Comparison of ovalbumin quantification using forward-phase protein microarrays and suspension arrays.
We employed ovalbumin (a simulant used for ricin and botulism toxins in biodefense applications) and its high affinity polyclonal antibody as a model system to examine the sensitivity, dynamic range, linearity, and reproducibility of forward-phase array results in comparison to suspension arrays. It was found that protein microarrays had a dynamic range of 4 orders of magnitude and a sensitivity of less than 1 pg/mL, respectively. The dynamic range and sensitivity of suspension arrays were close to 2 orders of magnitude and 0.25 ng/mL, respectively. The sensitivity we observed for the suspension arrays is comparable to that reported for enzyme-linked immunosorbent assays (ELISAs) in the literature. We used ovalbumin samples with two different purities, 38.0% and 76.0% (w/w), as determined by polyacrylamide gel electrophoresis (PAGE). These samples were used to evaluate the effect of impure samples on detection. The data obtained from the forward-phase protein arrays gave values that were consistent with the PAGE data. The data from the suspension arrays were not as consistent and may indicate that this format may not give as reliable data with impure samples. Knowledge of the advantages and disadvantages of the two proteomic methods would allow their more rational use in clinical diagnosis.
Wang
L
L
Biochemical Science Division, National Institute of Standards and Technology, Gaithersburg, Maryland 20899-8312, USA. lili.wang@nist.gov
Cole
K D
KD
He
Hua-Jun
HJ
Hancock
D K
DK
Gaigalas
A K
AK
Zong
Y
Y
D000818:Animals; D002645:Chickens; D010047:Ovalbumin / Q000032:analysis* / Q000302:isolation & purification; D040081:Protein Array Analysis / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D010047:Ovalbumin