Immunofluorescence Assay Using Monoclonal and Polyclonal Antibodies for Detection of Staphylococcal Enterotoxins A in Milk

2019 ◽  
Vol 13 (1) ◽  
pp. 137-145 ◽  
Author(s):  
Tzonka Godjevargova ◽  
Zlatina Becheva ◽  
Yavor Ivanov ◽  
Andrey Tchorbanov
Keyword(s):  
Monoclonal Antibody ◽  
Magnetic Nanoparticles ◽  
Polyclonal Antibody ◽  
Polyclonal Antibodies ◽  
Food Poisoning ◽  
Staphylococcal Enterotoxin ◽  
Staphylococcal Enterotoxin A ◽  
Fluorescence Immunoassay ◽  
Magnetic Separator ◽  
Milk Samples

Objectives: Staphylococcus aureus is a Gram-positive microorganism. S. aureus can grow in various foods and cause food poisoning by secreting enterotoxins. The most common enterotoxins involved in food poisoning are staphylococcal enterotoxin A and staphylococcal enterotoxin B, but Staphylococcal Enterotoxin A (SEA) is predominant. The main types of food contaminated with SEs are meat and meat products, poultry and eggs, milk and dairy products. The aim of this study was to develop a rapid and sensitive fluorescence immunoassay for detection of staphylococcal enterotoxin A in milk. Methods: Monoclonal and polyclonal antibodies for SEA were produced and characterized. Competitive fluorescence immunoassay based on Magnetic Nanoparticles (MNPs) was performed and optimized. MNPs were used as a solid carrier of the antibodies. The first step of the assay was immunoreaction between the immobilized antibody onto MNPs and SEA in milk sample. Then the fluorescein-SEA conjugate was added to the sample. Thus, competitive immunoreaction between MNP-mAb/MNP-pAb with SEA and SEA-FITC was performed. These immuno-complexes were separated by a magnetic separator and the obtained supernatants were analyzed. The fluorescent signal from the excess of conjugated SEA was proportional to the SEA contained in the milk. The assay duration was only 30 min. Results: The fluorescence immunoassays performed with polyclonal antibody had linear ranges from 5 pg/mL to 100 ng/mL SEA in a buffer, and from 50 pg/mL to 50 ng/mL SEA in spiked milk samples. While the same assays performed with monoclonal antibody had linear ranges from 1 pg/mL to 20 ng/mL SEA in buffer, and from 10 pg/mL to 10 ng/mL SEA in spiked milk samples. The detection limits of the developed immunoassays performed in milk were: 48 pg/mL with polyclonal antibody and 9 pg/mL with monoclonal antibody. Conclusion: A rapid and sensitive fluorescence immunoassay based on magnetic nanoparticles with a polyclonal and monoclonal antibody for determination of staphylococcal enterotoxin A in milk was developed.

scholarly journals Mass Outbreak of Food Poisoning Disease Caused by Small Amounts of Staphylococcal Enterotoxins A and H

2005 ◽  
Vol 71 (5) ◽  
pp. 2793-2795 ◽  
Author(s):  
Tetsuya Ikeda ◽  
Naoto Tamate ◽  
Keiji Yamaguchi ◽  
Sou-ichi Makino
Keyword(s):  
Food Poisoning ◽  
Staphylococcal Enterotoxin ◽  
Raw Material ◽  
Staphylococcal Enterotoxin A ◽  
Staphylococcal Enterotoxins ◽  
Reconstituted Milk

ABSTRACT It was believed that food poisoning in Osaka in 2000 was due to small amounts of staphylococcal enterotoxin A (SEA) in reconstituted milk. Results of this study clearly indicate that SEH was also present in the raw material of reconstituted milk, indicating that the food poisoning was caused by multiple staphylococcal enterotoxins.

scholarly journals Cloning, Sequencing and Production of Recombinant Polyclonal Antibodies against Egyptian Staphylococcal Enterotoxin A

2017 ◽  
Vol 5 (6) ◽  
pp. 131-137
Author(s):  
H. A. Nour El-Din ◽  
M. N. Abu El-Naga ◽  
M. E. El-Fouly ◽  
M. K. Ibrahim ◽  
E. H. El-Shatoury ◽  
...  
Keyword(s):  
Polyclonal Antibodies ◽  
Staphylococcal Enterotoxin ◽  
Staphylococcal Enterotoxin A

Superantigen-based immunotherapy: a phase I trial of PNU-214565, a monoclonal antibody-staphylococcal enterotoxin A recombinant fusion protein, in advanced pancreatic and colorectal cancer.

