FAQ

What is the difference between the competitive ELISA and the sandwich ELISA for CC10?

The competitive ELISA uses a single polyclonal anti-CC10 specific antibody coated onto the surface of the microtiter plate to capture the CC10 in the
sample. Prior to addition of the sample to the microtiter plate wells, a fixed amount of CC10 conjugated to HRP (horse radish peroxidase) is added
to the sample and mixed. The CC10 in the sample then competes with the CC10-HRP conjugate for binding sites in the plate well. The colorimetric
signal generated by the bound HRP therefore decreases as the concentration of CC10 in the sample increases.

The sandwich ELISA uses two different polyclonal anti-CC10 antibodies; one coated onto the surface of the microtiter plate to capture the CC10 in
the sample and one that is conjugated to HRP to bind and quantitate the captured CC10 in the plate well. The CC10 in the sample is thus
“sandwiched” between two CC10 specific antibodies and, therefore, requires that two sites on a single CC10 molecule can be recognized and
bound. The colorimetric signal generated by the bound HRP increases as the concentration of CC10 in the sample increases.

What are the advantages and disadvantages for the competitive ELISA versus the sandwich ELISA?

The competitive ELISA typically requires no sample processing, beyond centrifugation or filtration to remove particulates, and is significantly less
sensitive to sample dilution and sample matrix effects than the sandwich assay format. The competitive ELISA, therefore, typically provides much
tighter data with lower variability between duplicate samples and lower variability between assays. The competitive assay generally has greater
precision, accuracy, and reproducibility, but lower overall sensitivity and specificity than the sandwich assay.

Note: The limit of sensitivity for the competitive ELISA is 2-5 ng/ml of CC10, which is comparable to, or well below, the typical physiological range of
CC10 found in most human fluid and tissue samples (Levine, et al., 2005).

What are the advantages and disadvantages for the sandwich ELISA versus the competitive ELISA?

The sandwich ELISA may require sample processing and clean up to yield reproducible results. It is significantly more sensitive to sample dilution
and to sample matrix effects than the competitive assay. It is also more specific than the competitive assay since it involves two antibody recognition
steps, not one. Since CC10 has been found in both covalent and non-covalent complexes in vivo (Antico, et al., 2006; Zheng, et al., 2000), which are
presumably in equilibrium with each other and with other proteins in the milieu, it is not surprising that a dilution may result in a redistribution of the
absolute quantities of the various non-covalently CC10 complexes and species that may then be trapped and quantified in the sandwich.

Don’t the competitive and sandwich ELISAs both measure the same thing?

They both measure CC10 but they each may measure a different set of species of CC10 in biological samples. The competitive ELISA requires only
a single binding interaction with CC10 and is, therefore, more likely to measure a larger set of CC10 species in a sample compared to the sandwich
ELISA. The competitive ELISA may measure the total soluble CC10 in a sample. The sandwich format requires two binding interactions with CC10
and is, therefore, more likely to measure a subset of all CC10 species in a sample, including homodimers and multimers, or “free CC10”.

What is meant by “free CC10”?

We refer to CC10 that is not bound to other non-CC10 proteins in a complex as “free CC10”.

What is meant by “total CC10”?

We refer to the maximum measurable CC10 in a sample, requiring a single binding interaction, as total CC10. Total CC10 includes both bound and
unbound CC10.

What are the molecular forms of CC10?

The most easily recognized form of CC10 is a homodimer, although monomer, tetramer, and higher multimers also exist. In the presence of tissue
transglutaminase, purified CC10 forms a ladder of different sized multimers in vitro (Manjunath, 1984; Mantile, 1993). In vivo, CC10 forms many
species of covalent complexes (Antico, 2006). CC10 also forms a non-covalent complex with IgA and fibronectin (Zheng, et al., 2000). The vast
majority of molecular species of CC10 in vivo have not yet been characterized.

Have any other secretoglobins been found in complexes with other proteins?

Yes. Mammoglobin has been observed in complex with BU101 in serum (Colpitts, et al., 2001).

What is a covalently-linked complex?

A covalently linked complex is a protein complex that contains at least two different proteins that are held together by covalent bonds and do not
disassociate in the presence of chaotropic agents, detergents, extremes of temperature or pH, or altered salt concentration. Covalent complexes
that are held together by disulfide bonds disassociate in the presence of a reducing agent such as dithiothreitol (DTT) or beta-mercaptoethanol
(BME). Covalent complexes may be detected by SDS-PAGE, Western blot, competitive ELISA, and immunoprecipitation.

What is a non-covalently-linked complex?

A non-covalently-linked complex is a protein complex that is held together by non-covalent interactions such as ionic and hydrophobic interactions.
Non-covalent complexes disassociate in the presence of chaotropic agents, detergents, extremes of temperature or pH, or altered salt
concentration. Non-covalent complexes cannot be detected by standard SDS-PAGE and Western blot but may be isolated by immunoprecipitation
and then characterized.