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Updated: Aug 17, 2026
Immuno-fluorescence Assay of Leptospiral Surface-exposed Proteins
Published on: July 1, 2011
High immunoreactivity of lactoferrin contaminating commercially purified myeloperoxidase
M A Audrain1, T A Baranger, C M Lockwood
1Laboratoire d'Immunologie, Hotel Dieu, Nantes, France.
Abstract:
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.
Insights
Commercial myeloperoxidase (MPO) preparations contain contaminating lactoferrin (LF), leading to inaccurate antibody specificity testing. Researchers found antibodies reacted with LF instead of MPO, highlighting the need for careful reagent validation.
Area of Science:
- Immunology
- Biochemistry
Background:
- Myeloperoxidase (MPO) is a key enzyme in neutrophil function.
- Accurate detection of anti-MPO antibodies is crucial for diagnosing autoimmune diseases.
- Commercial MPO preparations are often used as reference standards.
Purpose of the Study:
- To evaluate the quality of commercially available MPO preparations.
- To assess the specificity of anti-MPO reagents, including monoclonal and polyclonal antibodies.
- To investigate potential cross-reactivity with contaminating proteins.
Main Methods:
- Immunization of mice with purified MPO.
- Generation and characterization of monoclonal antibodies using ELISA and immunoblotting.
- Testing antibody reactivity under various buffer and pH conditions.
- Evaluation of polyclonal anti-MPO and anti-lactoferrin (LF) reagents.
Main Results:
- Monoclonal antibodies raised against MPO cross-reacted significantly with lactoferrin (LF).
- Contaminating LF in MPO preparations showed high immunoreactivity.
- Buffer and pH conditions influenced MPO and LF reactivity.
- Polyclonal anti-MPO reagents also showed cross-reactivity with LF.
Conclusions:
- Caution is advised when determining anti-neutrophil cytoplasm specificities using ELISA.
- The presence of even low concentrations of contaminating LF in MPO preparations can lead to misidentification of antibody targets.
- Standardization of MPO preparations and validation of anti-MPO reagents are essential for reliable diagnostic and research applications.
