Preparation Of Hybridoma Cell Strains Secreting Monoclonal Antibodies Against Okadaic Acid

ObjectivePreparation of hybridoma cell strains secreting monoclonal antibodies (McAb) against okadaic acid (OA) using B lymphocytes hybrid technique, which make a base for on a large scale production of McAb against OA and development a method of enzyme-linked immu-nosorbent assay (ELISA) to detect okadaic acid (OA) in the furture.Methods1. Preparation of antigenAccording to carbodiimide method, OA, bovine serum albumin (BSA) or ovalbumin (OVA), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride ( EDC) and N-hydroxy sulfosuccmimide (Sulfo-NHS) are dissolved in phosphate buffered saline (PBS, pH7. 4) at 4 in the ratio of 1 ;3 :1:1, respectively. After centrifugated, antigens of OA-BSA as immunogen and OA-OVA for antibody detection are collected.2. Immunization of animals and determination of antibodies a-gainst OA in seraSix to eight week old female BALB/c mice are given initial subcutaneous (s. c. ) inoculation of 80 l of OA-BSA in PBS emulsified with an equal volume of complete Freund's adjuvant ( CFA). All sub-sequent injections are made in an equal volume of incomplete Freund' s adjuvant (IFA). At 4 weeks, two mice receive a second subcutaneous injection of 200 jxl of OA-BSA in PBS, followed on the seventh week by an identical third injection given intraperitoneally (i. p. ). At 9 weeks, they are intraperitoneally injected 160jjJ of OA-BSA in PBS. Two weeks later, mice are boosted intraperitoneally with 160jjj of OA-BSA in PBS three days prior to cell fusion.Before final immunization, antibodies against OA in sera are determined by ELISA using OA-OVA as an antigen for analysis.3. Cell fusion and selective cell cultureThe cell fusion is carried out by mixing splenic lymphocytes of immunized mice and the myeloma cells ( P3X63-Ag. 8.653 ) using polyethylene glycol ( PEG) . After the cell fusion, hybridoma cells are selected in a HAT medium ( a medium containing hypoxanthine, aminopterin, thymidine and fetal bovine serum).4. Selection and cloning of hybridoma cellsThe antibodies against OA are determined by ELISA in the hybridoma cells culture supernatant. The hybridoma cells in wells in which antibodies are positive are cloned by a limiting dilution method. Then the monoclonal antibodies against OA are not tested by ELISA until they are all positive in the hybridoma culture supernatant.5. Further culture and stored of hybridoma cellsThe hybridoma cells, which secrete positive antibodies and grow well, are further cultured. When those cells grow up to certain a-mount, they are stored in ampoules (2ml) and freezed.6. Statistic MethodThe values of optical density (OD) of the antibodies against OA in sera are analyzed according to ONE WAY ANOVA method.Results1. Preparation of antigenAccording to carbodiimide coupling method, antigens of OA-BSA and OA-OVA are successfully prepared from minute amount OA of hapten. OA-BSA and OA-OVA are used for immunization of animals and determination of antibodies against OA, respectively.2. Effect of immunizationTwo BALB/c mice are immunized by administering OA-BSA with a minute dose, amounting to five times and total of eleven weeks. These antibodies against OA are detected by ELISA in the sera diluted to 50 and 200 times in two mice.3. Cell fusion and selection of hybridoma cellsAfter cell fusion, 549 wells come out clones in the first selection and the rate of clones is 30. 1%. The clones that antibodies are positive are 9 wells and the rate of positive clones is 1. 6%.In the first selection of hybridoma cells, 6 culture plates are seeded and there are 29, 30, and 26 wells that come out clones in the second, fourth and fifth culture plates, respectively. After the super-natants of those wells are determined by ELISA, there are 11, 12, and 10 wells in which antibodies are positive, respectively. The rates of positive clones are 37. 9% , 40. 0% , and 38. 5% , respectively. Although there were some cells growth in the other 3 plates, the antibodies are all negative in the culture...