apc011

This kit contains all special reagents needed to perform a competitive ELISA to measure total human CC10 in
a 96 well microtiter plate format up to 40 samples plus 8 point standard curve (all in duplicate). The assay is
colorimetric and is read at an absorbance of 450 nm. This assay utilizes a single capture antibody and
typically produces higher concentration readings of CC10 in samples than a sandwich format assay. It is less
sensitive to matrix effects. In vivo CC10 is incorporated into numerous covalently-linked and non-covalent
complexes (Antico, et al. 2006), so the requirement for a single binding site in the competitive assay format,
rather than two binding sites in the sandwich assay format provides a greater probability of measuring the
total CC10 (the sum of complexed and free homodimer.)

No significant cross-reactivity with porcine, ovine (sheep), bovine, rabbit, mouse, or rat CC10 protein.
Recognizes chimpanzee and monkey CC10.
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ASSAY DESCRIPTION

In this competitive ELISA format, an anti-CC10 antibody is used as the capture reagent for CC10 in the sample. A conjugate of horse radish peroxidase
(HRP) to recombinant human CC10 is captured by the anti-CC10 antibody coated in the wells and generates a signal (A450) proportional to the amount of
CC10-HRP conjugate bound. The CC10-HRP conjugate is pre-mixed with the sample to be assayed (which may be pre-diluted with PBS, if necessary).
Typically, two different dilutions (1:2-3 and 1:10) of each sample are run in duplicate, using between 25-110 microliters of sample. The assay thus
measures a decrease in signal as the CC10 in the sample competes the CC10-HRP conjugate for binding sites (see figure). A standard curve, using
carefully prepared CC10 calibrators, is always run in duplicate with each set of samples to assess reproducibility and accurately quantitate CC10 in
samples. X-axis is CC10 concentration in nanograms/ml and Y-axis is absorbance at 450 nm. Greatest accuracy occurs in the linear range between 10-
100 ng/ml. Samples measuring higher than 200 ng/ml should be diluted and retested. Coefficients of Variation (CV’s) are typically less than 15%.

Sensitivity and Specificity. The competitive CC10 ELISA is sensitive to about 5 ng/ml and has been used to measure human CC10 in a number of
different types of background matrices from humans and other species. Normal plasma levels of endogenous CC10 in healthy humans are about 10-
100 ng/ml. Under the conditions used in the ELISA, no cross-reactivity has been observed with non-human CC10 in plasma samples from pigs, rats,
mice, sheep, and rabbits. Native CC10 in BAL of healthy individuals ranges from 1-25 microgram/ml, and up to 0.5-1.0 milligram/ml in sputum. Some
cross-reactivity with other species has been observed in BAL samples as measured by relatively high “background” (10-25 ng/ml). It may not be possible
to distinguish between human CC10 and native CC10 in other primates.

A competitive ELISA was used to measure human CC10 in the following publications:

1.        Chen J, et al. Clin Cancer Res. 2008 14(5):1590-7.
2.        Chen J, et al. Cancer Epidemiol Biomarkers Prev. 2007 16(3):577-83.
3.        Miller TL, et al. Pediatr Crit Care Med. 2007 8(1):40-6.
4.        Miller TL, et al. Biol Neonate. 2006;89(3):159-70.
5.        Shashikant BN, et al. J Appl Physiol. 2005 Dec;99(6):2204-11.
6.        Levine CR, et al. Pediatr Res. 2005 58(1):15-21.
7.        Miller TL, et al. Pediatr Crit Care Med. 2005;6(6):698-706.
8.        Nosratabadi AR, et al. Exp Lung Res. 2003;29(7):455-73.
9.        Chandra S, et al. Pediatr Res. 2003;54(4):509-15.