title: Development of a cell-based qualitative assay for detection of neutralizing anti-human interleukin-1 receptor antagonist (hIL-1Ra) antibodies in rats.
abstract: To determine the incidence of the positive neutralizing anti-human interleukin receptor antagonist (anti-IL-1Ra), a novel assay based on the proliferation of human melanoma A375.S2 cells was developed and validated. In the presence of a growth-limiting concentration of IL-1β, A375.S2 cells were able to regain proliferation following the addition of IL-1Ra in a concentration-dependent manner. This dose-response effect enabled the validation of a standard curve for calculation of the concentration of IL-1Ra or, inversely, the concentration of neutralizing anti-IL-1Ra antibodies in cell culture medium or sera. The assay used CCK-8 as an indicator of proliferation. The dose-response relationship between rhIL-1Ra (dose range of 5-75 ng/ml rhIL-1Ra) and A375.S2 cell proliferation was sigmoidal and fitted a four-parameter logistic model. The percent coefficients of variation (%CVs) of quality control samples were 12.5 and 11.9% for intra-assay repeatability and 14.5 and 19.5% for inter-assay repeatability, while the total accuracy was in the range of 97.2-103.6%. For the neutralization assay, the optimal sample dilution factor was found to be 40-fold and the reasonable standard for positive and negative decision was calculated to be 59.4% neutralization rate. The %CVs of quality control samples were 12.7 and 24.0% for intra-assay repeatability and 11.6 and 30.0% for inter-assay repeatability. Analysis using the assay showed that rats could produce neutralizing anti-IL-1Ra antibodies after repeated intramuscular injection with rhIL-1Ra, and this response was not significantly dependent on the dose injected.
authors.affiliation: School of Pharmacy, Shanghai Jiao Tong University , Shanghai , China .
authors.forename: Jin
authors.initials: J
authors.lastname: Gao
authors.forename: Jingjing
authors.initials: J
authors.lastname: Li
authors.forename: Minmin
authors.initials: M
authors.lastname: Yang
authors.forename: Mingyuan
authors.initials: M
authors.lastname: Wu
authors.forename: Ping
authors.initials: P
authors.lastname: Tu
authors.forename: Yan
authors.initials: Y
authors.lastname: Yu
authors.forename: Wei
authors.initials: W
authors.lastname: Han
mesh_terms: D000818:Animals; D057134:Antibodies, Neutralizing / Q000097:blood* / Q000235:genetics / Q000494:pharmacology; D001681:Biological Assay / Q000379:methods*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D006801:Humans; D053583:Interleukin-1beta / Q000097:blood*; D016015:Logistic Models; D008297:Male; D009500:Neutralization Tests; D017208:Rats, Wistar; D017472:Receptors, Interleukin-1 / Q000037:antagonists & inhibitors*; D011994:Recombinant Proteins; D012680:Sensitivity and Specificity
chemical_list: D057134:Antibodies, Neutralizing; D053583:Interleukin-1beta; D017472:Receptors, Interleukin-1; D011994:Recombinant Proteins
keywords: A375.S2; apoptosis; cell proliferation; human interleukin-1; neutralizing antibodies; recombinant human interleukin-1 receptor antagonist