I am planning/performing ELISA assays for the first time. Using a commercially available kit, that has been (semi-)validated by other researchers for the same matrix as I am testing, but from a different species. The sample matrix is stripped by TCA precipitation and sep-pak C18 column (but I assume some matrix components will still be in the sample after..)
I want to perform a partial validation and have planned out a plate layout which is below. My question is: Is this layout correct? In other words, by performing the test outlined below, will I be able to determine:
Limit of detection (LOD)
Limit of quantization (LOQ)
Another question is: is it generally ok to combine accuracy, precision and spike-recovery in to a single series (as seen below). Also can LOD and LOQ be combined?
Appreciate any help!!
ELISA Plate layout: Please see attachment - didn't cut/past well Advise for ELISA validation.docx 14.98KB 194 downloads
1 2 3 4 5 6 7 8 9 10 11 12 A Blk Std1 Std5 AC/PR/SR25 AC/PR/SR100 AC/PR/SR500 LOD5 LOD10 LOQ5 LOQ20 LIN1:4 LIN1:16 B Blk Std1 Std5 AC/PR/SR25 AC/PR/SR100 AC/PR/SR500 LOD5 LOD20 LOQ5 LOQ20 LIN1:4 Neat C TA Std2 Std6 AC/PR/SR25 AC/PR/SR100 LOD1 LOD5 LOD20 LOQ5 LOQ20 LIN1:4 Neat D TA Std2 Std6 AC/PR/SR50 AC/PR/SR100 LOD1 LOD5 LOD20 LOQ10 LOQ20 LIN1:8 Neat E NSB Std3 Std7 AC/PR/SR50 AC/PR/SR100 LOD1 LOD10 LOD20 LOQ10 LOQ20 LIN1:8 Neat F NSB Std3 Std7 AC/PR/SR50 AC/PR/SR500 LOD1 LOD10 LOD20 LOQ10 LIN1:2 LIN1:8 Neat G B0 Std4 AC/PR/SR25 AC/PR/SR50 AC/PR/SR500 LOD1 LOD10 LOQ5 LOQ10 LIN1:2 LIN1:16 N/A H B0 Std4 AC/PR/SR25 AC/PR/SR50 AC/PR/SR500 LOD5 LOD10 LOQ5 LOQ10 LIN1:2 LIN1:16 N/A
AC: Accuracy: closeness of mean test results to the true concentration. Concentrations tested: 25, 50, 100 and 500pg/mL analyte
PR: Precision (intra-assay): closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogenous sample. The closeness of individual measures is determined by applying the procedure repeatedly to multiple aliquots of a single homogeneous volume of biological matrix. Concentrations tested: 25, 50, 100 and 500pg/mL analyte.
SR: Spike-recovery: used to determine if the assay is affected by the difference between the diluent used to prepare the standard curve and the sample matrix. Concentrations tested: 25, 50, 100 and 500pg/mL of analyte.
LOD: Limit of detection: LOD1, LOD5, LOD10, LOD20 (1, 5, 10 and 20pg/ml of analyte added to neat sample)
LOQ: Limit of quantization: LOQ5, LOQ10, LOQ15 (5, 10 and 15 pg/ml analyte added to neat sample)
LIN: Linearity: Tests the effects of matrix components on measurement of the analyte, by serially diluting the extract in assay buffer.
Linearity tests determine the extent to which the dose-response of the analyte is linear in particular diluents. Dilutions to use: 1:2, 1:4, 1:8, 1:16
Neat: Neat (non-spiked) milk sample processed same way as all spiked samples (less spike)
(All the above have 5 replicates, except LIN, which has 3 replicates)
TA: Total Activity. Positive control - that ensures that the conjugate still has activity
NSB: Non-Specific Binding (Background, Neg contr). Contains standard dilutant, buffer, conjugate, substrate, stop soln.
B0: “zero standard”. Contains: standard diluent, conjugate, antibody, substrate, stop solution
Std1-7: Diluted standard in buffer: 1000, 500, 250, 125, 62.5, 31.25, 15.6 pg/ml.
Advise for ELISA validation.docx 14.98KB 194 downloads
Edited by Nicolai, 25 March 2014 - 03:42 PM.