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[ TEST PRINCIPLE ]
The microtiter plate provided in this kit has been pre-coated with an antibody specific to IL34. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody preparation specific for IL34. Next, Avidin conjugated to Horseradish Peroxidase （HRP） is added to each microplate well and incubated. After TMB substrate solution is added, only those wells that contain IL34, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substratereaction is terminated by the addition of sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450nm ± 10nm. The concentration of IL34 in the samples is then determined by comparing the O.D. of the samples to the standard curve.
[ CALCULATION OF RESULTS ]
Average the duplicate readings for each standard, control, and samples and subtract the average zero standard optical density. Create a standard curve on log-log graph paper, with IL34 concentration on the y-axis and absorbance on the x-axis. Draw the best fit straight line through the standard points and it can be determined by regression analysis. Using some plot software, for instance, curve expert 1.30, is also recommended. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.
[ TYPICAL DATA ]
In order to make the calculation easier, we plot the O.D. value of the standard （X-axis） against the known concentration of the standard （Y-axis）, although concentration is the independent variable and O.D. value is the
dependent variable. However, the O.D. values of the standard curve may vary according to the conditions of assay performance （e.g. operator, pipetting technique, washing technique or temperature effects）, plotting log of the data to establish standard curve for each test is recommended. Typical standard curve below is provided for reference only.
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