Development of an Enzyme-Linked Immunosorbent Assay (ELISA) for Detection of IgM and IgG Antibodies to Lipopolysaccharide of Escherichia coli O157

An enzyme-linked immunosorbent assay (ELISA) system was developed using polymyxin immobilized in the wells of a microtiter plate as a high-affinity adsorbent for Escherichia coli O157 lipopolysaccharide (LPS) antigens. Extracts from cell suspensions were reacted with polymyxin-coated microwells followed by immunoenzymatic detection of captured LPS antigens using a commercially available anti–E. coli O157 antibody–peroxidase conjugate and a 3,3′,5,5′-tetramethylbenzidine substrate. The polymyxin ELISA was highly sensitive and specific for E. coli strains bearing the O157 antigen. When this ELISA was combined with enrichment, results were in complete agreement with those of standard culture techniques for the detection of this pathogen in a variety of artificially inoculated and naturally contaminated foods. The polymyxin ELISA is a simple and inexpensive assay for E. coli O157 with a 96-well microtiter plate format, making this system ideally suited for processing large numbers of samples.

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Optimization of a Fluorescence Sandwich Enzyme-Linked Immunosorbent Assay for Detection of Escherichia coli O157:H7 in Apple Juice

Journal of Food Protection ◽

10.4315/0362-028x-67.12.2756 ◽

2004 ◽

Vol 67 (12) ◽

pp. 2756-2759 ◽

Cited By ~ 10

Author(s):

CYNTHIA NYQUIST-BATTIE ◽

LAURA E. FRANK ◽

DEANNA LUND ◽

DANIEL V. LIM

Keyword(s):

Escherichia Coli ◽

Immunosorbent Assay ◽

Fluorescence Intensity ◽

Apple Juice ◽

Enzyme Linked Immunosorbent Assay ◽

Escherichia Coli O157 ◽

Dependent Manner ◽

Background Fluorescence ◽

Assay Performance ◽

Phosphate Buffered Saline

Sandwich enzyme-linked immunosorbent assay, especially when coupled with biosensor technology, is a simple methodology that can rapidly screen juices for Escherichia coli O157:H7 contamination. However, sampling directly from apple juice and ciders has been postulated to reduce immunoassay sensitivity. In fluorescence sandwich enzyme-linked immunosorbent assays using commercially available polyclonal or monoclonal antibodies, sampling pasteurized apple juice spiked with E. coli O157:H7 compared to spiked phosphate-buffered saline shifted the range of detection. The spiked apple juice range of detection was 104 to 106 CFU/ml, whereas that for spiked phosphate-buffered saline was 106 to 108 CFU/ml, representing a hundredfold difference in sensitivity. Apple juice also increased background fluorescence intensity (P < 0.001) while reducing the net fluorescence intensity per CFU (P < 0.001). The addition of the polymer polyvinylpyrrolidone to apple juice significantly improved assay performance by increasing sensitivity and net fluorescence intensity per CFU and by reducing background fluorescence. Adjusting pH of apple juice from 3.9 to 7.4 improved assay performance but not to the degree seen with phosphate-buffered saline or polyvinylpyrrolidone-treated apple juice samples. The apple juice polyphenol, epicatechin, reduced net fluorescence intensity in a concentration-dependent manner, a change that was reversed by polyvinylpyrrolidone. Taken all together, these results suggest that polyvinylpyrrolidone can improve detection of O157:H7 in juices by reducing the effect of polyphenols on fluorescence sandwich enzyme-linked immunosorbent assay performance.

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A novel enzyme-linked immunosorbent assay for detection of Escherichia coli O157:H7 using immunomagnetic and beacon gold nanoparticles

Gut Pathogens ◽

10.1186/1757-4749-6-14 ◽

2014 ◽

Vol 6 (1) ◽

pp. 14 ◽

Cited By ~ 47

Author(s):

Zhiqiang Shen ◽

Nannan Hou ◽

Min Jin ◽

Zhigang Qiu ◽

Jingfeng Wang ◽

...

Keyword(s):

Escherichia Coli ◽

Gold Nanoparticles ◽

Immunosorbent Assay ◽

Enzyme Linked Immunosorbent Assay ◽

Escherichia Coli O157

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Development and Validation of 13-plex Luminex-Based Assay for Measuring Human Serum Antibodies to Streptococcus pneumoniae Capsular Polysaccharides

mSphere ◽

10.1128/msphere.00128-18 ◽

2018 ◽

Vol 3 (4) ◽

pp. e00128-18 ◽

Cited By ~ 9

Author(s):

Danka Pavliakova ◽

Peter C. Giardina ◽

Soraya Moghazeh ◽

Shite Sebastian ◽

Maya Koster ◽

...

