Why Detect Total Collagen?
Collagen is the most abundant structural protein in the extracellular matrix, providing tensile strength to skin, bone, cartilage, tendon, and blood vessels. Alterations in total collagen levels—whether due to fibrosis, wound healing, metabolic bone disease, or tumor invasion—are key indicators in a wide range of pathological processes. Quantifying total collagen with a single human collagen ELISA kit allows researchers to monitor matrix remodeling, evaluate antifibrotic therapies, and study organ fibrosis (liver, lung, kidney) without the need for complex histological scoring. A pan‑collagen detection approach, as delivered by this Human Collagen Type (Col) ELISA Kit, captures the overall collagen turnover and deposition, making it an efficient screening tool for preclinical and translational studies.
A Unique Advantage – Free ELISA Testing Service
Yanda Bio operates a fully equipped professional laboratory that not only develops kits but also provides ELISA testing services to the research community. When you purchase our Human Collagen Type (Col) ELISA Kit, you unlock a complimentary elisa testing service: simply send us your samples, and our experienced technicians will perform the assay for you. The entire procedure is video‑recorded, and you receive all raw data, standard curves, and calculated concentrations. This service eliminates the need to train personnel or invest in additional equipment, and it is especially valuable when you require high‑throughput analysis or immunogenicity ELISA testing services for biologics evaluation. We also offer standalone immunogenicity elisa testing services for drug development programs. Take advantage of our free experiment‑running offer and accelerate your research with confidence.
Assay Principle
This Human Collagen Type (Col) ELISA Kit employs a quantitative double‑antibody sandwich technique. A purified polyclonal antibody specific for human total collagen is pre‑coated onto the microplate. Standards and samples are added, and collagen present in the sample binds to the immobilized antibody. After washing, an HRP‑conjugated detection antibody is introduced, forming an antibody‑antigen‑enzyme complex. Following another wash step, TMB substrate is added. The HRP catalyzes a color change from blue to yellow after acid stopping, and the absorbance at 450 nm is directly proportional to the concentration of total collagen. A standard curve is generated, and sample concentrations are interpolated.
Kit Performance Characteristics
| Parameter | Specification |
|---|---|
| Detection Range | 50 μg/L – 1800 μg/L |
| Sample Types Validated | Serum, plasma, urine, cell culture supernatant, tissue homogenate, pleural/ascitic fluid, cerebrospinal fluid |
| Accuracy | Standard curve linear regression R ≥ 0.990 |
| Precision | Intra‑assay CV < 9%; inter‑assay CV < 11% |
| Specificity | Detects total human collagen; minimal cross‑reactivity with other structural proteins |
| Shelf Life | 6 months at 2–8°C |
| Recovery & Linearity | Within accepted ranges for biological samples |
Sample Collection & Preparation Guidelines
Adhere strictly to the following to avoid HRP inhibition and ensure reliable quantification:
- Serum: Allow blood to clot naturally at room temperature for 10–20 min. Centrifuge at 2000–3000 rpm for 20 min and collect the supernatant. Re‑centrifuge if precipitate forms during storage.
- Plasma: Use EDTA or sodium citrate as anticoagulant (do not use heparin that may interfere). Mix for 10–20 min, centrifuge as above, and harvest supernatant.
- Urine, pleural/ascitic fluid, cerebrospinal fluid: Collect in sterile tubes, centrifuge at 2000–3000 rpm for 20 min, and retain the supernatant. Repeat centrifugation if needed.
- Cell culture supernatant (secreted proteins): Collect with sterile tubes, centrifuge to remove debris. For intracellular collagen, resuspend cell pellet in PBS (pH 7.2–7.4) at ~10⁶ cells/mL, lyse by repeated freeze‑thaw cycles, centrifuge, and collect supernatant.
- Tissue homogenate: Weigh tissue, add PBS (pH 7.4), freeze rapidly in liquid nitrogen. Thaw while keeping at 2–8°C, homogenize thoroughly, centrifuge at 2000–3000 rpm for 20 min, and save the supernatant. Aliquot and freeze the remainder.
- Storage: Extract samples as soon as possible after collection. If immediate testing is not possible, store at -20°C and avoid repeated freeze‑thaw cycles. Do not use samples containing NaN₃, as it irreversibly inhibits HRP activity.
Brief Assay Protocol (Detailed Instructions Provided)
- Equilibrate the kit to room temperature for 15–30 min. Prepare wash buffer by diluting the 30× (or 20× for 48T) concentrate.
- Add 50 µL standards or samples (sample well: 40 µL sample diluent + 10 µL sample, resulting in a 5‑fold dilution) to the coated plate. Leave blank wells without sample or enzyme conjugate.
- Cover and incubate at 37°C for 30 min. Wash 5 times with wash buffer, soaking 30 seconds each cycle.
- Add 50 µL HRP‑conjugated detection antibody to each well (except blank). Incubate at 37°C for 30 min, then wash as before.
- Add 50 µL Chromogen A and 50 µL Chromogen B, mix gently, incubate at 37°C in the dark for 10 min.
- Add 50 µL Stop Solution. Read absorbance at 450 nm within 15 min.
- Construct a standard curve (concentration vs. OD). Multiply the readout by the dilution factor (×5) to obtain the actual sample concentration.
Note: For samples with very high collagen content (OD exceeds the highest standard), further pre‑dilute with sample diluent and multiply by the total dilution factor (×n ×5).
Important Operational Notes
- Allow the sealed plate to reach room temperature before opening. Return unused strips to the foil pouch with desiccant and store at 2–8°C.
- Concentrated wash buffer may form crystals; warm to dissolve completely before dilution.
- Use calibrated pipettes and add samples within 5 minutes for optimal accuracy. For large sample numbers, a multi‑channel pipette is recommended.
- Run a fresh standard curve in duplicate with every experiment.
- Seal plate membrane is single‑use to prevent cross‑contamination.
- Protect TMB substrate from light.
- All specimens and waste should be treated as potentially infectious.
- Do not mix components from different lot numbers.
Storage & Expiry
Store the kit at 2–8°C. The shelf life is 6 months from the date of manufacture.
Related ELISA Kits for Extracellular Matrix Research
Pair this total collagen assay with the following focused kits to dissect fibrosis and tissue remodeling:
- Human Apolipoprotein E (ApoE) ELISA Kit -The precise, seTnsitive quantification of apolipoprotein E in a variety of biological samples.
- Human Collagen Type III (COL3A1) ELISA Kit – An early marker of soft tissue fibrosis.
- Human CC Chemokine Ligand 12 (CCL12) ELISA Kit– For immunology and cancer studies
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For broader matrix profiling, explore our range of MMPs, TIMPs, and hyaluronic acid ELISA kits.
Disclaimer: This kit is for research use only. It is not intended for clinical diagnosis or therapeutic applications. Follow the protocol as provided; deviations are the responsibility of the user. Yanda Bio shall not be liable for consequences arising from improper use.
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