Production and characterization of monoclonal antibodies detecting chicken interleukin-2 and the development of an antigen capture enzyme-linked immunosorbent assay.
Eleven monoclonal antibodies (mAbs) which are specific for chicken interleukin-2 (chIL-2) were produced and characterized by enzyme-linked immunosorbent assay (ELISA), Western blotting and neutralizing assays. These mAbs were used to develop a mAb-based antigen capture ELISA specific for chicken IL-2 detection. Anti-IL-2 mAbs bound specifically to E. coli-derived rchIL-2 in ELISA and identified a 16kDa IL-2 polypeptide band in Western blot. Several mAbs were shown to neutralize the biological activities of both rchIL-2 and native chicken IL-2 as measured by concanavalin A (ConA)-induced lymphocyte proliferation assay, IL-2 bioassay, and natural killer cell assay. Among the neutralizing mAbs, the mAb chIL-2/11 was most potent in neutralizing IL-2 activity. To develop a sensitive ELISA for the detection of chicken IL-2, an antigen capture ELISA was developed using the mAb chIL-2/16 as the antigen capture antibody and rabbit anti-IL-2 peptide antibody as the detection antibody. Using the mAb-based antigen capture ELISA, significant correlation between the level of IL-2 detected in bioassays and in ELISA was observed. These results showed that the mAb-based antigen capture ELISA is less time-consuming and more reliable compared to a conventional IL-2 bioassay for chicken IL-2. These neutralizing mAbs will facilitate basic immunobiological studies of the role of IL-2 in normal and disease states in chickens.
Miyamoto
T
T
BARC-East, Building 1040, Parasite Biology, Epidemiology and Systematics Laboratory, Animal and Natural Resources Institute, USDA/Agricultural Research Service, Beltsville, MD 20705, USA.
Lillehoj
H S
HS
Sohn
E J
EJ
Min
W
W
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D000918:Antibody Specificity; D001681:Biological Assay; D002645:Chickens / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D007376:Interleukin-2 / Q000032:analysis* / Q000737:chemistry / Q000276:immunology*; D007694:Killer Cells, Natural / Q000276:immunology; D008213:Lymphocyte Activation; D051379:Mice; D008970:Molecular Weight; D009500:Neutralization Tests; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology
D000911:Antibodies, Monoclonal; D007376:Interleukin-2; D010446:Peptide Fragments; D011994:Recombinant Proteins