1997 ◽  
Vol 15 (5) ◽  
pp. 1994-2007 ◽  
Author(s):  
B J Giantonio ◽  
R K Alpaugh ◽  
J Schultz ◽  
C McAleer ◽  
D W Newton ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Single Dose ◽  
Fusion Protein ◽  
Performance Status ◽  
Staphylococcal Enterotoxin ◽  
Tnf Alpha ◽  
Soluble Antigen ◽  
Staphylococcal Enterotoxin A ◽  
Fab Fragment ◽  
Grade 3

PURPOSE To establish the maximum-tolerated dose (MTD) and define the toxicities of a single-dose infusion of PNU-214565, a recombinant Escherichia coli-derived fusion protein of Staphylococcal enterotoxin A (SEA) and the Fab-fragment of the C242 monoclonal antibody in patients with advanced colorectal and pancreatic carcinomas. To investigate the capability of PNU-214565 to induce a superantigen (SAg) response resulting in cytokine production and tumor regression. PATIENTS AND METHODS Twenty-one patients (age range, 39 to 76 years; median, 64; 12 men, nine women; 18 colorectal, three pancreatic cancers) were treated with a single 3-hour infusion of PNU-214565, with doses ranging from 0.01 to 1.5 ng/kg. All patients had prior chemotherapy and a good performance status Eastern Cooperative Oncology Group [ECOG] performance status [PS] = 0 [n = 10]; PS = 1 [n = 11]), 10 had prior radiation, and 18 had prior surgery. RESULTS Fever and hypotension were the most common toxicities. Fever of any grade occurred in 16 of 21 patients (76%): four of 21 (19%) with grade 2 and two of 21 (9.5%) with grade 3. Hypotension of any grade occurred in 13 of 21 (62%): four of 21 with grade 2 and one of 21 (5%) with grade 3. Interleukin-2 (IL-2) and tumor necrosis factor alpha (TNF alpha) induction correlated with toxicity. In the two patients with grade 3 fever, peak IL-2 and TNF alpha levels were 2.9 IU/mL and 165 pg/mL, and 8.3 IU/mL and 245 pg/mL, respectively. Transient, > or = 50% decreases in circulating monocytes were observed in 17 of 21 patients as early as 0.5 hours (median time, 2 hours) from the start of infusion. Decreases (mean 33%) in circulating lymphocytes were observed in seven of 21 patients. All three patients with grade 3 toxicity were treated at the 0.5-ng/kg dose. The significance of baseline anti-SEA, human antimouse antibody (HAMA), CA242-soluble antigen levels, and T-cell receptor variable beta region (TCR V beta) subsets and histocompatibility leukocyte antigen-DR (HLA-DR) genotypes was assessed as possible predictors of toxicity. All toxicities were transient and easily managed. No grade 3 toxicity occurred at the higher dose levels. CONCLUSION PNU-214565, a SAg-based tumor targeted therapy, is safe when given as a single 3-hour infusion at doses up to 1.5 ng/kg. The MTD for a single dose was not determined. The safety of a repeated dose schedule is currently under investigation, beginning with doses determined to be safe in this trial.

scholarly journals Monoclonal Antibody-Based Sandwich ELISA for the Detection of Staphylococcal Enterotoxin A

2013 ◽  
Vol 10 (4) ◽  
pp. 1598-1608 ◽  
Author(s):  
Hua Kuang ◽  
Wenbing Wang ◽  
Liguang Xu ◽  
Wei Ma ◽  
Liqiang Liu ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Sandwich Elisa ◽  
Staphylococcal Enterotoxin ◽  
Staphylococcal Enterotoxin A

