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ELISA samples should be measured in two replicates or three replicates. This provides sufficient data for statistical verification of the results. There are many computer programs available to process ELISA results in this manner.
Calculating the mean absorbance value measured is repeated standard and sample replicates per group. The deviation of each value of the repeated measurement from the average should be in the range of 20%.
standard curve line
Average absorbance of X -axis, the concentration of the target protein on the Y axis of the target protein standard curve. Draw the best fit curve from each point in the graph (recommended using the relevant computer program).
We recommend adding standards to each plate to ensure that each plate can be used to make a standard curve.
We recommend using a sample of known concentration as a positive control. For effective and accurate results, the concentration of the positive control sample should be within the linear region of the standard curve.
Target protein concentration in the sample
To determine a target protein concentration of each sample, first we need to obtain an average absorbance value of the sample. Then draw a horizontal line that intersects the standard curve from this value on the Y- axis of the standard curve.
For example, if the absorbance reading is 1 , draw this line (a) from that point on the Y- axis :
At the intersection, draw a line perpendicular to the X axis and read the corresponding concentration (b) .
Samples with absorbance values outside the range of the standard curve
To get accurate results, these samples should be diluted before proceeding with ELISA staining. If such a sample is used, the concentration obtained from the standard curve must be multiplied by the dilution factor when analyzing the results.
The coefficient of variation (CV) is the ratio of the standard deviation σ to the mean μ:
C v = σ
This data is the percentage of the variance as a percentage, representing the inconsistency and inaccuracy of the results. The larger the variance, the worse the consistency and accuracy of the results. Some computer programs are capable of calculating the CV value of an ELISA result.
Large CV can be caused by the following reasons:
The spiked recovery rate determines the effect of sample components on antibody antigen detection. For example, a large amount of protein contained in the tissue culture supernatant may hinder antibody binding and increase the signal-to-noise ratio, resulting in a concentration lower than the actual value.
A known concentration of protein is added to the sample matrix and standard diluent. The spiked protein was quantified using the same assay and the results of the sample matrix and standard diluent were compared.
If the results are the same, the sample matrix can be considered to be suitable for the assay procedure. If the recovery rates are different, the components in the sample matrix interfere with analyte detection.
What if the spike recovery experiment indicates that the sample matrix has an effect on the results?
We recommend diluting the standard in the sample matrix to obtain a standard curve. All effects of the sample matrix on the results are also present in the standard, so the comparison between the standard curve and the sample is more accurate. Many of our ELISA kits contain standard serum diluents specifically for this purpose.
Another solution is to change the sample matrix. For example, if an undiluted biological sample is used, the sample can be diluted in a standard diluent. With this approach, however, you need to ensure that the dilution factor is taken into account when analyzing the results and that the concentration remains within the linear region of the standard curve.