Introduction
This study aimed to confirm that the Type II Collagen ELISA kit (Cat # 6018) from Chondrex, Inc. detects only native type II collagen and not denatured collagen. The sample preparation involved heat denaturation of type II collagen and the use of proteolytic enzymes such as pepsin. Pepsin is used for extracting native type II collagen from tissues as atelocollagen molecules by cleaving specific peptide bonds near telopeptide sections. In contrast, heating collagen above 42°C denatures it by disrupting its triple helix structure, turning it into single collagen chains (Figure 1). The resistance of collagen to degradation is guaranteed by its original triple helical conformation. Once this structure is disturbed, the single alpha chains become susceptible to nonspecific proteolysis and rapidly degrade into small peptides. This degradation can be determined by gel electrophoresis analysis, showing the presence of denatured collagen in samples.
Protocol for Sample Preparation and Analysis
Chondrex, Inc.'s Cat # 20012 Chick Type II Collagen at 2 mg/ml, 5 ml was used in this study to determine both pre and post-pepsin treatment of collagen. Two samples were prepared, each 2.5 ml in volume. One of these samples was boiled for 10 minutes to prepare denatured collagen. After boiling, both the samples were mixed according to the proportions outlined in Table 1. Each sample contained 2 mg/ml collagen, corresponding to 1 mg of collagen per sample.
For pre-pepsin sample preparation, samples were stored at 4°C for gel electrophoresis and ELISA. For the pepsin treatment, pepsin was added to the remaining samples, with each sample containing 0.5 mg of pepsin. The samples were incubated for 2 hours at 4°C. After the pepsin incubation, the samples were store at 4°C for gel electrophoresis.
For ELISA analysis, pre-pepsin samples were diluted with kit Solution B to obtain a concentration of 30 ng/ml. Finally, the prepared samples were analyzed using Cat # 6018 Type II Collagen ELISA Kit according to the kit protocol.
Results
Figure 2 presents the gel electrophoretic pattern of six samples taken from pre- and post-pepsin digestion samples. Before pepsin digestion, all samples showed almost identical bands (alpha chain). After pepsin digestion, 100% denatured collagen showed almost no band and 25% denatured collagen showed a reduced band. However, there were no visible differences among 50%, 75%, and 100% native collagen, which displayed the same band as native collagen without pepsin digestion.
Figure 2. 6% gel electrophoresis analysis under non-reducing conditions of collagen solutions with a constant collagen concentration, but different amounts of native collagen as detailed in Table 1. CII: ELISA Grade Chick Type II Collagen (Cat # 2011).
The collagen concentration in the pre-pepsin samples were assayed by the kit (Table 2). The collagen levels were highly correlated with the native collagen ratio of each sample in Table 2. These results suggest that the kit detects only the native form of type II collagen, not the denatured form.
Conclusion
These results confirm that the kit can specifically detect native type II collagen. While the denaturing methods and pepsin digestion conditions may need optimization, the provided data is sufficient to understand the kit's functionality in detecting native type II collagen.
For more information, please refer to the Type II Collagen Detection Kit (Cat # 6018)