Keyword(s):

Human Serum ◽

Lower Limit ◽

Immunosorbent Assay ◽

Enzyme Linked Immunosorbent Assay ◽

Igg Antibodies ◽

Igg Antibody ◽

Correlate Of Protection ◽

Immune Correlate ◽

Relative Standard ◽

Limit Of Quantitation

ABSTRACT A Luminex-based direct immunoassay (dLIA) platform has been developed to replace the standardized pneumococcal enzyme-linked immunosorbent assay platform. The multiplex dLIA simultaneously measures the concentration of serum immunoglobulin G (IgG) antibodies specific for pneumococcal capsular polysaccharide (PnPS) serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F. The assay uses poly-l-lysine (PLL)-conjugated PnPS, chemically coupled to spectrally distinct Luminex microspheres. Assay validation experiments were performed using residual human serum samples obtained from 13-valent pneumococcal conjugate vaccine (13vPnC) clinical studies. Assay results are expressed as IgG antibody concentrations in micrograms per milliliter using the international reference serum, 007sp. The lower limit of quantitation (LLOQ) for all serotypes covered in the 13-plex dLIA fell within the range of 0.002 to 0.038 µg/ml serum IgG. The difference between the lower limit and upper limit of the assay range was >500-fold for all serotypes, and assay variability was <20% relative standard deviation (RSD) for all serotypes. IgG antibody measurements were shown to be serotype-specific (some cross-reactivity was observed only between the structurally related serotypes 6A and 6B as well as 19A and 19F), and no interference was observed between the serotypes when the assay was performed in the 13-plex format compared to the singleplex assays. The 13-plex dLIA platform developed by Pfizer Inc. generates up to 143 test results in a single 96-well plate and is a suitable replacement of the enzyme-linked immunosorbent assay (ELISA) platform for evaluating vaccine clinical trials. IMPORTANCE The pneumococcal enzyme-linked immunosorbent assay (ELISA) measures IgG antibodies in human serum, and it is an important assay that supports licensure of pneumococcal vaccines. The immune correlate of protection, 0.35 µg/ml of IgG antibodies, was determined by the ELISA method. Pfizer has developed a new Luminex-based assay platform to replace the ELISA. These papers describe the important work of (i) validating the Luminex-based assay and (ii) bridging the immune correlate of protection (0.35 µg/ml IgG) to equivalent values reported by the Luminex platform.

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An enzyme-linked immunosorbent assay to detect IgG antibodies to cell surface components of Pseudomonas cepacia in patients with cystic fibrosis

Serodiagnosis and Immunotherapy in Infectious Disease ◽

10.1016/0888-0786(88)90062-5 ◽

1988 ◽

Vol 2 (5) ◽

pp. 349-356 ◽

Cited By ~ 1

Author(s):

P.M. Zadik

Keyword(s):

Cystic Fibrosis ◽

Cell Surface ◽

Immunosorbent Assay ◽

Enzyme Linked Immunosorbent Assay ◽

Pseudomonas Cepacia ◽

Igg Antibodies

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Measles antibodies and response to vaccination in children aged less than 14 months: implications for age of vaccination

Epidemiology and Infection ◽

10.1017/s0950268811002184 ◽

2011 ◽

Vol 140 (9) ◽

pp. 1599-1606 ◽

Cited By ~ 16

Author(s):

E. BORRÀS ◽

L. URBIZTONDO ◽

J. COSTA ◽

J. BATALLA ◽

N. TORNER ◽

...

Keyword(s):

Immunosorbent Assay ◽

Maternal Antibodies ◽

Passive Immunity ◽

Enzyme Linked Immunosorbent Assay ◽

Igg Antibodies ◽

Blood Samples ◽

Measles Outbreak ◽

Measles Antibodies ◽

Measles Vaccination

SUMMARYPassive immunity against measles decreases during the first months of life. The objective of this study was to determine titres of measles antibodies in children aged 9–14 months and their mothers before vaccination, and the children's response to vaccination. Blood samples were collected by capillary puncture before and 28 days after vaccination. Samples were obtained between February and June 2007 during an ongoing measles outbreak. Titres of specific measles IgG antibodies were determined by enzyme-linked immunosorbent assay. Seroconversion was defined as the presence of antibodies after vaccination in subjects without antibodies before vaccination. Maternal antibodies were present in 37·7% of all 69 children included and in 45·1% of children aged 9 months. Of the 51 children in whom a second sample was obtained, 31 (60·8%) were seronegative before vaccination and 61·3% seroconverted. Interference of maternal antibodies was 30%. Advancing the first dose of measles vaccination from 15 to 12 months is a correct strategy, given the increase in the time of susceptibility of infants to measles.

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