反应详情

ELISAs were performed in polyvinyl 96-well microtiter plates (Dynatech, Alexandria, Va.); 200-μl volumes were used for each step. Wells were coated with a cell envelope preparation (10 μg/ml) of nontypable H. influenzae strain 3524 prepared by the method of Johnston, "Immunobiology of Neisseria gonorrhoeae", American Society for Microbiology, 1978, 121-9. Plates were incubated at 37° C. for 1 hour followed by overnight incubation at 4° C. Wells were washed three times with PBS (phosphate buffered saline) plus 0.05% Tween 20® surfactant between each step. Unbound sites on the plastic were blocked with 3% bovine serum albumin in PBS for 2 hours at 37° C. Tissue culture supernatants (or dilutions of mouse ascites fluid in subsequent experiments) containing monoclonal antibody were incubated in the wells overnight at 4° C. Rabbit antibody to mouse IgG and IgM was then incubated for 2 hours at 37° C. followed by protein A-peroxidase for 2 hr at 37° C. Two hundred microliters of substrate was then added to each well. Substrate was prepared by dissolving 10 mg of o-phenyl-enediamine in 1 ml of methanol and adding this solution to 99 ml of citrate-phosphate buffer, pH 5.0, plus 0.1 ml of 3 % H2O2. After the substrate was incubated for 45 min in the dark at room temperature, the reaction was stopped with 50 233 μl of 4N H2SO4. The OD490 was measured. Each set of ELISAs was performed with a control in which NS-1 tissue culture supernatant or ascites fluid was used in place of the monoclonal antibody being tested. On the basis of the results of ELISA screening, selected clones were propagated by subsequent transfer to larger tissue culture wells. Large quantities of antibody were produced in tissue culture and by ip injection of 105 hybridoma cells into pristane-primed BALB/c mice. The resulting ascitic fluid was harvested in three to four weeks and tested for specificity.

1. customWells were coated with a cell envelope preparation (10 μg/ml) of nontypable H
2. custominfluenzae strain 3524 prepared by the method of Johnston, "Immunobiology of Neisseria gonorrhoeae", American Society for Microbiology, 1978, 121-9
3. waitfollowed by overnight
4. customat 4° C
5. washWells were washed three times with PBS (phosphate buffered saline) plus 0.05% Tween 20® surfactant between each step
6. waitwere blocked with 3% bovine serum albumin in PBS for 2 hours at 37° C
7. additionTissue culture supernatants (or dilutions of mouse ascites fluid in subsequent experiments) containing monoclonal antibody
8. customwere incubated in the wells overnight at 4° C
9. waitRabbit antibody to mouse IgG and IgM was then incubated for 2 hours at 37° C.
10. waitfollowed by protein A-peroxidase for 2 hr at 37° C
11. additionTwo hundred microliters of substrate was then added to each well
12. customSubstrate was prepared
13. waitAfter the substrate was incubated for 45 min
14. customdark at room temperature
15. customOn the basis of the results of ELISA screening