment ◽  
Hen’S Egg ◽  
Peptone Water ◽  
Culture Stage ◽  
Egg Shell

Fifty locally laying hen’s eggs random samples were collected from different markets of Baghdad city in order to investigate the presence of Salmonellae Spp. in shell of those eggs. The samples were collected during the period from March 2012 to May 2012.The samples were directly transferred to the Food hygiene laboratory and analyzed immediately without further storage.The isolation and identification methods include: (pre-enrichment) culture stage by peptone water then, (Selective enrichment) culture stage by selenite broth after that culturing on sold (Selective media) which was Bismuth Sulphate agar. The biotyping by using API strip according to the API 20E miniaturized identification system for Salmonella SPP.. The isolated Salmonella strains were transferred on TSI agar to undergone sereotyping at the Institute of Public Health,Baghdad,Iraq. Data revealed that 15 out of the total 50 (30%) of the eggs samples were contaminated with Salmonella spp. Salmonella typhimurium and Salmonella enteritidis were the two serotypes that have been found in this study. Nine from 15 (60%) of the isolates was belong to Salmonella enteritidis serotypes while 6 from 15 (40%) of the isolates was belong to Salmonella typhimuriumserotype.

scholarly journals Detection of Vibrio spp., Salmonella spp., and Shigella spp. among the frozen food samples employing enrichment culture technique

2016 ◽  
Vol 5 (1) ◽  
pp. 26-29 ◽  
Author(s):  
Tahmina Shammi
Keyword(s):  
Enrichment Culture ◽  
Bacterial Load ◽  
False Negative ◽  
Food Samples ◽  
Food Preservation ◽  
Salmonella Spp ◽  
Potential Health ◽  
Frozen Food ◽  
Shigella Spp ◽  
Vibrio Spp

Freezing has long been an established method for food preservation. Freezing temperature may act as a stress factor for microbial cells, transforming the cells into injured or dormant state. Upon inoculation, these debilitated cells cannot grow on solid media and hence produce false negative results. Foods contaminated with injured cells of pathogenic bacterial strains are of potential health risk. Employing enrichment cultivation technique, present study attempted to detect such injured, dormant or viable but non culturable (VBNC) cells in different frozen food samples, collected from local markets and super-shops of Dhaka metropolis. Compared to the conventional cultivation means, the enrichment procedure revealed a significant increase in bacterial burden as well as increase in the pathogenic load. A maximum of 3 log increase in case of total bacterial load while 4 log, 5 log and 2 log increase in case of Vibrio spp., Salmonella spp. and Shigella spp., consecutively were observed. These findings clearly demonstrated the presence of injured cells in frozen foods which could be lethal under normal condition thereby posing public health risk.Stamford Journal of Microbiology, Vol.5(1) 2015: 26-29

Comparative study of assays detecting circulating immune complexes and specific antibodies in patients infected with Toxocara canis

1987 ◽  
Vol 61 (3) ◽  
pp. 196-202 ◽  
Author(s):  
C. Aguila ◽  
C. Cuéllar ◽  
S. Fenoy ◽  
J. L. Guillén
Keyword(s):  
Monoclonal Antibodies ◽  
Comparative Study ◽  
Immune Complexes ◽  
Sandwich Elisa ◽  
Toxocara Canis ◽  
Circulating Immune Complexes ◽  
Soluble Antigen ◽  
Elisa Method ◽  
Active Infection ◽  
Specific Antibodies

ABSTRACTA sandwich ELISA method using previously described E/S antigen-specific monoclonal antibodies has been developed to detect circulating immune complexes in patients infected with Toxocara canis. This technique could be used for the study of the dynamics of the parasite-host relationship, as we believe the detection of immune complexes and/or soluble antigen to be an improvement over detection of antibodies only. In this parasitosis, antibodies may be present in residual levels for prolonged periods after active infection.

Evaluation of invA Diversity among Salmonella Species Suggests Why Some Commercially Available Rapid Detection Kits May Fail To Detect Multiple Salmonella Subspecies and Species

2019 ◽  
Vol 82 (4) ◽  
pp. 710-717 ◽  
Author(s):  
ARIEL J. BUEHLER ◽  
MARTIN WIEDMANN ◽  
ZEINA KASSAIFY ◽  
RACHEL A. CHENG
Keyword(s):  
Foodborne Pathogens ◽  
Rapid Detection ◽  
Limit Of Detection ◽  
False Negative ◽  
Detection Methods ◽  
Test Results ◽  
Salmonella Spp ◽  
Development And Validation ◽  
In Silico Analyses

