Review of
Comparison of Three Enzyme-Linked
Immunosorbent Assays for Detection
of Immunoglobulin G Antibodies to Tetanus
T...
Author(s)
• Karen H. van Hoeven,
• Connie Dale,
• Phil Foster
• and Barbara Body
Introduction
 Accurate determination of the concentrations of
immunoglobulin G (IgG) antibody to tetanus toxoid to
evalua...
 The gold standard assay for the determination of specific
IgG antibodies to tetanus toxoid is the in vivo toxin neutrali...
• Serially diluted NIBSC 76/589 and TE-3 human tetanus IgG
immunoglobulin international reference standards were
analyzed ...
 IgG antibodies to tetanus toxoid were measured in 83
deidentified serum specimens using each manufacturer’s ELISA.
 Eac...
 Using the recommended cutoff for each ELISA
(<0.10 IU/ml), the overall agreement of all of the
ELISA methods was 78%.
 ...
MATERIALS AND METHODS
Reference materials:
 NIBSC reagent 76/589 was supplied by NIBSC
in a lyophilized vial containing ...
It was then further diluted to 10 IU/ml by
adding 50 l of the initially reconstituted
solution to 550 l of sterile distil...
• Serially diluted NIBSC 76/589 and TE-3 human tetanus IgG
immunoglobulin international reference standards were
analyzed ...
Serum samples
The ELISAs were compared using 83 serum
samples collected in 2007 and submitted for
diagnostic testing.
Th...
Antibody assays
Each of the ELISAs detects IgG antibodies to
tetanus toxoid by an indirect technique.
 Testing was perfor...
 Serially diluted NIBSC standards were run in
quadruplicate at each dilution.
 Serum samples were assayed once in
accord...
Analysis of data
• Intra-assay imprecision was calculated as means
with a coefficient of variation for the
quadruplicate r...
Results
 Results of controls provided by all manufacturers’
ELISAswere within acceptable limits.
 Intra-assay imprecisio...
The Binding Site ELISA yielded nonprotective
antibody concentrations in only these 3
samples, whereas 19 samples (22.9%)
...
Results of 21 samples showing nonprotective antibody
levels
Scimedx
(cutoff 0.10
IU/ml)
The Binding Site Euroimmun
<0.10
<...
testing for tetanus antitoxin levels is frequently
used for academic purposes or to test the
immunogenicity of vaccines t...
The present study provides valuable
comparative data for laboratories that are
evaluating different manufacturers’ produc...
THANK YOU
Overview  2014
Overview  2014
Overview  2014
Overview  2014
Upcoming SlideShare
Loading in …5
×

Overview 2014

334 views

Published on

Published in: Technology, Business
0 Comments
2 Likes
Statistics
Notes
  • Be the first to comment

No Downloads
Views
Total views
334
On SlideShare
0
From Embeds
0
Number of Embeds
5
Actions
Shares
0
Downloads
4
Comments
0
Likes
2
Embeds 0
No embeds

