Development & validation of an ELISA for quantification of soluble IFN-β receptor Research Article


M1.2.


200 116


45 66 97


31


14 21


IFNAR2.3-6xHN 2.5


0.0 0.5 1.0 1.5 2.0


110100 Concentration


STD#1 (Standards: Concentration vs mean OD val.


Y = ((A - D)/(1 + (x/CB)) + D: AB CD R2 0.118 1.12 415.276 6.91


1


Figure 1. Detection of sIFNAR2 by western blot and standard curve of the ELISA. (A) Purified recombinant sIFNAR2 was resolved in 12% SDS-PAGE (1.), followed by western blot analysis using IFNAR2 purified MaxPab rabbit polyclonal and antibody and phosphatase alkaline-coupled goat anti-rabbit IgG. A colorimetric reaction using NBT/BCIP solution was used for visualization, which showed the 30 kDa band corresponding to the size of purified sIFNAR2 (2.). (B) Representative standard curve of sIFNAR2. The correlation coefficient was equal to 1, y-intercept (A) was equal to 0.118 (the response value at 0 standard concentration), and the slope of the regression line (B) was 1.12. The estimated response at infinite standard concentration (D) was 6.91. M: Molecular weight marker.


ELISA This protocol describes the ELISA procedure used for the validation process. Concentrations of capture and secondary antibodies, as well as crossreactivity, pH, incubation temperature and time were optimized previously (not shown). Background signal obtained with all the possible


combinations of the antibodies (capture, detection and enzyme-linked secondary antibodies), in the absence of sample, is shown in the Supplementary Figure 3, along with a standard curve and a negative control. The strategy and steps followed for the develop- ment and validation of


the ELISA are shown in


Supplementary Figure 4. Amino surface 96-well microtiter plates were coated


with 0.2 μg rabbit polyclonal antihuman IFNAR2 anti- body (Abnova) in 100 μl/well coating buffer (0.05 M carbonate-bicarbonate, pH 9.6) and incubated (over- night, 4°C). Wells were then emptied, washed four- times with 300 μl/well washing buffer (WB:Tris-buff- ered saline + 0.05% Tween-20) and blocked with 200 μl/well casein blocking buffer (2 h, RT). The plate was washed four-times with WB, and 50 μl standard curve or serum samples (diluted1:2) were added to wells in duplicate and incubated (1 h, RT). Blocking buffer was used as a blank. Plates were washed and 50 μl mouse


future science group nm in a VERSAmax microplate reader. SO4


polyclonal antihuman IFNAR2 antibody (1 μg/ml in assay buffer; Abnova) added per well and incubated (1 h, RT). After three washes, 50 μl horseradish per- oxidase (HRP)-conjugated goat antimouse IgG (H+L) adsorbed against human immunoglobulins (diluted 1/10,000 in blocking buffer; Southern Biotech) was added to each well and incubated (1 h, RT). After three additional washes, 100 μl/well TMB One Component HRP Microwell Substrate (BioFX Laboratories) was added and incubated (10–15 min, RT, in the dark). Color development was terminated by adding 50 μl/well 1 N H2


. Optical density (OD) was measured at 450


Sample analysis sIFNAR2 concentration in human serum samples was evaluated in duplicate. Each assay included a standard curve with seven concentrations, two quality controls and a negative control. The sIFNAR2 concentration was determined by OD interpolation from the samples and controls in the standard curve. The calibration curve was established using a four-parameter curve fitting model (Softmax Pro, Molecular Devices). Sample measurement was considered acceptable


if the coefficient of variation (CV) of the duplicates was <10% and the interassay CV of the standard was


www.future-science.com 2871 1000


Standard curve


Mean OD value


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  |  Page 63  |  Page 64  |  Page 65  |  Page 66  |  Page 67  |  Page 68  |  Page 69  |  Page 70  |  Page 71  |  Page 72  |  Page 73  |  Page 74  |  Page 75  |  Page 76  |  Page 77  |  Page 78  |  Page 79  |  Page 80  |  Page 81  |  Page 82  |  Page 83  |  Page 84  |  Page 85  |  Page 86  |  Page 87  |  Page 88  |  Page 89  |  Page 90  |  Page 91  |  Page 92  |  Page 93  |  Page 94  |  Page 95  |  Page 96  |  Page 97  |  Page 98  |  Page 99  |  Page 100  |  Page 101  |  Page 102  |  Page 103  |  Page 104  |  Page 105  |  Page 106  |  Page 107  |  Page 108  |  Page 109  |  Page 110  |  Page 111  |  Page 112  |  Page 113  |  Page 114  |  Page 115  |  Page 116  |  Page 117  |  Page 118  |  Page 119  |  Page 120  |  Page 121  |  Page 122  |  Page 123  |  Page 124  |  Page 125  |  Page 126  |  Page 127  |  Page 128  |  Page 129  |  Page 130  |  Page 131  |  Page 132  |  Page 133  |  Page 134  |  Page 135  |  Page 136  |  Page 137  |  Page 138  |  Page 139  |  Page 140  |  Page 141  |  Page 142  |  Page 143  |  Page 144  |  Page 145  |  Page 146  |  Page 147  |  Page 148  |  Page 149  |  Page 150  |  Page 151  |  Page 152  |  Page 153  |  Page 154