Production of monoclonal antibodies to calcitonin and development of a two-site enzyme immunoassay.
A procedure is described for the production of calcitonin-specific hybridomas which involves primary and secondary immunization of Balb/c mice in the hind footpads with free, synthetic human calcitonin and cell fusion of lymphocytes from popliteal lymphonodes with a P3 x 63 myeloma line. This protocol offers the following advantages: (a) it is short and easy to perform, (b) it requires small amounts of unconjugated antigen, and (c) it gives a high yield of antigen-specific IgG-secreting hybridomas. Routine screening was carried out by ELISA on solid phase calcitonin and binding of the monoclonals to free antigen was studied with calcitonin linked to biotin through a 13 carbon atom spacer. Monoclonal couples capable of simultaneously binding calcitonin in solution were found by pairing in all possible combinations 25 purified antibodies, in their unlabelled and biotinylated form, in a checkerboard matrix experimental system. Of the over 30 positive pairs identified, four were used in a one-step enzyme immunoassay for calcitonin determination on microtiter plates in a concn range between 0.1 and 5 ng/ml. With the detecting monoclonal directly conjugated to peroxidase with a heterobifunctional crosslinker, the range of the assay with one monoclonal pair was between 25 and 1000 pg/ml with an 18 hr incubation at 4 degrees C.
Section of Biochemistry and Immunology, RECORDATI SpA, Milano, Italy.
G
G
Racchetti
G
G
Fossati
R
R
Comitti
S
S
Putignano
Y M
YM
Galante
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D002116:Calcitonin / Q000032:analysis / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C
D000911:Antibodies, Monoclonal; D002116:Calcitonin