Methodology Research Of Indirect ELISA Test Kit For Measuring Serum Free β-HCG

Objective1. An indirect-coated ELISA of serum freeβ-HCG detection method.2. To discuss the method of decreasing non-specificity coloration in ELISA.3. Evaluation the significance of freeβ-HCG test in the diagnosis and treatment of related diseases.Method1. To establish an indirect-coated ELISA assay of serum freeβ-HCG (1)To prepare BBC enzyme reaction plate, (2) Biotinlation of anti-free P-HCG monoclonal antibody, (3)Label horseradish peroxidase by NaIO4 methods to the anti-free P-HCG polyclonal antibody), (4)Preparation and evaluation of indirect-coated ELISA assay of serum freeβ-HCG.2. To discuss the method of decreasing non-specificity coloration in ELISA (1)To replacement the standard dilution,(2)To adsorbent enzyme labeled polyclonal antibody through affinity column which connected with coated antibody,(3) Added Fc segment of coated antibody in the enzyme-labeled antibody in advance.3. The clinical research of serum freeβ-HCG and its related diseases (1) serum freeβ-HCG test in normal pregnancy and ectopic pregnancy diagnosis, (2) The clinical significance of serum free P-HCG in gestational trophoblastic disease.Result1. To establish an indirect-coated ELISA assay of serum freeβ-HCG:the optimization of various reaction conditions, we determine the optimal working conditions:the optimal concentration of biotin antibody and enzyme-labeled antibody is 1:1000 and 1:12000, the method has better stability, sensitivity 0.1ng/ml, recovery range is 95.92%～102.51%, within-difference is 7.54%～11.12% and between-difference is 12.51%～18.11%, the cross-reaction rate with FSH, TSH, LH,α-HCG<0.1%, detection range 0.1～500ng/ml, and the relevance with imported kits was not good. (r2=0.7892). 2. To discuss the method of decreasing non-specificity coloration in ELISA:The results showed that replacement of the standard dilution and purification of HRP-labeled antibody by affinity column does not effected,but.adding Fc of coated-antibody to HRP-labeld antibody has obvious effect in this experiment.3. The clinical research of serum freeβ-HCG and its related diseases:the serum freeβ-HCG levels of normal pregnancy maternal were significantly higher than ectopic pregnancy patient(p<0.05),and the gestational trophoblastic disease of serum freeβ-HCG levels were significantly higher than normal pregnancy(p<0.05).Conclusion1. We developed an indirect detection method of serum freeβ-HCG, the introduction of the biotin-avidin system to enhance the utilization of antibodies and reduce the "hook effect" in a large extent, has high sensitivity and specificity, good stability and detection range, suitable for clinical application.2. This article discusses the three method to lower non-specific coloration in ELISA, all have some value, especially adding Fc of coated-antibody to the HRP-labeled antibody method, There are still no relevant reports, pending further study.3. Serum freeβ-HCG determination provide an important basis for the diagnosis of the ectopic pregnancy and gestational trophoblastic diseases.