ABSTRACTinvA is a common molecular target for Salmonella-specific detection methods and is recommended by the U.S. Food and Drug Administration Bacteriological Analytical Manual as a target for PCR confirmation of putative Salmonella isolates. Novel assays designed for the rapid detection of foodborne pathogens are often validated according to guidelines provided by validation schemes, such as the AOAC International or the International Organization for Standardization. However, these validation guidelines allow for flexibility in the validation study experimental design, which may inflate the assay's ability to detect foodborne pathogens, especially for foodborne pathogens such as Salmonella, exhibiting tremendous species diversity with >2,600 confirmed serovars. This study was conducted to (i) describe the sequence diversity of invA, across a diverse set of Salmonella serovars and (ii) evaluate the ability of two commercially available, AOAC International–validated rapid detection assays to detect a diverse collection of Salmonella spp. strains. In silico analyses identified 362 of 2,058 nucleotide sites that were variable among invA sequences from a diverse collection, representing 86 unique serovars spanning all species and subspecies. Not surprisingly, the majority of variable sites (308 of 2,058) occurred in non–Salmonella enterica subsp. enterica strains, including Salmonella bongori and the other S. enterica subspecies. In vitro testing showed that both rapid detection assays, examined here, failed to detect all Salmonella strains at 1 log above the limit of detection, with assay A failing to detect S. enterica subsp. salamae, and assay B failing to detect S. bongori. Both strains were eventually detected at 100,000 times the limit of detection. Taken together, our study highlights the need to include non–subsp. S. enterica strains in the development and validation of rapid detection methods to limit false-negative test results.HIGHLIGHTS

Application of Random Amplified Polymorphic DNA Analysis for Detection of Salmonella spp. in Foods

1998 ◽  
Vol 61 (7) ◽  
pp. 785-791 ◽  
Author(s):  
TAKAHISA MIYAMOTO ◽  
HUAI ZE TIAN ◽  
TAKASHI OKABE ◽  
SUDSAI TREVANICH ◽  
KAORU ASOH ◽  
...  
Keyword(s):  
Dna Analysis ◽  
Random Amplified Polymorphic Dna ◽  
Culture Method ◽  
Cell Number ◽  
Oligonucleotide Primer ◽  
Salmonella Spp ◽  
Initial Inoculum ◽  
Selective Enrichment ◽  
Conventional Culture ◽  
Raw Foods

The random amplified polymorphic DNA (RAPD) band patterns from 23 Salmonella spp. produced by use of an oligonucleotide primer (called du primer) designed on the basis of the N-terminal sequence of dulcitol 1-phosphate dehydrogenase (5'-GTGGTGACCCAGGATGGCCAGGTG-3') were different from those from 16 non-Salmonella spp. The bands at 460 and 700 bp were produced in all Salmonella strains tested. These RAPD fragments obtained from Salmonella typhimurium strongly hybridized with the corresponding RAPD bands from the other strains of Salmonella, but not with those from non-Salmonella spp. in Southern blot analysis. The RAPD bands were detected by ethidium bromide staining even when genomic DNA prepared from as few as 2.8 × 103 cells was used. The minimum detectable cell number in the initial inoculum of S. typhimurium was 4 × 10 −1 CFU/25 g of raw beef after the preenrichment in Enterobacteriaceae enrichment mannitol (EEM) broth for 6 h and the selective enrichment in dulcitol-magnesium chloride-pyridinesulfonic acid-brilliant green-novobiocin (DMPBN) medium for 18 h at 42°C. Seven raw foods inoculated with S. typhimurium at numbers from 4 × 10−1 to 2.6 × 102 CFU/25 g of food were positive in both the RAPD analysis and the conventional culture method.

Relative Effectiveness of Selective Plating Agars for Recovery of Salmonella Species from Selected High-Moisture Foods

1995 ◽  
Vol 78 (3) ◽  
pp. 679-690 ◽  
Author(s):  
Patricia S Sherrod ◽  
Rene Miguel Amaguana ◽  
Wallace H Andrews ◽  
Geraldine A June ◽  
Thomas S Hammack
Keyword(s):  
False Positive ◽  
Elevated Temperatures ◽  
Relative Effectiveness ◽  
False Negative ◽  
High Moisture ◽  
Salmonella Spp ◽  
Pork Sausage ◽  
Selective Enrichment ◽  
Egg Yolks ◽  
Selective Plating

