Figure 6. SP-D levels in 98 samples from the ECLIPSE cohort. SP-D
levels were measured in serum samples by sandwich ELISA with 4
different antibodies ((A) 246–4, (B) 1F4, (C) 246–2 and (D) 245–1).
CV: inter-assay coefficient of variation. Inserted graph: Representative
standard curve. Low/high: Samples were chosen for their low/high SP-D levels according to previous measurements. Bars represent the average values. The mean values for the high and low samples were 83.431
(±37.1) and 46.899 (±14.16) ng/ml for monoclonal antibody 246–4
(A), 128.602 (± 56.21) and 82.172 (±43.37) ng/ml for monoclonal antibody 1F4 (B), 133.858 (± 62.2) and 102.348 (± 78.13) ng/ml for monoclonal antibody 246–2 (C) and 0.021 (±0.0152) and 0.018 (±0.0109)
units for monoclonal antibody 245–1 (D). Monoclonal antibody 245–1
did not recognise purified full length SP-D in ELISA and thus, no standard curve was constructed. Eight serum samples were added to each
ELISA plate to control for intra-plate variation.
COPD is a complex disease that is characterized by only partially reversible airflow obstruction (28). However, dyspnoea,
quality of life and frequency of exacerbations do not correlate well with the standard measures of lung function, such
as FEV1, that are used to classify the disease. SP-D is a pulmonary protein that is involved in the innate immune response (7) and in inflammation (10, 11). Serum levels are
raised in current and former smokers but are even higher in
individuals with COPD (20).
Serum SP-D does not correlate with the severity of disease as assessed by FEV1 and CT scores of emphysema. However, raised levels have a weak association with exacerbations
of COPD (20). The aim of the current study is to establish
whether existing or novel monoclonal antibodies can improve the utility of serum SP-D as a biomarker of COPD. Support for this hypothesis comes from the demonstration that
SP-D can be cleaved by elastase in the lung in response to inflammation (22, 23). This then leaks in the circulation. Antibodies to small fragments may be more sensitive in reporting
components of the COPD phenotype. We have therefore produced monoclonal antibodies against full length and cleaved
SP-D and assessed their utility in individuals with COPD.
Antibodies were generated that recognized full length as
well as cleaved SP-D. However, 1F4 was of a particular interest as it had no affinity for cleaved SP-D and was specific
for full length SP-D. Monoclonal antibody 1F4 was compared
to several commercially available antibodies (246–2, 246–4,
245–1) as well as a polyclonal antibody in Western blot analysis in order to characterize any potential differences between
these antibodies. 1F4 had a similar recognition pattern to all
existing monoclonal antibodies. They all recognized the full-length form of SP-D. The only exception was 245–1, which
recognized the full length and cleaved fragment, which was
similar to the specificity of the polyclonal antibody.
We then used these monoclonal antibodies to measure
SP-D in 98 serum samples from individuals with COPD recruited to the ECLIPSE cohort. Blood samples are taken regularly in patients in this study. Samples obtained from the first
visit were used to measure serum SP-D with a commercially
available ELISA (20). We have used samples taken from patients 6 months after the start of the study. Samples were selected from individuals with serum levels of SP-D that were in
the highest and lowest quartile. This is reasonable as serum
levels of SP-D are relatively stable over time in individuals
with stable disease (20).
The concentrations of SP-D measured with our novel
monoclonal antibody were compared with the values obtained with the commercial monoclonal antibodies. Although average values of the low and high quartile samples
retain the same profile of low and high levels of SP-D, there
were wide differences with some of the antibodies. Monoclonal antibodies 246–4 and 1F4 reported a larger difference
between the high and low SP-D levels than monoclonal antibodies 246–2 and 245–1. There was no correlation between
the value of SP-D reported by any of the antibodies and either age, GOLD stage of COPD, the number of pack-years of