Immunoassays with collagen: Tips to optimize anti-collagen antibody performance

Dr Yong Zhang, Ph.D. is a senior scientist and R&D Manager for antibody generation and assay development at Rockland Immunochemicals, Inc. who are recognised and renowned for their high quality primary antibodies, secondary antibodies and many other reagents for Life Sciences.

Dr Yong Zhang’s special focus is on collagen related products, and I’m sure you’ll find his expert tips and tricks that he kindly shares in this post very useful if you’re working with collagen immunoassays. Read on to the end, where you’ll find a selection of high quality, reliable collagen related reagents that may be very useful for your projects.

Collagen proteins are essential components of the extracellular matrix and constitute the chief structural component of skin. Collagens are the most abundant protein in the body, representing approximately 30% of its dry weight. The use of collagen has become increasingly prevalent in various medical applications such as implants, organ replacement, tissue equivalents, arterial plugs, cosmetic surgery, surgical sutures, and surgical dressings for wounds and burns. These advances were predicated on the use of anti-collagen antibodies to allow researchers to detect collagens, including type-specific collagens, in cell biology research. The following tips help researchers in optimizing the performance of anti-collagen antibodies to improve experimental protocols and results.

Know your Collagen

Variations in collagen structure create functional diversity essential for distinct biological features in the various types of tissues of the body. Based on their supramolecular structures, collagens are divided into two main classes: fibril-forming collagens (type I, II, III, and V) and non-fibril-forming collagens (type IV and VI). In humans, there are more than 20 unique procollagens that are subjected to various post-translational modifications resulting in great diversity between collagen types.

Precise insights into the behaviour and physiology of cells can be made by researchers, especially when using type-specific anti-collagen antibodies to study collagen’s bio-distribution, interactions between associated cells, mechanisms of cell adhesion, and cell development and differentiation. Because of the high specificity of type-specific anti-collagen antibodies, which often differentiate one collagen type from another by subtle differences in the triple-helical structure of native collagen molecules, it is important to select the correct antibody that is “fit-for-purpose” to detect a specific type of collagen in the right tissue and immunoassay.

Know your Antibody

It cannot be understated that the appropriate controls should be included in experimental design when type-specific anti-collagen antibodies are used to ensure the success of the experiment and data reproducibility. Only highly purified and type-specific collagen proteins should be used as controls that are themselves free of impurities, including serum proteins, other collagen proteins, and non-collagen extracellular matrix proteins. Standards must not only be physically pure for collagen but also be immunologically pure for the type of collagen to be used as controls in experiments of this type. Type-specific anti-collagen antibodies should be selected that are appropriate for the detection of collagens present in the tissue to be tested, and these antibodies should be used with protocols designed to optimize antibody performance for the collection of high-quality data ultimately intended to help better understand the biology of these proteins.

Production of antibodies capable of targeting type-specific collagens can present unique challenges for antigen design, screening strategies, and antibody validation especially if the goal is to generate an antibody capable of detecting collagen in its native, non-denatured form and recognize collagens in a type-specific manner. Denatured collagen and native collagen present immunologically distinct epitopes to the immune system for recognition. It is not possible to generate type-specific anti-collagen antibodies when denatured epitopes are used as immunogens because the features that make types of collagen distinct from one another are lost when the collagen is physically denatured.

Type-specific anti-collagen antibodies must first be affinity purified against the target type-specific collagen immobilized to beads, e.g. type I, and the resultant antibody must be sequentially cross adsorbed against each of the other type-specific collagens, e.g. types II-VII, to render an antibody with the desired specificity. An additional challenge is to accomplish the purification and cross adsorption steps while maintaining the immobilized collagen in its native form so that only antibodies recognizing collagen’s three-dimensional structure will be purified, which greatly enhances the antibody’s type-specific binding properties. Extreme pH, high salt concentration, and detergents must be avoided at all costs as these conditions will permanently disrupt the collagen structure. Nevertheless, highly functional anti-collagen antibodies can be used in assays like Western blotting when the recommended protocols are precisely followed by researchers.Western blotting when the recommended protocols are precisely followed by researchers.