Production of a Monoclonal Antibody by Simultaneous Immunization of Staphylococcal Enterotoxin A and B

2011 ◽  
Vol 164 (6) ◽  
pp. 831-840 ◽  
Author(s):  
Bin Liang ◽  
Yongxia Zhang ◽  
Aiping Liu ◽  
Youxiang Zhou ◽  
Fusheng Chen ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Staphylococcal Enterotoxin ◽  
Staphylococcal Enterotoxin A

scholarly journals Two-site immunofluorometric assay of intact salmon calcitonin with improved sensitivity

1997 ◽  
Vol 43 (1) ◽  
pp. 71-75 ◽  
Author(s):  
Haiqin Rong ◽  
Leonard J Deftos ◽  
Hong Ji ◽  
Elisabet Bucht
Keyword(s):  
Monoclonal Antibody ◽  
Detection Limit ◽  
Polyclonal Antibody ◽  
Salmon Calcitonin ◽  
Polyclonal Antibodies ◽  
Pharmacokinetic Studies ◽  
Human Calcitonin ◽  
Plasma Samples ◽  
Serum Concentrations ◽  
Capture Antibody

Abstract We recently developed a two-site immunofluorometric assay (IFMA) of salmon calcitonin (SCT) by DELFIA (dissociation enhancement lanthanide fluoroimmunoassay) technique using the same polyclonal antibodies both for “catching” the antigen and for signaling. In the present study we used a monoclonal antibody to SCT 1–11 as the capture antibody. This antibody was biotinylated before use in streptavidin-coated microtitration plates. The polyclonal antibody labeled with Eu chelate was used as a signaling marker. This combination of antibodies resulted in an assay that was three to four times more sensitive than the previous IFMA, with a detection limit of 0.3 pmol/L serum. Intact SCT 1–32 was detected by the assay (recoveries 94–96%), but not the fragments SCT 1–11 and SCT 10–32 or human calcitonin. Dilutions of plasma samples containing SCT were parallel to the calibration curve of SCT 1–32. Pharmacokinetic studies of SCT, 100 IU administered intramuscularly to 10 men, indicated peak serum concentrations of 32–128 pmol/L within 10–20 min with apparent half-life of 56 ± 18 min (mean ± SD). This new assay will allow study of the pharmacokinetics of new calcitonin preparations that do not require injection.

Expression and characterization of single-chain variable fragment antibody against staphylococcal enterotoxin A in Escherichia coli

2014 ◽  
Vol 60 (11) ◽  
pp. 737-743 ◽  
Author(s):  
Weifeng Chen ◽  
Li Hu ◽  
Aiping Liu ◽  
Jinquan Li ◽  
Fusheng Chen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Food Poisoning ◽  
Staphylococcal Enterotoxin ◽  
Enzyme Linked Immunosorbent Assay ◽  
Staphylococcal Enterotoxin A ◽  
Single Chain Variable Fragment ◽  
Single Chain

The staphylococcal enterotoxins (SEs) are potent gastrointestinal exotoxins synthesized by Staphylococcus aureus, which is responsible for various diseases including septicemia, food poisoning, and toxic shock syndrome, as well as bovine mastitis. Among them, staphylococcal enterotoxin A (SEA) is one of the most commonly present serotypes in staphylococcal food poisoning cases. In this study, the stable hybridoma 3C12 producing anti-SEA monoclonal antibody was established with an equilibrium dissociation constant (KD) of 1.48 × 10−8 mol·L−1, its ScFv-coding genes were obtained and then the anti-SEA single chain variable fragment (ScFv) protein was expressed in Escherichia coli. Characterization of the expressed target ScFv protein was analyzed by sodium dodecyl sulfate – polyacrylamide gel electrophoresis, Western blot, and enzyme-linked immunosorbent assay. The results demonstrated that the recombinant anti-SEA ScFv protein retained a specific binding activity for SEA, and the KD value of the soluble ScFv was about 3.75 × 10−7 mol·L−1. The overall yield of bioactive anti-SEA ScFv in E. coli flask culture was more than 10 mg·L−1.

Sign in / Sign up

Export Citation Format

Share Document