ELISA: Spectrophotometric Determination of Unknown Bovine Serum Albumin

ELISA or Enzyme-Linked Immunosorbent Assay is a biochemical technique that is used for detecting and quantifying antibodies or antigens. In reading the colorimetric reaction, an equipment called spectrophotometer is used to measure the absorbance of a solution as light of a specified wavelength is passed through it. In this study, Bovine Serum Albumin or BSA which is a serum albumin protein isolated from cows was used, specifically in the determination of its unknown concentration. Serial dilutions of 6 different concentrations (50 mg/mL, 25 mg/mL, 12.5 mg/mL, 6.25 mg/mL, 3.125 mg/mL & 1.5625 mg/mL) of BSA sample is performed, together with one sample containing the unknown BSA concentration and another sample containing water (H20) which served as the negative control. Thereafter, triplicate samples were delicately transferred using a micropipette into an ELISA microplate and each of it received 180 µL of Biuret reagent. After 5 minutes of storage and incubation, absorbance of each sample was read by placing the microplate in the Biotek ELx800 ELISA reader. By further calculations of the average absorbance of each samples for the wavelengths of 450nm, 490nm and 630nm using the MS Excel file in the computer as well as by plotting the calculated absorbance and concentration values of each known samples in a graph, the R2 value for the wavelength of 630nm is used in comparison since it has the highest value. The result gathered after computing the value of x using the equation x = was 25.42 or 25, so the researcher arrived at a conclusion that 25 mg/mL, more or less is the concentration of the unknown BSA sample.