No notes for slide

Overview 2014

  1. 1. Review of Comparison of Three Enzyme-Linked Immunosorbent Assays for Detection of Immunoglobulin G Antibodies to Tetanus Toxoid with Reference Standards and the Impact on Clinical Practice
  2. 2. Author(s) • Karen H. van Hoeven, • Connie Dale, • Phil Foster • and Barbara Body
  3. 3. Introduction  Accurate determination of the concentrations of immunoglobulin G (IgG) antibody to tetanus toxoid to evaluate the immunogenicity of tetanus toxoid vaccines, determine immune competence in individual patients, and measure the prevalence of immunity in populations.  The performance of three commercially available enzyme- linked immunosorbent assays (ELISAs) for IgG antibodies to tetanus toxoid were evaluated.  Scimedx  The Binding Site  Euroimmun
  4. 4.  The gold standard assay for the determination of specific IgG antibodies to tetanus toxoid is the in vivo toxin neutralization test. But have many Disadvantage:  time-consuming,  relatively expensive,  subjective, and raises ethical issues regarding the use of live Mammals. The End point of INVIVO TOXIN Neutralization assay in mice is death or paralysis  The use of accurate and automated in vitro assays  is desirable for ethical, clinical, and economic reasons.  highly reproducible, sensitive.  specific in vitro testing improves the efficiency of the clinical laboratory.
  5. 5. • Serially diluted NIBSC 76/589 and TE-3 human tetanus IgG immunoglobulin international reference standards were analyzed in quadruplicate using ELISAs manufactured by The three assays. Standard and sample(s) Transfer Vol Starting concn (IU/ml) Amt (l) of sterile distilled water Working concn (IU/ml) TE-3 1 2 to 8 480 7.00 360 0 120 7.00 5.25, 3.94, 2.95, 2.21, 1.66, 1.25, and 0.93, respectively NIBSC 76/589 9 10 to 16 300 1.00 180 0 120 1.00 0.60, 0.36, 0.33, 0.13, 0.08, 0.05, and 0.03, respectively The National Institute for Biological Standards and Control (NIBSC)
  6. 6.  IgG antibodies to tetanus toxoid were measured in 83 deidentified serum specimens using each manufacturer’s ELISA.  Each ELISA provided linear results when evaluated with the reference preparations.  The Binding Site ELISA provided results that closely corresponded to the reference preparations (y 1.09x 0.08), whereas the Scimedx ELISA gave results that were consistently lower (y 0.21x 0.07) and the Euroimmun ELISA gave results that were consistently higher (y 1.5x 0.30) than the reference preparation concentrations.
  7. 7.  Using the recommended cutoff for each ELISA (<0.10 IU/ml), the overall agreement of all of the ELISA methods was 78%.  serum samples demonstrated inadequate immunity with all three assays.
  8. 8. MATERIALS AND METHODS Reference materials:  NIBSC reagent 76/589 was supplied by NIBSC in a lyophilized vial containing 9.2 IU.  It was reconstituted in 9.2 ml of sterile distilled water to yield a working concentration of 1IU/ml. Serial dilutions of NIBSC 76/589 were performed to yield final concentrations. An ampoule of the first International Standard for human tetanus immunoglobulin, coded TE-3, was also obtained from the NIBSC. TE-3 was supplied lyophilized at 120 IU, reconstituted in 1 ml of sterile distilled water to yield a working concentration of 120 IU/ml.
  9. 9. It was then further diluted to 10 IU/ml by adding 50 l of the initially reconstituted solution to 550 l of sterile distilled water. TE-3 was then rediluted to a starting concentration of 7 IU/ml by adding 350 l of the previously diluted fluid to 150 l of sterile distilled water.  Serial dilutions of TE-3 were performed to yield final concentrations . One set of dilutions was made and tested with all three ELISAs.
  10. 10. • Serially diluted NIBSC 76/589 and TE-3 human tetanus IgG immunoglobulin international reference standards were analyzed in quadruplicate using ELISAs manufactured by The three assays. Standard and sample(s) Transfer Vol Starting concn (IU/ml) Amt (l) of sterile distilled water Working concn (IU/ml) TE-3 1 2 to 8 480 7.00 360 0 120 7.00 5.25, 3.94, 2.95, 2.21, 1.66, 1.25, and 0.93, respectively NIBSC 76/589 9 10 to 16 300 1.00 180 0 120 1.00 0.60, 0.36, 0.33, 0.13, 0.08, 0.05, and 0.03, respectively
  11. 11. Serum samples The ELISAs were compared using 83 serum samples collected in 2007 and submitted for diagnostic testing. These samples would have been discarded but instead were stored at 20°C prior to testing.
  12. 12. Antibody assays Each of the ELISAs detects IgG antibodies to tetanus toxoid by an indirect technique.  Testing was performed on thawed serum samples and reconstituted reference materials in strict accordance with the manufacturers’ specifications using reagents that were supplied with the kits.
  13. 13.  Serially diluted NIBSC standards were run in quadruplicate at each dilution.  Serum samples were assayed once in accordance with general laboratory procedures.  Each run included high and low control solutions provided by the manufacturers.
  14. 14. Analysis of data • Intra-assay imprecision was calculated as means with a coefficient of variation for the quadruplicate runs. The recovery percentage was calculated by dividing the final result with the expected result and multiplying by 100. Linear regression analysis was performed by using the mean of the quadruplicate results of each reference standard dilution for each ELISA.
  15. 15. Results  Results of controls provided by all manufacturers’ ELISAswere within acceptable limits.  Intra-assay imprecision ranged from 1.22 to 16.68%  for the Scimedx ELISA, from 0.00 to 9.11%  for The Binding Site ELISA, and from 2.96 to24.62% for the Euroimmun ELISA.  Reproducibility of ELISA tests with dilutions of reference standards at levels near the protective antibody titer of 0.1 IU/ml
  16. 16. The Binding Site ELISA yielded nonprotective antibody concentrations in only these 3 samples, whereas 19 samples (22.9%) according to the Scimedx ELISA and 6 samples (7.2%) according to the Euroimmun ELISA demonstrated nonprotective Concentrations .
  17. 17. Results of 21 samples showing nonprotective antibody levels Scimedx (cutoff 0.10 IU/ml) The Binding Site Euroimmun <0.10 <0.10 <0.10 <0.10 <0.10 <0.10 0.15 0.1 <0.10 <0.10 <0.10 <0.10 <0.10 <0.10 <0.10 <0.10 <0.10 <0.10 <0.10 <0.10 <0.10 0.07 2.09 0.15 0.89 0.28 0.19 0.14 0.19 0.10 0.11 0.08 0.06 0.31 0.32 1.06 0.25 0.54 0.44 0.62 2.06 2.23 0.05 3.64 0.13 1.29 0.25 0.26 0.08 0.06 0.07 0.12 0.07 0.05 0.80 1.17 4.18 0.84 2.19 1.88 3.63 >10.0 >10.0 6 samples Non protected Ab level 7.2% 3 samples only Non protected Ab level 3.6% 19 samples Non protected Ab level 22.9%
  18. 18. testing for tetanus antitoxin levels is frequently used for academic purposes or to test the immunogenicity of vaccines that are in commercial development, specific recommendations have been issued for their appropriate uses in clinical practice. In the 2006 Recommendations of the Advisory Committee on Immunization Practices, there are several guidelines that describe when testing for serum tetanus levels would be warranted.
  19. 19. The present study provides valuable comparative data for laboratories that are evaluating different manufacturers’ products for routine use in their own centers. Each product that was evaluated here performed well relative to internal precision, linearity, and internal controls.
  20. 20. THANK YOU

×