High immunoreactivity of lactoferrin contaminating commercially purified myeloperoxidase.
Three sets of experiments were performed to investigate the quality of myeloperoxidase (MPO) preparations and anti-MPO reagents. In the first experiment, two groups of three and four mice were immunized with commercially purified MPO (Calbiochem). Immunization was performed in PBS in the first group and in acetate buffer in the second. From the first group, five monoclonals were raised, and their specificities examined by ELISA and immunoblotting. Surprisingly, these antibodies reacted with lactoferrin (LF) and not MPO. In the second group, 13 monoclonals were raised; six of these reacted with MPO and seven reacted with LF. In a second set of experiments, MPO and LF reactivity were tested in different buffer conditions in the ELISA procedure. Slight variations in the detection of contaminating LF were found. In a third experiment, polyclonal reagents directed against MPO and LF were tested in MPO immunoblotting studies. A polyclonal anti-MPO reagent reacted not only with MPO but also with contaminating material including LF. The anti-MPO polyclonal reagent also reacted with LF on immunoblotting. We conclude that: (i) caution should be exercised when defining anti-neutrophil cytoplasm specificities of human sera and monoclonals by ELISA, (ii) the low concentration of contaminating LF in the commercially purified reference MPO preparation should be taken into consideration since it appears to have high immunoreactivity, (iii) changes in MPO immunoreactivity may occur under different buffer and pH conditions.
Audrain
M A
MA
Laboratoire d'Immunologie, Hotel Dieu, Nantes, France.
Baranger
T A
TA
Lockwood
C M
CM
Esnault
V L
VL
D000818:Animals; D019268:Antibodies, Antineutrophil Cytoplasmic; D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D001323:Autoantibodies / Q000276:immunology; D002021:Buffers; D003429:Cross Reactions; D004340:Drug Contamination; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D007781:Lactoferrin / Q000276:immunology*; D051379:Mice; D009195:Peroxidase / Q000276:immunology* / Q000302:isolation & purification; D011817:Rabbits
D019268:Antibodies, Antineutrophil Cytoplasmic; D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D002021:Buffers; D009195:Peroxidase; D007781:Lactoferrin