Introduction
A ELISA kit detects only native type II collagen and not denatured collagen. The sample preparation approach involves heat denaturation of collagen and the use of proteolytic enzymes such as pepsin. Pepsin is employed for extracting collagen from tissues by cleaving a limited number of peptide bonds near covalent crosslinks, thus releasing components from both inter- and intramolecular crosslinks. For insoluble collagen, this process renders the product susceptible to extraction in its native form. In contrast, heating collagen above 42°C denatures it by disrupting its triple helix structure, turning it into single collagen chains. The resistance of collagen to degradation is guaranteed by its original triple helical conformation. Once this structure is disturbed with single alpha chains, alpha chains become susceptible to nonspecific proteolysis and, after administration, is rapidly degraded into small peptides. This degradation can be determined by gel electrophoresis analysis, showing existing denatured collagen in samples.
Figure 1. Collagen structure changes with pepsin digestion and heating treatment
Protocol for Sample Preparation and Analysis
Chick type II collagen 2 mg/ml, 5 ml was used for test samples. To begin the sample preparation, first prepare two samples, each with a volume of 2.5 ml. Boil one of these samples for 10 minutes. After boiling, mix the samples according to the proportions outlined in Table 1. Each sample can have a concentration of 2 mg/ml, corresponding to 1 mg of collagen per assay. The specific volumes for mixing native and denatured samples are detailed in the table.
For pre-pepsin sample preparation, samples have been stored for gel electrophoresis. For the pepsin treatment, pepsin is added to the pre-pepsin samples, ensuring that each assay contains 0.5 mg of pepsin. Incubate the samples for 2 hours at 4°C. After the incubation period, the samples were stored for ELISA and gel electrophoresis.
For ELISA analysis, samples were prepared with a buffer to obtain a concentration of 30 ng/ml. Finally, the prepared samples were analyzed using an ELISA kit.
Table 1. Sample preparation
| Sample # | 1 | 2 | 3 | 4 | 5 | 6 |
| Native % | 0 | 25 | 50 | 75 | 100 | 100(no pepsin) |
| Native | 0 | 125 ul | 250 ul | 375 ul | 500 ul | 500 ul |
| Denatured(Boiled) | 500 ul | 375 ul | 250 ul | 125 ul | 0 | 0 |
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, and they showed the same band as native collagen without pepsin digestion.
Figure 2. 6% gel electrophoresis analysis under non-reducing conditions of collagen solutions of constant collagen concentration but different content of native collagen as Table 1.
The samples were assayed by a type II collagen ELISA kit. Table 2 shows that the concentration of native collagen detected by the kit was almost identical to the ratio of denatured and native collagen prepared.
Table 2: Collagen detection rate of samples received pepsin treatments
| Sample # | Native collagen% | (ng/ml) | Detection rate |
| 1 | 0 | 1.4 | 4.6% |
| 2 | 25 | 7.1 | 23.4% |
| 3 | 50 | 15.6 | 51.5% |
| 4 | 75 | 21.8 | 71.6% |
| 5 | 100 | 30.4 | 100.0% |
| 6 | No pepsin | 27.7 |
Conclusion
The results confirm that the kit can specifically detect native collagen. While the denaturing methods and pepsin digestion conditions may need optimization, the data provided are sufficient to understand the kit’s functionality in detecting native collagen.