Bovine Albumin ELISA Assay

Additional Information

Assay Background

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This Bovine Albumin ELISA Assay Kit furthermore allows quantification of albumin in bovine urine to estimate kidney function. When blood is filtered in glomeruli of kidneys, highly molecular substances are held back. There­fore, urine is nearly protein free. In case of kidney damages, small proteins like albumin appear in urine.  Research has demonstrated that albumin therefore serves as a marker in moni­toring kidney damages.

Assay Principle

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The determination of albumin is carried out as direct competitive ELISA. Bovine albumin has been pre-coated onto a microplate. During incubation the binding of an enzyme-linked anti-albumin (bovine)-antibody to the wells is inhibited by albumin from samples/standards. After washing away any unbound antibody, a substrate solution is added to the wells and color develops in proportion to the amount of antibody conjugate. The absorption at 450 nm is reverse proportional to the albumin con­cen­tration.

Assay Procedure

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1. Prepare all reagents, standard curve and samples as directed in the previous section.
2. Pipette 50 µl of samples, standards or diluent (as negative control) into the wells.
3. Immediately add 50 µl of HRP conjugate to each well.
4. Mix gently.
5. Seal wells with adhesive strip and incubate for 2 hours at room temperature.
6. Aspirate fluid from wells and wash three times with 300 µl wash buffer.
7. After the last wash, invert the plate and tap on a clean paper towel.
8. Dispense 100 µl of TMB substrate solution into each well.
9. Incubate for 20 minutes at room temperature in the dark.
10. Add 100 µl of stop solution to each well.
11. Determine the absorbance within 30 minutes at 450 nm. A reference wavelength of 620 nm/690 nm is recommended.

Typical Standard Curve

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