A kinetic enzyme immunoassay for the quantitation of antibodies to a humanized monoclonal antibody in human serum.

A kinetic enzyme immunoassay was developed and validated to quantitate human antibodies to the humanized monoclonal antibody CAMPATH1-1H (C1H) in human serum. The assay was configured using C1H-coated 96-well plates which were blocked with bovine serum albumin, and incubated with dilutions of human serum containing anti-C1H antibody. Antibody was detected using biotinylated C1H followed by streptavidin-conjugated alkaline phosphatase and p-nitrophenyl phosphate. Absorbance data were collected for 10 min, and mOD min-1 data were exported to MultiCalc data analysis software. A 4-parameter logistic-log algorithm was shown to model the data through the range of the standard curve within 15% of nominal values. The overall assay performance coefficient of variation by ANOVA was 9.2%. The lower limit of detection was defined at 160 Units ml-1. The anti-idiotype antibody standard stock solution is stable at 4 degrees C and at -80 degrees C for at least 11 months in buffer. The anti-idiotype antibody controls are stable for at least seven freeze-thaw cycles and at least 6 months in human serum stored at -20 degrees C. A strategy was devised by which to establish the specific antibody potency for any given batch of anti-C1H antibody standard relative to the Reference Standard. This EIA has been used to quantify and characterize anti-C1H antibody in human serum in support of clinical safety and efficacy studies.

Plasmid composition of Staphylococcus species.

A total of 342 staphylococci representing 13 different recognized species were screened for plasmid composition using agarose gel electrophoresis techniques. Plasmids of molecular weight (MW) 30 x 10(6) or larger in size were uncommon in staphylococci. Moderately large plasmids of MW 15 x 10(6) to 29 x 10(6) were not or only occasionally found in the species Staphylococcus sciuri, S. intermedius, S. hyicus, or S. simulans, but were common in S. aureus (55%) and members of the S. epidermidis (79%) and S. saprophyticus (86%) species groups. Small plasmids were common in most of the species. They produced more complex profiles in simian subspecies or biotypes than in human subspecies or biotypes, e.g., in the species S. warneri, S. haemolyticus, S. cohnii, S. xylosus, and S. aureus. Although some of the species appeared to have rather unique plasmid patterns, we would currently hesitate to use this feature as a taxonomic character, considering the occurrence of exceptional strains and possible plasmid transfer between species.