2008 PBL Cat_mech_r.qxd:PBL Catalog.mech 11/29/07 1:50 PM Page 19
Application Note
Plasma studies using the VeriKine
TM
Human Interferon Alpha Multi-Subtype Serum ELISA Kit
Yognandan Pandya, Laura Sheridan, Thomas B. Lavoie, Ph.D. and William A. Clark, Ph.D.
PBL InterferonSource, 131 Ethel Road West, Suite 6, Piscataway, NJ 08854
Abstract
Accurate detection of Interferons (IFN) in serum or plasma samples by Enzyme-Linked Immunosorbent Assay
(ELISA) is affected by interference from components in serum or plasma such as heterophilic antibodies and
clotting factors. In such cases, to evaluate the performance of an ELISA kit, a known concentration of IFN is
added into IFN-free plasma and the percentage difference between the concentration detected by ELISA and
the actual amount is determined. This is known as Spike Recovery. In this technical note, we report our results
on Spike Recovery of Human IFN-α using varying dilutions of human plasma.
Introduction
The ELISA is based on the binding of a target molecule (analyte/ assay, the plate was read at 450 nm on a Vmax plate reader (Molecular
antigen) to antibodies which specifically recognize the target. The Devices Corporation, CA). The Mean OD @ 450nm on Y-axis was plotted
presence of an antigen-antibody complex is detected using a against the actual concentration in pg/ml on X axis for each curve. The
secondary enzyme-conjugated antibody. Detection is obtained by curves were generated using non-linear 4-Parameter fit (Figures 1 and
addition of a substrate which yields a quantifiable signal. Standard 2). Based on the curve fit equation and the Mean OD value for the data
curves are prepared by performing a dilution series of a known point, the extrapolated concentrations (backfit concentrations) were
concentration of the analyte using different sample diluents. The non- determined for each data point. All data was analyzed using SoftMax Pro
linear 4-Parameter fit of the curves are then used to calculate the version 5 (Molecular Devices Corporation, CA).
concentration of an unknown sample in the similar diluent. The analyte
to be measured can be present in different diluents, such as tissue Results
culture media with serum, neat serum, neat plasma, and salt buffers. In From Tables 2-4, it is seen that the LLOQ and MSL values for all matrices
cases where the analyte to be measured is in serum or plasma, (curves) are lower than the mean OD values for the lowest point on the
interference in the assay may arise due to presence of serum proteins, curve (S1). This indicates an excellent curve fit for standard curves
heterophilic antibodies, and/or clotting factors. This may lead to prepared using the three different plasma matrices at all dilutions.
detection of false positives, false negatives, or high backgrounds. In Additionally, this separation between the OD values for blanks and the OD
this study we show that by using PBL InterferonSource VeriKine
TM
value for the prescribed lowest point on the curve indicates that there is
Human Alpha Multi-Subtype Serum Sample ELISA kit (PBL product no adverse effect of plasma components on the sensitivity of the assay
#41110), greater than 90% of actual concentration of Human Interferon for the indicated range. It is notable that the OD values for data points on
(IFN) Alpha can be detected in diluted human plasma. Additionally, we standard curves prepared using plasma dilutions are ∼25% lower than for
show that false positives and false negatives at low concentrations of the corresponding data points on curve prepared using Sample Diluent.
IFN are not seen, and high signals for blanks are absent. However, all curves are within internal QC specifications.
From table 1, the mean spike recovery of IFN-α from Human Plasma
Methods and Analysis with Na-EDTA is greater than 90%. This coupled with the data from tables
Standard curves were prepared according to the kit protocol in Sample 2-4 and figure s 1 and 3 (which show similar standard curves for
Diluent provided in the ELISA Kit (PBL product #41110); and in Human Human Plasma with Na-EDTA, Na-Heparin and Na-Citrate dilutions)
Plasma (Na-EDTA, Na-Heparin and Na-Citrate preparations), each indicate that the spike recoveries using Human Plasma with Na-
diluted to 75%, 50% and 25% in Sample Diluent. Additionally, Human IFN Heparin and Na-Citrate will also be ∼90%.
Alpha was spiked into 75% and 50% Human Plasma with Na-EDTA to Note: On repeated freeze thaw (FT) cycles, we observed increased
achieve independent concentrations of 25, 250 and 500 pg/ml. The aggregation of plasma. However, one FT cycle of diluted spiked plasma
ELISA was performed as per the kit protocol. After completion of the did not affect adversely the detection of the spike (data not shown).
www.interferonsource.com Tel: +1 732-777-9123
I
Fax: +1 732-777-9141
19
Page 1  |  Page 2  |  Page 3  |  Page 4  |  Page 5  |  Page 6  |  Page 7  |  Page 8  |  Page 9  |  Page 10  |  Page 11  |  Page 12  |  Page 13  |  Page 14  |  Page 15  |  Page 16  |  Page 17  |  Page 18  |  Page 19  |  Page 20  |  Page 21  |  Page 22  |  Page 23  |  Page 24  |  Page 25  |  Page 26  |  Page 27  |  Page 28  |  Page 29  |  Page 30  |  Page 31  |  Page 32  |  Page 33  |  Page 34  |  Page 35  |  Page 36  |  Page 37  |  Page 38  |  Page 39  |  Page 40  |  Page 41  |  Page 42  |  Page 43  |  Page 44  |  Page 45  |  Page 46  |  Page 47  |  Page 48  |  Page 49  |  Page 50  |  Page 51  |  Page 52  |  Page 53  |  Page 54  |  Page 55  |  Page 56  |  Page 57  |  Page 58  |  Page 59  |  Page 60  |  Page 61  |  Page 62