الفهرس 
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Abstract
Conclusion:
During the present study, the radioimmunoassay kit for human chorionic gonadotropin was prepared and can be used to estimate the concentration of HCG in human serum with high efficiency. The validity of this specific, sensitive, precise, and accurate method should be suited for routine laboratory use. It has been used effectively in monitoring pregnancy and also can be used as a decisive diagnostic kit for gestational trophoblastic diseases in the hope of early diagnosis and treatment.
Recommendation:
The study presented here may be of interest particularly in developing countries, which may prefer our procedures to be used for HCG estimation in human serum, instead of using the more expensive diagnostic kits available from commercial sources. It is felt that this locally developed RIA technique will be of great use for HCG assay in clinical laboratories.
SUMMARY
This thesis aims to produce a valid technique with a low-cost radioimmunoassay system (RIA) to measure the glycoprotein so-called human chorionic gonadotropin (HCG) in human serum. Estimation of HCG in human serum is a very important tool in monitoring pregnancy and enables the early diagnosis, treatment and follow-up of gestational trophoblastic diseases.
Human chorionic gonadotropin is a glycoprotein hormone, that can be considered the wonder of today’s sciences, there are several variants of HCG each one play a critical function in pregnancy, among these variants regular HCG, has many functions; maintenance of corpus leutum that allows continuous production of progesterone during early pregnancy, blastocyte formation, hemochorial placentation, angiogenesis, and fetus protection against immunological attack from maternal blood. Hyperglycosylated HCG plays a critical role in implantation and invasion of the blastocyte into the uterus and has a major role in malignancy of most cancers, recently the new trend for the potential cure of cancer is the use of specific antibodies for hHCG.
The problem of nowadays market HCG immunometric assay kit is that almost all of them use monoclonal antibodies for HCG C- terminal peptide, where not all HCG variants share CTP, this will cause underestimation of HCG, the invaluable advantage of HCG-radioimmunoassay is that it uses polyclonal antibodies for βHCG that ensure the selective binding to HCG without crossreactivity with homologous glycoproteins and bind with coreβ1 and coreβ2 epitopes that found in almost all HCG variants, make the locally prepared HCG-RIA sensitive and 100% specific for HCG estimation.
Human chorionic gonadotropin, the raw material of development of HCG-RIA, was extracted and purified from urine of pregnant women at the 10th week of gestation, using centrifugation, acetone and ethanol precipitation, dialysis using a semi-permeable membrane with a cutoff of 14 kDa, then captured using weak anion exchange chromatography DEAE Sepharose FF, and polished by purification using gel filtration chromatography Sephacryl S200.
Specific βHCG polyclonal antibodies were produced by injecting rabbits with βHCG subunits that dissociated from HCG using urea treatment method, specific separating agent goat anti-rabbit IgG 2ry antibody were prepared by injecting goats with rabbit IgG during four months of immunization; one primary and four subsequent boosters were injected and antisera were characterized by measuring the antibody titer and displacement.
Radiolabelling of HCG with radioactive iodine (125I) using the previously optimized chloramine-T method to produce 125I-HCG tracer with high purity and specific activity.
HCG standard solutions were prepared in assay buffer matrix and calibrated to be used in HCG-RIA standard curve setup. Finally, the optimum conditions for radioimmunoassay were studied and formulated. The validity of this locally prepared HCG-RIA system was confirmed to accurately estimate the level of HCG in human serum.
The results showed that, human chorionic gonadotropin was purified with purity greater than 98.2%, recovery rate greater than 60% and molecular weight of 36.7 KDa as documented by SDS-PAGE analysis, polyclonal antibodies for βHCG were produced as a component of radioimmunoassay system with high specificity and displacement capability between the different standards, low, medium and high with high efficiency. HCG radioiodinated tracer was prepared with radiochemical yield equal to 71.3%, a radiochemical purity equal to 99.2 %, and specific activity 170.42 µCi/µg. The result revealed that the locally prepared standard solutions of HCG in assay buffer matrix is matched with the international standards obtained from (TOSOH Kit) with correlation coefficient ‘r’=0.998.
The results of validation tests revealed that high sensitivity 0.5mIU/ml, high specificity for HCG without any cross-reactivity with related glycoproteins FSH, LH, and TSH, high precision with low coefficient of variation %, and high accuracy. Locally prepared HCG-RIA system was compared with the commercially available Kit (TOSOH), the results revealed strong positive correlation, where ‘r’ value equal to 0.998.
Using the locally prepared low cost HCG-RIA system, HCG in human serum could be estimated with high sensitivity, specificity, accuracy, and precision, for monitoring pregnancy and for diagnosis of gestational trophoblastic diseases.