scholarly journals A Quantitative ELISA Protocol for Detection of Specific Human IgG Against the SARS-CoV-2 Spike Protein

Vaccines ◽  
2021 ◽  
Vol 9 (7) ◽  
pp. 770
Author(s):  
Rémi Vernet ◽  
Emily Charrier ◽  
Julien Grogg ◽  
Nicolas Mach
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Immunosorbent Assay ◽  
Spike Protein ◽  
Quantitative Detection ◽  
Enzyme Linked Immunosorbent Assay ◽  
Igg Antibodies ◽  
Human Igg ◽  
Microtiter Plates ◽  
Global Priority ◽  
Humoral Responses

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a worldwide pandemic with at least 3.8 million deaths to date. For that reason, finding an efficient vaccine for this virus quickly became a global priority. The majority of vaccines now marketed are based on the SARS‑CoV‑2 spike protein that has been described as the keystone for optimal immunization. In order to monitor SARS‑CoV‑2 spike-specific humoral responses generated by immunization or infection, we have developed a robust and reproducible enzyme-linked immunosorbent assay (ELISA) protocol. This protocol describes a method for quantitative detection of IgG antibodies against the SARS‑CoV‑2 spike protein using antigen-coated microtiter plates. Results showed that antibodies could be quantified between the range of 1.953 ng/mL to 500 ng/mL with limited inter- and intra-assay variability.

scholarly journals Evaluation of the EUROIMMUN Anti-SARS-CoV-2 ELISA Assay for detection of IgA and IgG antibodies

2020 ◽  
Author(s):  
Scott M. Matushek ◽  
Kathleen G. Beavis ◽  
Ana Abeleda ◽  
Cindy Bethel ◽  
Carlissa Hunt ◽  
...  
Keyword(s):  
Clinical Microbiology ◽  
Spike Protein ◽  
Quantitative Detection ◽  
Enzyme Linked Immunosorbent Assay ◽  
Elisa Assay ◽  
Plasma Samples ◽  
Igg Antibodies ◽  
Human Coronavirus ◽  
Human Coronaviruses ◽  
The University

AbstractAs the Coronavirus 2019 (COVID-19) pandemic evolves, the development of immunoassays to help determine exposure and potentially predict immunity has become a pressing priority. In this report we present the performance of the EUROIMMUN enzyme-linked immunosorbent assay (ELISA) for semi-quantitative detection of IgA and IgG antibodies in serum and plasma samples using recombinant S1 domain of the SARS-CoV-2 spike protein as antigen. Specimens from patients, with and without COVID-19 infection, were tested at the University of Chicago Clinical Microbiology and Immunology Laboratory. Of 57 samples from COVID-19 PCR-negative patients, including 28 samples positive for common human coronavirus strains, 53 tested negative and 4 tested positive for IgA (93.0% agreement) while 56 tested negative and 1 tested positive for IgG (98.2% agreement). For COVID-19 PCR-positive patients, 29 of 30 (96.7%) samples collected ≥3 days after positive PCR were positive for IgA, and 28 of 28 samples collected ≥4 days after positive PCR were positive for IgG.The EUROIMMUN Anti-SARS-CoV-2 ELISA Assay demonstrates excellent sensitivity for detection of IgA and IgG antibodies from samples collected ≥3 days and ≥4 days, respectively, after COVID-19 diagnosis by PCR. This assay did not demonstrate cross reaction in any of the 28 samples from patients with common human coronaviruses, including types HKU1, NL63, CV229E, and OC43.

scholarly journals Recombinant Protein-Based Enzyme-Linked Immunosorbent Assay and Immunochromatographic Tests for Detection of Immunoglobulin G Antibodies to Severe Acute Respiratory Syndrome (SARS) Coronavirus in SARS Patients

2004 ◽  
Vol 11 (2) ◽  
pp. 287-291 ◽  
Author(s):  
Ming Guan ◽  
Hsiao Ying Chen ◽  
Shen Yun Foo ◽  
Yee-Joo Tan ◽  
Phuay-Yee Goh ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Recombinant Proteins ◽  
Immunosorbent Assay ◽  
Immunoglobulin G ◽  
Predictive Value ◽  
Rapid Test ◽  
Enzyme Linked Immunosorbent Assay ◽  
Igg Antibodies ◽  
Kappa Value ◽  
Sensitivity Specificity

