The CL-11 concentration in serum from two CL-11-deficient individuals was below the lower working limit Ribociclib mouse of the assay. Thus, the CL-11 concentration in these sera was lower than 2.1 ng/ml when the dilution of the samples was taken into account (Fig. 4A). Similar observations were made for normal plasma depleted by affinity chromatography on anti-CL-11 MAb columns. The stability of CL-11 in serum and plasma upon storage at different temperatures was examined using matched serum and plasma samples from five blood donors.
Storage of the samples for up to 1 week at room temperature, 4 °C or − 20 °C did not affect the concentration of CL-11 significantly. The CL-11 concentration was not influenced by up to eight freeze/thaw cycles (data not shown). A quantitative sandwich CL-11 ELISA was developed using two different MAbs and using culture supernatant
containing recombinant CL-11 as the calibrator. The current ELISA was thoroughly validated and appears very reliable and robust. Our validation is summarized in Table 1 and Table 2, and based on our observations, the ELISA is unaffected by the presence of rheumatoid factors or differences in storage conditions of samples. The ELISA allows also measured serum and plasma concentrations to be directly compared with each other. check details We observed excellent parallelism between our calibrator made of recombinant CL-11 and plasma and serum samples in all our experiments. We have previously estimated the mean CL-11 concentration to approximately 2 μg/ml in the plasma of 10 healthy individuals based on an absolute correlation with OD280 measurements of purified recombinant CL-11 (Hansen et al., 2010). Our quantitative amino acid analysis in present work showed that this was highly overestimated, most likely due to the presence of glycosides that influences the absorbance measurements (not shown). Due to this discrepancy, seven different quantitative amino acid analyses were performed,
all and the average of these was used for final correlation to absolute concentrations. Antibody specificities were tested by means of Western blotting and we observed only immunoreactivity with bands that we expect to be CL-11. We speculate that the immunoreactive bands at approximately 28 and 160 kDa, in the reduced and nonreduced eluate, respectively, represent the reported CL-11 isoforms that lack parts of the collagen-like region (Keshi et al., 2006) or degraded CL-11 (Fig. 1A). Nonreduced, CL-11 appears to be made of dimers (200 kDa), trimers (300 kDa) and several multimers of subunits larger than the trimers (> 300 kDa). In comparison, previous analysis of recombinant CL-11 showed only the presence of dimers of subunits (Hansen et al., 2010), but judged from the careful studies of parallelism the discrepancy appears not to influence the ELISA.