Study On A Double-antigen Sandwich ELISA For Assay Of RHu IFN-ω In Blood On Quantity

As a recently found interferon with broad-spectrum antivirus function, enhancement facilitation on immunological function and depressant effect on cell growth, recombinant interferon-ω(IFN-ω)has important potential in disease treatment. It has been completely investigated in aspects of recombinant Human interferon-ω(rHu IFN-ω) gene expression, purification and nature. The investigation in clinical pharmacokinetics and pharmacodynamics is in progress currently. To achieve it, it becomes necessary to establish a dependable assay method. Compared to varies kinds of methods, enzyme linked immunosorbent assay (ELISA) is found to be accurate, sensitive, specific and simple. double-antigen sandwich ELISA to assay rHu IFN-ωin blood on quantity was established in this study. The research work is as following:①preparation and purification of rabbit polyclonal antibody of rHu IFN-ωNew-Zealand albino rabbits were immunized with rHu IFN-ωand Freund adjuvant to prepare antiserum. The antiserum was purified by tri-degree precipitation with saturated ammonium sulfate (50%, 40%, 33%), and then diethylaminoethyl (DEAE) column for further purification. High pure part was collected step by step .Its antibody titer was detected by indirect ELISA, while concentration by Lowy approach. Finally, the antibody for adouble-antigen sandwich ELISA was obtained.②p reparation of enzyme labeled antibodyThe purified antibodies were cross-linked with HRP (horse radish peroxidase) by glutaraldehyde double-step approach to gain enzyme labeled antibodies. Then the enzyme labeled antibodies were collected by Superdex-200 GEC step by step and separated by ELISA activity experiment as labeled and unlabeled antibodies, unlabeled enzymes. So the enzyme labeled antibodies for double-antigen sandwich ELISA were obtained.③the optimal condition for double-antigen sandwich ELISA and standard curveThe condition optimized by checkboard approach is: antibody coating concentration 50μg/mL, enzyme labeled antibody dilution 1:100. Three standard curves was established for rHu IFN-ωin original serum, 5 times diluted serum and10 times diluted serum. It was indicated that when the concentration of rHu IFN-ωis in the range of 15pg/mL~480pg/mL, standard rHu IFN-ωconcentration natural logarithm is favorably linear correlated with OD. All the standard curves'r are over 0.98. Furthermore, the linear correlation and specificity will increase as the dilution of serum grows.④investigation in methodologyThe double-antigen sandwich ELISA established was investigated in methodological aspect. Linear detect range: 15pg/mL~480pg/mL. Degree of precision in batch: rHu IFN-ωin different concentration were detected 8times, with CV (coefficient of variation) between 1.12% and 5.04%. Degree of precision between batches: rHu IFN-ωin different concentration were detected 7 days continuously, with CV between 3.04% and 115%. Recovery rate: rHu IFN-ωin high, medium and low concentration were detected 6 times, with recovery rate between 103.26%and105.47%, fulfilling the requirement for detection of rHu IFN-ωin blood.The double-antigen sandwich ELISA for assaying rHu IFN-ωin blood on quantity is accurate, sensitive, specific and simple. It can offer methodological base and experimental parameter for the pharmacokinetics and clinical research of rHu IFN-ω.