| Background and objectives: Hepatocellular carcinoma (HCC) is one of the most common malignancies in China. At present, surgical resection is still the main method to treat it. The key factors that influence the prognosis of HCC are early discovery, early diagnosis, and early treatment. The best way to discover patients of HCC early is the determination of the level of serum alpha-fetoprotein (AFP) and the image examination in the high risk of HCC population (chronic hepatitis B, chronic hepatitis C, and cirrhosis) at regular intervals.We established hybridoma lines which secret anti-AFP McAb by hybridization technique. After a series of identification, two monoclonal antibodies-3A8 and 1E6 which have high specificity, high affinity, and direct to different epitope of AFP were selected to make colloidal gold immunochromatographic assay (CIA) strip for qualitative analysis of serum AFP. The method is based on the "sandwich" assay format using monoclonal antibodies (McAbs) of two distinct specificities. McAb 1E6 was labeled to 25nm in diameter of colloidal gold granules while McAb 3Ag was immobilized to define detection zone on a porous nitrocellulose membrane (NCM). This method is a rapid, specific,sensitive, and convenient tool for diagnosis of HCC in hospital and survey spot.Method: 1.Establish of hybridoma lines which secrete anti-AFP McAb by fuse the spleen cells from immuned BALB/c mice with purified AFP and myeloma of mouse SP2/owith 50% polyethylene glycol (PEG, MW.1450). Hybridomas were screened by enzyme-link immunosorbent assay (ELISA). Hybridomas whose supernatants show stronger positive were subcloned by limited dilution methods 3-4 times, until the positive rate come up to 100%. The desired hybridomas were expanded and some of them are reserved in liquid nitrogen as reservoir, the others of them are injected into BALB/c mice by intraperitoneal injection, which were primed by intraperitoneal injection of 0.5ml liquid paraffin before 5-7 days. The ascitic fluid which contains McAb was collected 10-14 days after injection of the hybridoma cells. The specificity of McAb were identified by methods of competitive inhibition, immunohistochemistry, and western blotting. The affinity constant of McAb was determined with ELISA. The isotypes of the McAb were determined with a subisotype kit according to the manufacturer's instruction.2. The colloidal gold granules whose diameter is about 25nm were prepared by using trisodium citrate reduction of solution of chloroauric acid. The makers were prepared by labeling the colloidal gold granules with McAb 1E&. The mixture was absorbed to glassfibre and then was lyophilized.3. Another McAb against to AFP and rabbit anti-mouse immunoglobuin were diluted to 2mg/ml and 4mg/ml in 0.01M pH 7.2 PBS respectively. Then on NCM two lines were drawn with the two solutions between which the distance was about 4mm used as captive antibodies to capture AFP in sample and cAu-McAb 1E& respectively.4. The PVC plate, glassfiber containing cAb-McAb 1E&, NCM on which hadbeen coated McAb against to AFP and PcAb against to mouse immunoglobulin, and absorbent paper pad composed into a plate and was cut into 70mm X 3mmstrips.5. The temperature influence on the quality of strips of temperature wasobserved by putting the strips in 37 ℃ and 4℃ respectively and check themregularly.6. Dry the used strips in the room temperature and then scale them into smallplastic bags and put them into 4℃, and observe the time of preservation.7. Use the strips to test a series of panels obtained from the National Institute for the Control of Pharmaceutical and Biological Products in order to determine the lowest limit.8. Dilute the high concentration AFP sample in diluted series and then test it with strips to determine the testable rang.9.Use the CIA strips and ELISA kit to test 147 sera samples, which have been tested by RIA, including 72 positive and 75 negative to get the strips' sensitivity and specificity compared with RIA and ELISA.Result...
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