Application Of Biochip Methodology In Studies Of Specific Antigenecity And Quantitative Assay Of Hemoglobin

In this study, we established a protein chip system for detecting specific Hb and/or related derivatives by using protein chip technologies based on a specific antigen-antibody binding mechanism. This system had two formats, namely, hemoglobin antigen-based protein chip approach (Ag-chip) and anti-hemoglobin antibody-based protein chip approach (Ab-chip). In our studies, westrn blot and antigen-chip format were used to qualitatively and/or quantitatively study the specific antigenic nature of hemoglobin of human, bovine or porcine origin. Furthermore, the major properties of an antigen-chip based assay and an antibody-chip based method were compared.In order to understand the variations in dominant antigen determinants of hemoglobin of human, bovine or porcine origin, this study detected the antigen antibody cross-binding reaction between one specific anti-hemoglobin antibody and different hemoglobins and/or related derivatives using specific antigen-antibody binding mechanism, the degree of cross-binding reaction was also measured by antigen-chip based assay. The qualitative detecting of westrn blot with mouse anti-hemoglobin antibodies found that one specific mouse antibodies only weakly cross-reacted with hHb, bHb or pHb and/or related derivatives. Using the method of quantitative protein chip, it showed that the cross reaction between rabbit anti-hemoglobin antibodies and the Hb/PolyHb of other two species concerned was very weak. Rabbit antibodies against pPolyHb barely bound to hHb/hPolyHb or bHb/bPolyHb, cross-reaction rates was less than 3.5%; rabbit antibodies against bPolyHb also had very low cross-reacting rates (only 4% and 6% respectively) with hHb/hPolyHb and pHb/pPolyHb; but, the biggest rate of rabbit antibodies against hPolyHb cross-bound with bPolyHb could arrive 24.77%, and they cross-reacted with pHb/pPolyHb at a rate of less than 7.5%. Furthermore, the study of quantitative protein chip found that rat antibodies against hPolyHb, bPolyHb or pPolyHb only slighty or even did not cross-react with the Hb/PolyHb of other two species. We also discovered Mg²⁺ differentially inhibited specific binding interactions between a rabbit anti-hemoglobin antibody preparation and its cognate hemoglobin antigen of human, bovine or porcine origin. The inhibition was strongest to pHb, the inhibition ratio was more than 85%; next was bHb, the inhibition ratio was about 50%; and the last was hHb, less than 20%. This study indicated that hHb, bHb and pHb have independent dominant antigen determinants, despite very high degree of identity in their primary amino acid sequences.This study compared the properties of Ag-chip and Ab-chip in quantitative measurement of pPolyHb (such as the linearity, linear range, sensitivity) and applications in detecting pHb and related derivatives in body fluid show that, both Ag-chip and Ab-chip are highly specific, and it is the only method reported capable of measuring specific types of hemoglobin and related derivatives. The two protein-chip methods in quantitative measurement of pPolyHb had a linear range between 1μg/mL and 12μg/mL and sensitivity of 0.5μg/mL, which is 250 times higher than the reported most sensitive method, that is, LC-MS-MS. The Ag-chip demonstrated a good linearity and high-through-put in quantitative measurement of pPolyHb, but it was interfered by body fluid, the specimen must be diluted 50 times before use, it had a sensitivity of 25μg/mL. The Ab-chip was not interfered by body fluid, the specimen could used to detect without dilution, so the sensitivity was 50 times higher than Ag-chip, but with a relatively poor linearity.