ABSTRACT An enzyme-linked immunosorbent assay (ELISA) and a rapid immunochromatographic test for detection of immunoglobulin G (IgG) antibodies in severe acute respiratory syndrome (SARS) patients were developed by utilizing the well-characterized recombinant proteins Gst-N and Gst-U274. The ELISA detected IgG antibodies to SARS-CoV in all 74 convalescent-phase samples from SARS patients while weakly cross-reacting to only 1 of the 210 control sera from healthy donors. This finding thus led to a kit sensitivity, specificity, and accuracy of 100, 99.5, and 99.6%, respectively. The test thus provided a positive predictive value (PPV) of 98.7% and a negative predictive value (NPV) of 100%. In addition, the ELISA gave a positive delta of 5.4 and a negative delta of 3.6, indicating an excellent differentiation between positives and negatives. The same recombinant proteins were also applied to a newly developed platform for the development of a 15-min rapid test. The resulting rapid test has an excellent agreement of 99.6%, with a kappa value of 1.00, with the ELISA. Again, this rapid test was able to detect 100% of the samples tested (n = 42) while maintaining a specificity of 99.0% (n = 210). The PPV and NPV for the rapid test thus reached 95.3 and 100%, respectively.

An Enzyme-Linked Immunosorbent Assay (Elisa) for Measurement of Human Igg Subclass Levels in Serum

1990 ◽  
Vol 23 (6) ◽  
pp. 1017-1037 ◽  
Author(s):  
Kunihiko Hayashi ◽  
Hiroshi Miyasaka ◽  
Munekazu Tagawa
Keyword(s):  
Immunosorbent Assay ◽  
Enzyme Linked Immunosorbent Assay ◽  
Igg Subclass ◽  
Human Igg

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

mSphere ◽  
2018 ◽  
Vol 3 (4) ◽  
pp. e00128-18 ◽  
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.

scholarly journals Rubella-specific IgG subclass concentrations in sera using an enzyme-linked immunosorbent assay (ELISA): the effect of different sources of rubella antigen

1988 ◽  
Vol 101 (3) ◽  
pp. 599-604 ◽  
Author(s):  
H. I. J Thomas ◽  
P. Morgan-Capner
Keyword(s):  
Immunosorbent Assay ◽  
Enzyme Linked Immunosorbent Assay ◽  
Igg Subclass ◽  
Human Igg ◽  
Discrepant Result ◽  
Specific Igg ◽  
Antigen A ◽  
Different Sources ◽  
Positive Results

SUMMARYFive rubella antigens were evaluated in an antiglobulin enzyme-linked immunosorbent assay for rubella-specific IgG subclass antibody. One monoclonal anti-human IgG subclass antibody was used for each of IgG1, IgG2and IgG4, but two were compared for IgG3. A total of 101 sera were tested from cases of rubella in the distant past and from cases of primary rubella, reinfection and following immunization. Only one serum gave a discrepant result for specific IgG1, being positive with only one rubella antigen, a commercially prepared antigen coated on to microtitre wells (Enzygnost; Behringwerke). No sera contained detectable specific IgG2. Only four sera contained specific IgG4, and this was detectable only with Enzygnost antigen. For specific IgG3little difference was observed between the two monoclonal anti-human IgG3subclass antibodies; only two very weakly positive sera gave discrepant results. However, varying results were obtained for specific IgG3with the different antigens. Enzygnost gave more positive results for specific IgG3with most categories of sera.It is concluded that the differences between various reports of the rubella-specific IgG subclass profile cannot be explained entirely by the use of different rubella antigens.

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

2011 ◽  
Vol 140 (9) ◽  
pp. 1599-1606 ◽  
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.

scholarly journals Επιπολασμός λοίμωξης από ελικοβακτηρίδιο του πυλωρού σε ασθενείς με χρόνια αποφρακτική πνευμονοπάθεια

2005 ◽  
Author(s):  
Αναστάσιος Ρούσσος
Keyword(s):  
Immunosorbent Assay ◽  
Enzyme Linked Immunosorbent Assay ◽  
Igg Antibodies

