In vitro proliferation of human lymphocytes measured by an enzyme immunoassay using an anti-5-bromo-2-deoxyuridine monoclonal antibody.
A previously described, non radioactive method for the measure of in vitro mouse lymphocyte proliferation was applied to human lymphocyte proliferation assays. It involved incorporation into DNA, during cell multiplication, of 5-bromo-2-deoxyuridine (BUdR), a thymidine analogue. BUdR-DNA was then assayed by a sandwich enzyme immunoassay (BUdR-EIA) using an anti-BUdR monoclonal antibody (McAb 76-7). BUdR-DNA from crude cell extracts was first immobilized on microtitration plates coated with McAb 76-7. In a second step BUdR-DNA was reacted again with McAb 76-7 conjugated to horse radish peroxydase. The quantity of peroxydase in microtitration wells was then measured by the coloration of o-phenylenediamine (492 nm). Titration curves obtained with dilutions of crude extracts were compared to the curve obtained with a purified BUdR-DNA reference solution. Results were expressed as equivalent ng BUdR-DNA/ml. BUdR-EIA was compared to 3H-thymidine incorporating assay for the measure of lymphocyte proliferation induced by PHA mitogen, candidine and tuberculine antigens and mixed lymphocyte culture. Excellent correlation between both assays was observed for each experiments (r = 0.953 to 0.999). Overall correlation coefficient for the 5 experiments was 0.785, indicating greater variation of BUdR than 3H-thymidine incorporation, according to the mitogen or antigen used and the culture conditions. This could be due to that fact that BUdR-EIA measured only BUdR incorporated into DNA, while 3H-thymidine incorporation assay measured 3H-thymidine both incorporated into DNA, and stocked into the cell before DNA incorporation. BUdR-EIA would thus reflect cell proliferation more exactly than 3H-thymidine incorporation assay. The sensitivities of both techniques were comparable.(ABSTRACT TRUNCATED AT 250 WORDS)
Martinon
F
F
Laboratoire d'Immunologie et d'Histocompatibilité, Hôpital Saint Louis, Paris, France.
Rabian
C
C
Loiseau
P
P
Ternynck
T
T
Avrameas
S
S
Colombani
J
J
D000911:Antibodies, Monoclonal / Q000494:pharmacology; D001973:Bromodeoxyuridine / Q000276:immunology*; D004247:DNA / Q000032:analysis; D007124:Immunoenzyme Techniques; D008213:Lymphocyte Activation
D000911:Antibodies, Monoclonal; D004247:DNA; D001973:Bromodeoxyuridine