Study Of Selective And Quantitative Measurement Of Hemoglobin And/or Related Derivatives In The Body Fluid

Objectives:This study established a high selective, high sensitive and high-through-put qualitative and/or quantitative methodologies for detecting specific hemoglobin (Hb) and/or related derivatives in body fluid using a protein chip technology based on a specific antigen-antibody binding mechanism.Methods:With Western-blot method and a protein chip method which is in an assay format of solid phase-hemoglobin or other protein sample-Cy3-labbled anti-hemoglobin antibodies were used to evaluate the extent with which a sample of antibodies specific for porcine glutaraldehyde-cross-linked hemoglobin (pPolyHb) cross-reacted with human glutaraldehyde-cross-linked hemoglobin (hPolyHb) or bovine glutaraldehyde-cross-linked hemoglobin (bPolyHb) and human hemoglobin (hHb) or human plasma(HP). The properties of this method in quantitative measurement of specific hemoglobin was studied. antigen(Ag) chip and antibody(Ab) chip were used to detect the interferer of the human plasma and urine ,establishded the method of Selective and Quantitative Measurement of Hemoglobin and/or Related Derivatives in the body fluid;Explored the applied feasibility of protein chip in the pharcokinetics(PK) of glutaraldehyde-cross-linked hemoglobin (pPolyHb) in the rats.Results:The anti-pPolyHb-antibodies did not react with hPolyHb bPolyHb hHb and HP.The protein-chip method demonstrated a good linearity in quantitative measurement of pPolyHb with a linear range between 1ug/ml and 12 μg/mL and sensitivity of 0.0005 g/L. There were reasonable measurement deviations between the parallel samples spotted on different glass-slides in a single experiment. Including a reasonable number of parallel samples could increase the quality of the quantitativemeasurement. The errors between different experiments were random and difficult to control. The Ag-chip method demonstrated a good linearity in quantitative measurement of pPolyHb in body fluid with a linear range between 50 fig/mL and 600ug/ml and sensitivity of 0.025 g/L. The Ab-chip method demonstrated a bad linearity, as it was not interfered by the body fluid, with sensitivity of 0.0005 g/L. The protein chip method demonstrated a good application in the pharcokinetics(PK) of glutaraldehyde-cross-linked hemoglobin (pPolyHb) in the rats. The lO^-lZ* Tm rat serum which was detected by the Ab-chip was positive.ConclusionrThe anti-pPolyHb-antibodies has such a high quality to be used in methods based on specific antigen-antibody binding mechanism for detection of porcine hemoglobin and its derivatives;Ag- chip method and Ab-chip method may be used to establish a system for high selective, high sensitive and high through-put qualitative measurement of specific types of hemoglobin and related derivatives. The protein chip can be used as the detection method of the pharcokinetics(PK) ofglutaraldehyde-cross-linked hemoglobin (pPolyHb)...