Tips for Collagen Western Blotting

Collagens are insoluble in organic solvents, and water-soluble collagen represents only a small fraction of total collagen, depending on the age and type of tissue. Many procedures call for the use of limited proteolytic digestion followed by salt extraction while maintaining the collagen preparation on ice. These steps are intended to minimize unwanted collagen degradation and denaturation.

Optimized protocols for detecting collagen by Western blotting include several steps that can be adjusted to generate the highest quality reproducible data. For instance, sample buffer for SDS PAGE should be adjusted to contain 125 mM Tris-HCl, pH 6.8, 10% Glycerol, 5% SDS, and 0.007% bromide blue with or without 4% β-mercaptoethanol. Many researchers have achieved successful separation of collagens when 4M urea is included in the sample, which results in better separation of the collagens during migration through the running gel.

As collagen types, especially native collagen, display significant molecular size differences, selection of SDS-PAGE running gels with the proper percentage of acrylamide and cross linking will generate better separation of collagen in gels. In general, 6% acrylamide gels work well for collagen types I, II, and III, while 10% acrylamide gels are recommended for collagen IV, V, and VI. More information on collagen protein separation from Rockland can be found on the Rockland website.

For best imaging of blots, use PVDF membrane and block the membrane with buffers containing 3% BSA. Type specific anti-collagen antibodies should be diluted from stock concentrations in blocking buffer to 1 µg/mL prior to use. After incubating the blot with primary antibody, wash the membrane with PBST for 15 minutes with gentle agitation at room temperature then decant and repeat the wash three additional times. Never allow the blot to dry during processing. An optimized secondary antibody, like Rockland’s Anti-Rabbit IgG (H&L) Peroxidase Conjugated Pre-adsorbed (p/n 611-103-122), is recommended for use at a 1:50,000 dilution from stock concentrations. These steps generally re

Tips for Enhanced Collagen ELISA Sensitivity

Sandwich ELISA techniques are recommended for collagen studies because this method can maintain the native structure of collagen as denaturing and dissociating conditions are not required. Sandwich or capture ELISA is more sensitive than direct or tittering ELISA methods. We recommend this straightforward and optimized ELISA protocol for high-quality results:

Step 1—Coating:
  • Coat the wells with 100 µL capture antibody at a concentration of 1–10 µg/mL
  • Cover the plate with adhesive plastic and incubate the plate overnight at 4°C
  • Decant the coating solution and wash with 250 µL of 1X PBST three times.
Step 2—Blocking:
  • Block the coated wells with 200 µL/well of blocking buffer containing 3% BSA
  • Cover the wells with adhesive plastic
  • Incubate the plate at room temperature for 2 hours or at 4°C for overnight.
Step 3—Sample incubation:
  • Add 100 µL of collagen standards and test samples, incubate at 37°C for 60–90 minutes
  • Decant the sample, wash 3 times with PBST
Step 4—Detection and secondary antibody incubation:
  • Add 100 μL of detection antibody, according to manufacturer’s optimal dilution
  • Cover the wells using adhesive plastic and incubate at room temperature for 2 hours
  • Wash the plate with PBST 3 times
  • Process for signal development and read plate.

Horseradish peroxidase (HRP) and alkaline phosphatase (ALP) are recommended as detection enzymes due to their sensitivity and low levels of detection.enerally result in low background staining and minimize non-specific protein staining.

Tips for Collagen Immunohistochemistry

A comprehensive histological study of type-specific collagens assists in determining its distribution and abundance in each tissue sample. While many immunoassays designed to detect type-specific collagens can be challenging, including immunohistochemistry, high-quality and reproducible data can be generated when optimized protocols are used. As type-specific anti-collagen antibodies recognize native, non-denatured collagens, frozen section immunohistochemistry reliably produces excellent results.

Researchers have reported that the following recommendations for fixed tissues and cells will improve study outcomes. Fix tissue with formaldehyde, followed by blocking tissue with 2.5% horse serum for 1 hour at 25°C. Certain tissues may require antigen retrieval methods and additional optimization. Both heat-induced epitope retrieval and protease-induced epitope retrieval have been used by researchers successfully to unmask collagen epitopes and restore epitope-antibody binding.

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Isabelle Nobiron
Written by Isabelle Nobiron
Isabelle is a Product Manager at tebu-bio, and also the company's ISO Quality Manager.