Supplementary Methods.

The data described in this section supplements the main text. It provides information about the determination of analytical information for the modified ELISA protocol using standard curve analysis based on a four-parameter logistic in SigmaPlot 11 software; calculation of LOD; and analytical comparison of the developed protocol with the conventional protocol.

Standard curve analysis based on a four-parameter logistic in SigmaPlot 11 software

The procedureused, as described below, is useful approach for plotting ELISA graphs. The method of calculating EC50 is also specified.

The procedure for creating a standard curve (Supplementary Figure 1) for the modified ELISA protocol using SigmaPlot 11 software is described here:

[Click “Pharmacology” in the Sigmaplot 11 toolbar on top of the screen → click “Standard Curves Analysis”→ in “Equation”, use “Four Parameter Analysis” and tick the “Log X axis scale” and plot the graph.]

The software also generates a separate report file (as shown below), while plotting the graph, which contains the analytical information pertaining to Hillslope, EC50, and min and max of the plotted curve. The format of the report generated is shown below:

Equation: Standard Curves, Four Parameter Logistic Curve

f1 = min + (max-min)/(1 + (x/EC50)^(-Hillslope))

f = if(x<=0, if(Hillslope>0,min,max), f1)

R Rsqr Adj Rsqr Standard Error of Estimate

0.990.99 0.99 0.04

Coefficient Std. Error t P

min 0.0038 0.0215 0.1781 0.8631

max 2.8703 0.1000 28.7068 <0.0001

EC50 3331.2840 270.2485 12.3267 <0.0001

Hillslope 1.1322 0.0727 15.5807 <0.0001

(A similar procedure was used for the ELISAsproduced with passive adsorption of antibody)

Calculation of Limit of Detection (LOD).

The calculation of LOD is shown with an example below.

Table depicting the three repeats of a blank and two dilutions of sample ‘X’

Absorbance 1 / Absorbance 2 / Absorbance 3 / Average / Standard deviation
Blank / 0.0485 / 0.0397 / 0.0416 / 0.0432 / 0.0039
Sample dil A / 0.0496 / 0.0413 / 0.0429 / 0.0446 / 0.0083
Sample dil B / 0.0683 / 0.0649 / 0.0701 / 0.0678 / 0.0103

The LOD was calculated using the widely used formula

Absorbance corresponding to the LOD = Average absorbance of the blank from all the repeats + 3 standard deviations

For this data, the absorbance corresponding to the LOD will be

= 0.0432 + 3 (0.0039) = 0.0539

Therefore, the concentration corresponding to the absorbance of 0.0539 on the data curve will be the LOD.

The working/ dynamic range of the assay was determined from the standard curve discussed previously.

Analytical comparison of the developed protocol with the conventional protocol1

Developed protocol / Conventional protocol
DetectionRange (pg/mL) / 4.0-20, 000 / 151.0-20,000
LOD (pg/mL) / 9.0 / 312.0
Analytical Sensitivity (pg/mL) / 39.0 / 625.0
EC50 (pg/mL) / 3, 000 / 23, 000
% CV
Intra-day (n=5)
Inter-day (n=3) / 2.4-10.4
1.7-17.6 / 4.7-17.4
3.6-20.0
Assay duration (hours) / ~6 / ~20
Assays on various substrates / Yes / No

LOD = Limit of Detection; CV = Coefficient of Variation

Intra-day precision was calculated from the five repeats (n=5) of the same assay at different times on a single day, while inter-day precision was calculated from the three assay repeats on three different days (n=3). All the assays were carried out in triplicate. Analytical sensitivity was calculated using the formula [average absorbance of the blank+3(SDblank)].

References:

  1. Dixit, C.K., Vashist, S.K., O’Neill, F.T., O’Reilly, B., MacCraith, B.D. & O’Kennedy, R. Development of a high sensitivity rapid sandwich ELISA procedure and its comparison with the conventional approach, Anal. Chem.82, 7049 (2010).