A sandwich enzyme-linked immunoabsorbent assay for measurement of picogram quantities of murine granulocyte colony-stimulating factor.
Granulocyte colony-stimulating factor (G-CSF) stimulates the proliferation and differentiation of hematopoietic progenitor cells of the neutrophil lineage. Measurement of murine G-CSF levels will allow examination of its role in host defense using murine models. Therefore, we developed a sensitive sandwich enzyme-linked immunoabsorbent assay (ELISA) for murine G-CSF. A polyclonal antibody to recombinant murine G-CSF was produced in rabbits and isolated using a protein A column. This purified native IgG served as the capture antibody and a portion of the IgG was biotinylated to serve as the developing antibody. Specificity was verified by lack of reactivity to GM-CSF, IL-6, IL-3, prolactin, and growth hormone. The lower limit of sensitivity routinely extended to 16 pg/ml in multiple ELISAs. Intra-assay coefficient of variation (CV) ranged from 3.4 to 21.5% across the detection limits of the assay, with the greatest variance occurring near the standard curve maximum. Interassay CV ranged from 11.5 to 23.3%. The ability of the ELISA to detect G-CSF in different sample preparations was examined in RPMI 1640 with 10% FCS, Hanks balanced salt solution, PBS/Tween-20/2% FCS, and the dilution media for ELISA (10% BLOTTO/PBS/0.05% Tween-20). Average recovery in these media ranged from 98 to 107%. Heparin anti-coagulated normal mouse plasma had a suppressive effect on the ELISA that varied between individual mice. Recovery was also determined from liver, spleen, and lung homogenate suspensions at dilutions of 1:5, 1:10, and 1:20 in dilution buffer. Recovery from liver was optimal at the 1:10 and 1:20 dilutions at 105%, with that of the 1:5 dilution at 135%. Recovery from spleen ranged from 94 to 96%. Lung homogenate displayed enhanced recovery (139% or greater) across all dilutions. The ability of the assay to detect G-CSF was explored by measurement of G-CSF levels in peritoneal lavage following polymicrobial intra-abdominal infection. Peak levels of G-CSF production occurred at 16 h after cecal ligation and puncture surgery with 18- and 21-guage needles (75.7 ng/ml and 111.4 ng/ml, respectively) as compared to the sham animals (0.61 ng/ml). The assay was found to be specific, sensitive, and accurate for measurement of murine G-CSF in a variety of sample types.
Granger
J
J
Department of Anesthesiology, University of Michigan Medical Center, Ann Arbor 48109-0615, USA.
Remick
D
D
Call
D
D
Ebong
S
S
Taur
A
A
Williams
B
B
Nauss
M
M
Millican
J
J
O'Reilly
M
M
D000818:Animals; D003429:Cross Reactions; D004195:Disease Models, Animal; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005122:Exudates and Transudates / Q000382:microbiology; D016179:Granulocyte Colony-Stimulating Factor / Q000097:blood*; D007410:Intestinal Diseases / Q000382:microbiology; D051379:Mice; D008832:Microchemistry; D010533:Peritoneal Lavage; D011817:Rabbits; D012680:Sensitivity and Specificity
D016179:Granulocyte Colony-Stimulating Factor