By William C. Davis
Monoclonal Antibody Protocols offers researchers in biomedical, agricultural, and organic technology with a collection of designated, easy-to-follow equipment for constructing and utilizing monoclonal antibodies. The protocols emphasize innovations that optimize the outgrowth of hybridomas from basic cultures of fused cells and using an alternate, electric-field-mediated cellphone fusion strategy to bring up the yield of hybridomas. The booklet stresses antibodies produced in mice, yet comprises equipment of manufacturing xenogeneic hybrids that yield human, bovine, equine, and porcine monoclonal antibodies. With its designated directions, its reviews on easy methods to modify some of the steps of a protocol with a purpose to accommodate assorted fabrics, and its tricks and tips that frequently make the variation among good fortune and failure, Monoclonal Antibody Protocols provide you with a prepared and essential resource of knowledge for getting ready and utilizing monoclonal antibodies effectively on your laboratory.
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Alternatively, transfer cells to bioreactor casette for production of antibody (Chapter 17). 4. Notes 1. In our laboratory, the direct fusion between mouse myeloma cells (P63 or NS-1) and immune human cells results in heterohybridomas that produce specific human antibody directed against surface-exposedbacterial antigens. However these hybridomas are very unstable, and after 8-10 passages,the cells cease to produce antibody and ultimately die. This problem has been overcome by the described procedures.
Medium with 10% FBS (10% FSM): Mix 89 mL high-glucose DMEM, 10 mL FBS, 1 mL of 200 mM L-glutamme, and 50 pg/mL gentamicin. 8. 5 mL murme ascites derived from SP2/0 myeloma cells (see Note 5). 9. Freezing solution: 10 mL dimethyl sulfoxide (DMSO; Pierce, Rockford, IL) and 90 mL FBS. 3. 1. Immunization 1. Immunize three-way crossbred pigs (Duroc x Yorkshire x Hampshire; American Cyanamid, Princeton, NJ) subcutaneously with the antigen emulsified in complete Freund’s adjuvant followed by several booster inJections with the same antigen m incomplete Freund’s adJuvant.
7. 0 g/mL). Use undiluted. 8. , Gtbco). To prepare working soluttons, dilute 10 mL of the 10X stock with 90 mL of stertle, high-purity detonized water. 9. , BioWhittaker, Walkersvrlle, MD). MEM contains Earle’s balanced salt solution without L-glutamine and sodium bicarbonate. To prepare working solutions, dilute 10 mL of the 10X stock with 90 mL of sterile, high-purity deionized water. 10. 1N NaOH: Dissolve 4 g NaOH in 100 mL of deionized water and ftlter-sterilize. 11. 8 mL of MEM. Add 100 pL of gentamicin.