Chondrex, Inc., specializes in extracellular matrix (ECM) analysis, offering high-quality monoclonal antibodies (mAbs) and Enzyme-Linked Immunosorbent Assay (ELISA) kits designed for collagen detection. We also provide optimized collagen solubilizing protocols from samples based on efficient collagen purification protocols. These methods ensure highly reproducible and reliable results.
Recent reports indicate that consuming undenatured (native) type II collagen (UCII) may improve the composition of intestinal bacteria, potentially reducing inflammation in joints in human(1–3), dogs (4, 5), and horses(6). To support quality control analysis in food supplements, Chondrex, Inc’s type II collagen detection kit (Catalog # 6018, UCII kit) has been used to quantify UCII contents in products.
Chondrex, Inc., also offers UCII analysis services for samples or products. The service can assist in product validation and laboratory protocol establishment. Please contact us at support@chondrex.com for more information and a quote.
The following data are validation results of UCII kit for understanding how the kit works for collagen analysis.
1. Variability Assessment
2. Denatured Type II Collagen Detection
3. The Hook Effect
1.Variability Assessment
ELISA is an essential method for detecting and quantifying proteins. Validation data of ELISA kits ensures their accuracy, precision, and reliability, which is crucial for obtaining consistent and dependable results in research. Inter-assay variability assesses the consistency of results across different runs on separate days, indicating the kit's reproducibility over time. Intra-assay variability measures the precision within a single run, ensuring stable and repeatable results within the same experiment. Spiking tests involve adding known quantities of the target analyte to different sample matrices to evaluate the kit's accuracy and recovery rate.
The UCII kit with a one-step assay protocol was used for variability assessment . Samples with different concentrations of ELISA grade chick type II collagen were prepared. Inter-assay variability was assessed by testing samples on three different days. Intra-assay variability was assessed by testing five replicates of samples within the same assay run. Spiking tests were performed by adding known quantities of type II collagen to samples.
Table 1. Inter-Assay Variability
Table 2. Intra-Assay Variability
Table 3. Spiking Recovery Test
The inter-assay coefficient of variation (CV) values were 6.3- 9.6%, all below 10%. The intra-assay CV values were 2.1- 4.4%, also below 10%. Additionally, the spiking recovery tests, 85.7 – 97%. These results strongly suggest that UCII kit is a reliable tool in measuring undenatured type II collagen concentrations in samples.
2. Undenatured Collagen Detection
This study aims to confirm that UCII kit detects only undenatured (native) type II collagen and not denatured collagen. The sample preparation protocol 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 collagen from both inter- and intramolecular crosslinks. In the case of insoluble collagen, this process results in the extraction of collagen 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. Collagen's resistance to degradation by pepsin is due to its triple helical structure. Disrupting this structure results in single alpha chains that are susceptible to nonspecific proteolysis. These alpha chains are rapidly degraded into small peptides. This collagen degradation can be confirmed by a gel electrophoresis analysis.
The UCII kit was evaluated to ensure it detects only UCII. Samples were prepared with UCII and heat denatured collagen as outlined in Table 4 and were analyzed using gel electrophoresis in pre and post pepsin digestion. The concentration of UCII detected by the kit was compared to the ratio of prepared undenatured and denatured collagen.
Table 4. Sample Preparation
The gel electrophoretic pattern of six samples was obtained from pre- and post-pepsin digestion samples (Figure 2). 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 UCII as shown in Table 4.
The samples were assayed by the UCII kit. The concentration of type II collagen detected by the kit was almost identical to the ratio of UCII prepared.
Table 5: Collagen detection rate of samples received pepsin treatments
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 is sufficient to understand the kit's functionality in detecting UCII.
3. The Hook Effect
The hook effect, also known as the prozone effect or high-dose hook effect, is a phenomenon that can sometimes occur in sandwich ELISAs using a one-step assay protocol. It results in a falsely low or even negative signal when the concentration of the target analyte in the sample is extremely high. This can lead to misinterpretation of results, potentially causing flawed research conclusions.
Figure 3. The hook effect concept
To determine the hook effect, samples containing different concentrations of type II collagen were assayed by the UCII kit. The assay results were evaluated for the presence of the hook effect.
With the 1-step assay protocol, samples containing more than 3 µg/ml of type II collagen fall within the hook-effect range. Therefore, the 1-step assay protocol requires careful attention to avoid results caused by the hook effect. For samples with unknown concentrations, we recommend running multiple sample dilutions to see where they are in the reportable range and to avoid misunderstandings in the assay results. If samples contain more than 30 µg/ml of type II collagen, the hook effect may cause the results to show lower concentrations than the validated 200 ng/ml standards (Figure 4).
Alternatively, assays using the 2-step assay protocol do not require consideration of the hook effect. The assay results will be saturated if samples contain more than 200 ng/ml of type II collagen. If oversaturation occurs, re-assay the samples using higher dilutions to fit within the standard curve range.
Chondrex, Inc., proposes the two solutions below to avoid these issues.
1) Use the 2-step assay protocol
2) Make multiple sample dilutions to ensure samples within the reportable range, such as 1:10, 1:100, 1:1000, and 1:10000
Figure 4. A hook effect in UCII kit assays
References