The Anti-HSA Affibody® molecule can be used as capture reagent in a sandwich ELISA in combination with a rabbit anti-HSA antibody as the detection reagent. Titration of HSA gives a sigmoid curve with a sensitivity of 5 ng HSA/ml (defined as two times background value) and a measurement interval between 10 and 100 ng/ ml.
Sera from six different species, human, mouse, rat, goat, bovine and rabbit were titrated in 3-fold dilution series on Anti-HSA Affibody® molecule coated plates. As shown in figure 2, the Anti-HSA Affibody® molecule binds with high affinity to human albumin followed by rat and mouse albumin. Low binding was observed to bovine, goat and rabbit albumin. The species discrepancy is dependent on the binding properties of the Anti-HSA Affibody® molecule as well as the second step rabbit anti-HSA antibody.
The chromatograms represent run number 1, 50 and 300 after consecutive injections of 300 ul of five times diluted human serum on 0.37 ml SulfoLink® Coupling Gel with immobilized Anti-HSA Affibody® molecule.
The identical chromatograms and consistent peak areas of eluted fractions prove that the depletion procedure can be reproducibly repeated at least 300 times without loss of binding capacity.
The high specificity of the Anti-HSA Affibody® molecule is maintained through all the 300 consecutive injections. The capacity of this coupling gel allows for depletion of HSA from 800 ul of five times diluted human serum per ml gel, corresponding to 160 ul of undiluted human serum per ml gel. Lane 1: Untreated 5x diluted serum sample Lane 2: FT (flow through) run 1 Lane 3: FT run 75 Lane 4: FT run 150 Lane 5: FT run 225 Lane 6: FT run 300 Lane 7: eluate run 1 Lane 8: eluate run 300 Lane 9: HSA standard
The remaining HSA in samples from serum, depleted from HSA by passage through an Anti-HSA Affibody® molecule SulfoLink® Coupling Gel column (see "Protocols" link), was quantified using the Anti-HSA Affibody® ELISA. Samples from cycle 25, 150, 200 and 300 were analyzed and the concentrations of HSA in the samples recorded.