Abstract The relative effectiveness of 6 selective plating media were compared for effectiveness in recovery of Salmonella spp. from selected high-moisture foods. Three new plating agars (EF-18, Rambach, and xylose lysine Tergitol-4) and 3 selective plating agars (bismuth sulfite, Hektoen enteric, and xylose lysine desoxycholate) recommended by AOAC INTERNATIONAL and the Bacteriological Analytical Manual (BAM) were compared. The agars were streaked from cultures selectively enriched in selenite cystine broth, tetrathionate broth, and Rappaport–Vassiliadis medium. The high-moisture foods studied were naturally contaminated pork sausage, chicken parts, turkey parts, and frog legs and artificially contaminated shrimp, oysters, egg yolks, and lettuce. The relative effectiveness of each selective plating agar was determined by recovery of Salmonella spp. and enumeration of false-positive and false-negative reactions. Although the new selective plating agars compared favorably with the AOAC/BAM-recom mended agars, they offered no advantage. Incubation of selective enrichment broths at elevated temperatures decreased the numbers of false-positive and falsenegative reactions for all 6 selective plating agars.

scholarly journals Preparation, Characterization and Diagnostic Valuation of Two Novel Anti-HPV16 E7 Oncoprotein Monoclonal Antibodies

Viruses ◽  
2020 ◽  
Vol 12 (3) ◽  
pp. 333
Author(s):  
Renjian Hu ◽  
Zhen Dong ◽  
Kui Zhang ◽  
Guangzhao Pan ◽  
Chongyang Li ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Detection System ◽  
Early Stage ◽  
Sandwich Elisa ◽  
Quantitative Detection ◽  
Enzyme Linked Immunosorbent Assay ◽  
Elisa Method ◽  
Cervical Cancers ◽  
E7 Oncoprotein ◽  
Oncogenic Protein

At present, the clinical detection method of human papillomavirus (HPV) is mainly based on the PCR method. However, this method can only be used to detect HPV DNA and HPV types, and cannot be used to accurately predict cervical cancer. HPV16 E7 is an oncoprotein selectively expressed in cervical cancers. In this study, we prepared an HPV16 E7-histidine (HIS) fusion oncoprotein by using a prokaryotic expression and gained several mouse anti-HPV16 E7-HIS fusion oncoprotein monoclonal antibodies (mAbs) by using hybridoma technology. Two mAbs, 69E2 (IgG2a) and 79A11 (IgM), were identified. Immunocytochemistry, immunofluorescence, immunohistochemistry, and Western blot were used to characterize the specificity of these mAbs. The sequences of the nucleotide bases and predicted amino acids of the 69E2 and 79A11 antibodies showed that they were novel antibodies. Indirect enzyme-linked immunosorbent assay (ELISA) with overlapping peptides, indirect competitive ELISA, and 3D structural modeling showed that mAbs 69E2 and 79A11 specifically bound to the three exposed peptides of the HPV16 E7 (HPV16 E749–66, HPV16 E773–85, and HPV16 E791–97). We used these two antibodies (79A11 as a capture antibody and 69E2 as a detection antibody) to establish a double-antibody sandwich ELISA based on a horseradish peroxidase (HRP)-labeled mAb and tetramethylbenzidine (TMB) detection system for quantitative detection of the HPV16 E7-HIS fusion oncoprotein, however, it was not ideal. Then we established a chemiluminescence immunoassay based on a labeled streptavidin-biotin (LSAB)-ELISA method and luminol detection system—this was sufficient for quantitative detection of the HPV16 E7-HIS fusion oncogenic protein in ng levels and was suitable for the detection of HPV16-positive cervical carcinoma tissues. Collectively, we obtained two novel mouse anti-HPV16 E7 oncoprotein mAbs and established an LSAB-lumino-dual-antibody sandwich ELISA method for the detection of the HPV16 E7-HIS fusion oncogenic protein, which might be a promising method for the diagnosis of HPV16-type cervical cancers in the early stage.

scholarly journals Efficient detection of pre-proinsulin by double antibody sandwich ELISA

2020 ◽  
Author(s):  
Zhu Zhu ◽  
Guoliang Ma ◽  
Li Wang ◽  
Han Wang ◽  
Hengfang Tang ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Human Insulin ◽  
Limit Of Detection ◽  
Sandwich Elisa ◽  
Insulin Production ◽  
Recombinant Human Insulin ◽  
Elisa Method ◽  
Efficient Detection ◽  
Sds Page ◽  
Conventional Cell

