Development of an immunoassay for BMS-191352, a single-chain immunotoxin, and its application to toxicokinetic studies.
BMS-191352 is a single-chain fusion protein composed of the variable regions of chimeric BR96 monoclonal antibody and the binding defective form of Pseudomonas Exotoxin A (PE40). The immunotoxin exhibits potent cytotoxicity against tumor cells expressing the Lewis antigen. A sensitive and specific double antibody sandwich ELISA has been developed and validated for the determination of BMS-191352 in rat and dog EDTA plasma. A monoclonal anti-PE40 antibody (EXA2-1H8) was used to capture BMS-191352 in plasma samples. The captured BMS-191352 was then detected using a biotinylated monoclonal BR96 antiidiotypic antibody (757-4-1) followed by the addition of streptavidin-horseradish peroxidase conjugate and chromogen 3,3',5,5'-tetramethylbenzidine. The optical density was measured at 450 nm. The standard curve range in rat and dog plasma was 2-32 ng/mL. The RSD for the inter- and intra-assay precision was within 9.2% and the accuracy was greater than 89.0%. The ELISA method was applied to the analysis of BMS-191352 in plasma samples from toxicokinetic studies conducted in rats and dogs. These studies revealed that the systemic exposure of BMS-191352 was dose proportional and the kinetics of BMS-191352 were linear between the dose range of 1.8-7.2 mg/m2 in the dog.
Damle
B
B
Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ 08543, USA.
Hollenbaugh
D
D
Timoszyk
J
J
Tay
L
L
Kaul
S
S
D036002:ADP Ribose Transferases; D000704:Analysis of Variance; D000818:Animals; D000911:Antibodies, Monoclonal / Q000097:blood / Q000633:toxicity; D019540:Area Under Curve; D001427:Bacterial Toxins; D004285:Dogs; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005098:Exotoxins / Q000097:blood / Q000633:toxicity; D005260:Female; D000922:Immunotoxins / Q000097:blood* / Q000633:toxicity; D016015:Logistic Models; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000097:blood* / Q000633:toxicity; D012680:Sensitivity and Specificity; D057127:Single-Chain Antibodies; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D000911:Antibodies, Monoclonal; D001427:Bacterial Toxins; D005098:Exotoxins; D000922:Immunotoxins; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A; C115038:BMS-191352 protein, recombinant