An international comparability study to determine the sources of uncertainty associated with a non-competitive sandwich fluorescent ELISA.
BACKGROUND
Immunoassays allow the specific detection and quantitation of biological molecules in complex samples at physiologically relevant concentrations. However, there are concerns over the comparability of such techniques when the same assay is performed by different operators or laboratories. An international intercomparison study was performed to assess the uncertainty involved in the estimation of a protein cytokine concentration using a fluorescent ELISA.


METHODS
The intercomparison study method was based on a non-competitive sandwich immunoassay with an enhancement step to generate a fluorescent readout. The intercomparison was performed in two phases, with the uncertainty of the instrument determined separately from that of the assay. The 11 laboratories participating in the study represented national metrology institutes or nominated expert laboratories.


RESULTS
Participants were asked to determine an undisclosed concentration of interferon using a supplied standard. The mean participant estimate and experimental standard deviation of the mean was 3.54+/-0.22 mg/L, with the spread of data ranging around +/-35% of the mean. The quantitation range of the ELISA and of participants' instruments displayed large variation that contributed to the overall uncertainty.


CONCLUSIONS
Identified sources of uncertainty within the ELISA methodology included pipetting, data fitting, model selection and instrument/plate variation.
Noble
James E
JE
Analytical Science Group, National Physical Laboratory, Teddington, UK. james.noble@npl.co.uk
Wang
Lili
L
Cerasoli
Eleonora
E
Knight
Alex E
AE
Porter
Robert A
RA
Gray
Elaine
E
Howe
Chris
C
Hannes
Elisabeth
E
Corbisier
Philippe
P
Wang
Jing
J
Wu
Liqing
L
Altieri
Ilaria
I
Patriarca
Marina
M
Hoffman
Angelika
A
Resch-Genger
Ute
U
Ebert
Bernd
B
Voigt
Jan
J
Shigeri
Yasushi
Y
Vonsky
Maxim S
MS
Konopelko
Leonid A
LA
Gaigalas
Adolfas K
AK
Bailey
Marc J A
MJ
D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000032:analysis*; D011994:Recombinant Proteins / Q000032:analysis; D012680:Sensitivity and Specificity; D035501:Uncertainty
D000077190:Interferon alpha-2; D016898:Interferon-alpha; D011994:Recombinant Proteins