Type I collagen is the most abundant structural protein in connective tissues. Because collagen possesses a unique amino acid sequence, it tends to share high amino acid sequence homology across different species. Therefore, evaluating the cross-reactivity of monoclonal antibodies (mAbs) with various collagen species is essential for studying tissue fibrosis, scaffold degradation, and implant integration.
Chondrex, Inc. provides high-quality mAbs directed against type I collagen. Understanding antibody specificity is critical for accurate immunohistochemical analysis, particularly when tracking the tissue distribution of type I collagen. This is especially important in xenograft models, such as human cell implantations or biomaterial scaffolds evaluated in mice and rats. The following table displays mAb reactivity against type I collagen from various species, expressed as a ratio relative to the highest-reacting species. This information can guide you in choosing the correct antibodies for your studies and interpreting the experimental results accurately.
Table 1. Specificity of monoclonal antibodies against type I collagen
| Clone (mAb) Name | Subtypes/Subclasses | Type I Collagen Species | Catalog # Unconjugated (Biotinylated) | |||||
| Chicken | Bovine | Porcine | Rat | Human | Mouse | |||
| 42R | Rat IgM | 56% | 25% | 43% | 53% | 100% | 23% | 7025 (7026) |
| 8D4A1 | Rat IgG2a | 58% | 15% | 16% | 27% | 7% | 100% | 7041 (7042) |
| 1F10C2 | Mouse IgG2a | 11% | 29% | 9% | 100% | 0% | 0% | 7043 (7044) |
| 7G5D2 | Rat IgG2a | 12% | 100% | 76% | 32% | 0% | 87% | 7045 (7046) |
| 2H12B4 | Mouse IgG1 | 31% | 100% | 97% | 83% | 97% | 4% | 7066 (7067) |
| 222b | Rat IgM | 0% | 100% | 57% | 13% | 38% | 12% | 7086 (7087) |
| 1C1F8 | Rat IgG2a | 0% | 5% | 1% | 1% | 100% | 0% | 7135 |
| 3E9B3 | Mouse IgG2c | 19% | 76% | 63% | 15% | 100% | 92% | 7136 |
Materials and Methods
mAb specificity and inter-species cross-reactivity were evaluated using indirect enzyme-linked immunosorbent assays (ELISA). ELISA plates were coated with the ELISA grade type I collagens listed in Table 2.
Table 2. ELISA grade type I collagen for plate coating
| ELISA Grade Type I Collagen | Catalog # |
| Chicken | 1001 |
| Bovine | 1002 |
| Porcine | 1003 |
| Rat | 1004 |
| Human | 1005 |
| Mouse | 1006 |
| Collagen Dilution Buffer | 9003 |
1. Microtiter ELISA plates were coated with 5 µg/ml of each ELISA grade collagen (Table 2) diluted with collagen dilution buffer (Cat # 9003) and incubated overnight at 4°C.
2. Following incubation, plates were washed with PBS containing 0.05% Tween 20 (PBST) and then incubated for 2 hours at room temperature with anti-type I collagen mAbs diluted to 1 µg/ml in Solution B (Sample/Standard Dilution Buffer, Cat # 2072).
3. After washing with PBST, plates were incubated for 1 hour at room temperature with Secondary Antibody: Peroxidase-Conjugated Goat Anti-Mouse IgG (Cat # 7012) or Peroxidase-Conjugated Goat Anti-Rat IgG (Cat # 10214), both diluted in Solution C (Secondary Antibody Dilution Buffer, Cat # 2073). For assays utilizing rat IgM mAbs, detection was performed using a biotinylated anti-type I collagen mAb followed by Streptavidin Peroxidase (Cat # 9029) diluted in Solution D (Streptavidin Peroxidase Dilution Buffer, Cat# 9055).
4. After a final PBST wash, the colorimetric reaction was developed by adding TMB substrate (3,3',5,5'-tetramethylbenzidine) diluted with chromogen dilution buffer (Cat # 90023) and incubating for 25 minutes at room temperature.
5. The reaction was terminated with stop solution (Cat # 9016) and absorbance was measured at 450 nm, with 630 nm used as the reference wavelength.
6. Relative cross-reactivity was quantified by assigning a value of 100% to the highest recorded OD value among the tested species, and the reactivity of all other species was calculated and expressed as a percentage relative to this maximum value.