Abstract ObjectiveTo generate monoclonal antibodies against pre-proinsulin (PPI), and establish sandwich ELISA method to provide a basis for PPI detection in recombinant human insulin production.MethodsThe Balb /c mice were immunized with PPI, and the hybridomas secreting anti-PPI monoclonal antibodies were obtained by conventional cell fusion technique and ELISA screening.The antibody was purified using a Protein G gel column and identified for purity by SDS-PAGE. Pairing effect was found by the sandwich ELISA, and the specificity of the paired antibody was determined. A paired antibody with better specificity was selected to establish sandwich ELISA, and construct a quantitative curve, the accuracy and sensitivity of the method were evaluated.ResultsSix anti-PPI monoclonal antibodies were obtained, named P1, P2, P3, P4, P5 and P6, of which P5 had the highest titer. The sandwich ELISA method was established with P5 for plating and P2 for detection antibodie. The linear range of the quantitative curve of PPI by sandwich ELISA was 0. 645-82.5 pg/mL, the recovery was 89%–95%, and the limit of detection was 3.06 pg/mL.ConclusionSix monoclonal antibodies against PPI were generated and the sandwich ELISA method was established to detect PPI in process control and product release control for recombinant human insulin production.

scholarly journals Rigid monoclonal antibodies improve detection of SARS-CoV-2 nucleocapsid protein

2021 ◽  
Author(s):  
Curtis D. Hodge ◽  
Daniel J. Rosenberg ◽  
Mateusz Wilamowski ◽  
Andrzej Joachimiak ◽  
Greg L. Hura ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Nucleocapsid Protein ◽  
Point Of Care ◽  
Sandwich Elisa ◽  
Epidemiological Studies ◽  
Detection Sensitivity ◽  
Linear Arrangement ◽  
X Ray Scattering ◽  
Biological Phenomena ◽  
Flow Devices

AbstractMonoclonal antibodies (mAbs) are the basis of treatments and diagnostics for pathogens and other biological phenomena. We conducted a structural characterization of mAbs against the N-terminal domain of nucleocapsid protein (NPNTD) from SARS-CoV-2 using small angle X-ray scattering (SAXS). Our solution-based results distinguished the mAbs’ flexibility and how this flexibility impacts the assembly of multiple mAbs on an antigen. By pairing two mAbs that bind different epitopes on the NPNTD, we show that flexible mAbs form a closed sandwich-like complex. With rigid mAbs, a juxtaposition of the Fabs is prevented, enforcing a linear arrangement of the mAb pair, which facilitates further mAb polymerization. In a modified sandwich ELISA, we show the rigid mAb-pairings with linear polymerization led to increased NPNTD detection sensitivity. These enhancements can expedite the development of more sensitive and selective antigen-detecting point-of-care lateral flow devices (LFA), key for early diagnosis and epidemiological studies of SARS-CoV-2 and other pathogens.

scholarly journals Elisa Method For Serum Hepcidin Quantification In Bulgarian Population

2014 ◽  
Vol 41 (1) ◽  
pp. 22-29 ◽  
Author(s):  
V. Manolov ◽  
B. Atanasova ◽  
V. Vasilev ◽  
K. Tzatchev ◽  
M. Velizarova
Keyword(s):  
Monoclonal Antibodies ◽  
Iron Homeostasis ◽  
Sandwich Elisa ◽  
Active Form ◽  
Hemodialysis Patients ◽  
Healthy Population ◽  
Elisa Method ◽  
Limit Of Quantification ◽  
Elisa Format ◽  
Bulgarian Population

Summary Hepcidin is a 25-aminoacid cysteine-rich iron regulating peptide. Hepcidin quantification in human blood may provide further insights for the pathogenesis of disorders of iron homeostasis and might prove a valuable tool for clinicians for the differential diagnosis of anaemia. This study describes ELISA immunoassay for hepcidin quantification in human serum. We used a sandwich ELISA method from USCN Life Science inc., that consists of ready to use, pre-coated 96-well strip plate with 2 antihepcidin-25 monoclonal antibodies. A recombinant hepcidin in 16 μg/l concentration is used as a standard; it reconstitutes with 1.0 ml standard diluent to prepare a stock solution. We correlated ELISA results of hepcidin-25 measurements in healthy population with hemodialysis patients. The sandwich ELISA was highly specific for hepcidin-25, having a low limit of quantification of 0.020 μg/l. Hepcidin- 25 concentrations were increased in hemodialysis patients (median 33.05 μg/l, range 22.31 -60.98 μg/l, n = 10) compared with healthy individuals (median 12.41 μg/l, range 6.05-18.53 μg/l, n = 40). The use of 2 monoclonal antibodies in a sandwich ELISA format provides a robust, convenient and not very expensive method for measuring concentrations of the active form of hepcidin. It should help to improve our understanding of the role of hepcidin in regulating iron metabolism.

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