An enzyme-linked immunosorbent assay (ELISA) for quantification of mouse surfactant protein D (SP-D).
Surfactant protein D (SP-D) is a pattern recognition molecule of the collectin family of C-type lectins. It is found in the airways and at mucosal surfaces. SP-D is part of the innate immune system where it neutralizes and leads to elimination of microorganisms. It regulates the functions of other innate immune cells, such as macrophages and neutrophils. It also modulates the adaptive immune response by interacting with antigen-presenting cells and T cells. Monoclonal anti-mouse-SP-D antibodies were raised from SP-D deficient mice using recombinant SP-D as antigen. Ten monoclonal antibodies were characterized and validated for use in sandwich enzyme-linked immunosorbent assay (ELISA). Based on two of these, we established an ELISA that allows for measurements of mouse SP-D in various body fluids. The final ELISA was optimized and calibrated with a standard of purified recombinant mouse SP-D, which was analyzed by quantitative amino acid analysis. The ELISA was validated with respect to parallelism, recovery, intra- and inter-assay variation. The practical working range was estimated to be 1.9-200 ng/ml. We measured SP-D in lung lavage to an average concentration of 435 ng/ml (3-ml lavage), and in mouse vaginal fluid (1-ml lavage) to an average concentration of 94 ng/ml, but could not detect SP-D in the serum or plasma of healthy mice (<3.8 ng/ml). With this ELISA, it will be possible to study the regulation of SP-D in various mouse models and upon various stimuli.
Hansen
Soren
S
Department of Immunology & Microbiology, University of Southern Denmark, Winsloewparken 21-1, Odense, Denmark. shansen@health.sdu.dk
Schmidt
Vivi
V
Steffensen
Maria Abildgaard
MA
Jensen
Pia Hoennerup
PH
Gjerstorff
Morten
M
Thiel
Steffen
S
Holmskov
Uffe
U
D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D001826:Body Fluids / Q000276:immunology*; D001992:Bronchoalveolar Lavage Fluid / Q000276:immunology*; D016466:CHO Cells; D002138:Calibration; D006224:Cricetinae; D003412:Cricetulus; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods* / Q000592:standards; D005260:Female; D008297:Male; D051379:Mice; D011489:Protein Denaturation; D037663:Pulmonary Surfactant-Associated Protein D / Q000032:analysis* / Q000097:blood / Q000235:genetics / Q000276:immunology; D011994:Recombinant Proteins / Q000276:immunology; D015203:Reproducibility of Results; D014162:Transfection; D014621:Vagina / Q000276:immunology*; D044364:Vaginal Douching
D000911:Antibodies, Monoclonal; D037663:Pulmonary Surfactant-Associated Protein D; D011994:Recombinant Proteins