Ασθενείς με πεπτικό έλκος εμφανίζουν αυξημένο επιπολασμό Χρόνιας Αποφρακτικής Πνευμονοπάθειας (ΧΑΠ) . Το κάπνισμα το οποίο αποτελεί κοινό προδιαθετικό παράγοντα και για τα δύο αυτά νοσήματα έχει ενοχοποιηθεί για τη συσχέτιση αυτή Όμως, πρόσφατες μελέτες έδειξαν ότι η ασθενείς με χρόνια βρογχίτιδα, ίσως, παρουσιάζουν αυξημένο επιπολασμό Η. pylori λοίμωξης Είναι γνωστό ότι σε ορισμένους ασθενείς η λοίμωξη από Η. pylori ενεργοποιεί την απελευθέρωση μίας σειράς προφλεγμονωδών κυτοκινών. Υπεύθυνα για την παραγωγή αυτών των κυτοκινών θεωρούνται τα ιδιαίτερα λοιμογόνα στελέχη Η. pylori τα οποία παράγουν την πρωτεΐνη CagA. Οι προφλεγμονώδεις αυτές κυτοκίνες εμπλέκονται και στην παθογένεια της ΧΑΠ, πιθανότατα προάγοντας τη μη ειδική φλεγμονή του βρογχικού δέντρου. Σκοπός της παρούσας μελέτης ήταν η διερεύνηση του επιπολασμού της Η. pylori λοίμωξης και ιδιαίτερα των CagA θετικών λοιμογόνων στελεχών σε ασθενείς με ΧΑΠ. Επίσης, επιχειρήθηκε η συσχέτιση της οροθετικότητας για το Η. pylori και για τα CagA θετικά στελέχη του με κλινικοεργαστηριακές παραμέτρους της ΧΑΠ (σπιρομετρικός έλεγχος και βαρύτητα της νόσου) Συνολικά μελετήθηκαν 126 ασθενείς με ΧΑΠ (88 άντρες and 38 γυναίκες με ηλικία 61.3 ± 8.1 έτη) και 126 μάρτυρες, προτυποποιημένοι κατά φύλο ηλικία και κοινωνικοοικονιμικό επίπεδο. Όλα τα άτομα τα οποία συμπεριελήφθησαν στη μελέτη (ασθενείς και μάρτυρες) υποβλήθηκαν σε ενζυμική ανοσοπροσροφητική μέθοδο προσδιορισμού [enzyme-linked immunosorbent assay (elisa)] των IgG αντισωμάτων για το Η. pylori και την πρωτεΐνη CagA. Επίσης, οι ασθενείς με ΧΑΠ υποβλήθηκαν σε σπιρομετρικό έλεγχο (FEV1, FEV1/FVC) και σε συνακόλουθη σταδιοποίηση της βαρύτητας της νόσου Ο εττιπολασμός της FI pylori λοίμωξης ήταν υψηλότερος στους ασθενείς με ΧΑΠ συγκριτικά με τους μάρτυρες. [77.8% έναντι 54.7% αντίστοιχα (ρ<0.001)]. Ο επιπολασμός της λοίμωξης από CagA (+) Η pylori στελέχη ήταν υψηλότερος στους ασθενείς με ΧΑΠ συγκριτικά με τους μάρτυρες [53.9% έναντι 29.3% αντίστοιχα (ρΟ.ΟΟΙ)]. Επίσης, οι ασθενείς με ΧΑΠ είχαν σημαντικά υψηλότερη μέση συγκέντρωση τόσο anti-FI. pylori IgG (118.3±24.4 vs 61.9±12.9 U/ML, ρ<0.001) όσο και anti-CagA IgG antibodies (33.8±3.4 vs 19.0±1.5 U/ML, ρ<0.001). Οι σπιρομετρικές παράμετροι δεν διέψεραν σημαντικά μεταξύ των Fi pylori (+) και Η pylori (-) ασθενών με ΧΑΠ (FEV1:61.5±18.9%, FEV1/FVC 63.0± 4.9%, έναντι FEV1:63.5±17.9%, FEV1/FVC: 63.9± 4.6%, αντίστοιχα, ρ>0.05). Παρομοίως, οι σπιρομετρικές παράμετροι δεν διέφεραν σημαντικά μεταξύ των CagA(+) και CagA (-) ασθενών με ΧΑΠ (FEV1: 59.1 ±9.7%, FEV1/FVC 62.4± 5.1%, έναντι FEV1: 65.4±6.3%, FEV1/FVC: 64.3± 4.3%, αντίστοιχα, ρ>0.05) Τέλος, δεν αναδείχθηκε σημαντική διαφορά μεταξύ των ποσοστών αντί- FI pylori IgG θετικότητας και αντι-CagA IgG θετικότητας στα διάφορα στάδια ΧΑΠ. Με δεδομένο τον υψηλό επιπολασμό της ελικοβακτηριδιακής λοίμωξης σε ασθενείς με ΧΑΠ και ιδιαίτερα των λοιμογόνων Caga (+) στελεχών τα οποία ενοχοποιούνται για την παραγωγή προφλεγμονωδών κυτοκινών (όμοιες με αυτές που εμπλέκονται στην παθογένεια της ΧΑΠ) θεωρείται πιθανός ο παθογενετικός ρόλος του ελικοβακτηριδίου στη ΧΑΠ. Αντίθετα, περιορισμένος φαίνεται να είναι ο ρόλος τον οποίο διαδραματίζει το ελικοβακτηρίδιο στην εξέλιξη της νόσου, καθώς δε διαπιστώθηκε συσχέτιση της Η pylori οροθετικότητας και της CagA οροθετικότητας με τη βαρύτητα της ΧΑΠ Σίγουρα στο μέλλον απαιτούνται μελέτες σε μεγαλύτερο αριθμό ασθενών οι οποίες να ελέγχουν την ορθότητα των ευρημάτων μας.

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