Optimized Methods for the Surface Immobilization of Collagens and Collagen Binding Assays

Summary

This work presents an optimized protocol to reproducibly immobilize and quantify type I and III collagen onto microplates, followed by an improved in vitro binding assay protocol to study collagen-compound interactions using a time-resolved fluorescence method. The subsequent step-by-step data analysis and data interpretation are provided.

Abstract

Fibrosis occurs in various tissues as a reparative response to injury or damage. If excessive, however, fibrosis can lead to tissue scarring and organ failure, which is associated with high morbidity and mortality. Collagen is a key driver of fibrosis, with type I and type III collagen being the primary types involved in many fibrotic diseases. Unlike conventional protocols used to immobilize other proteins (e.g., elastin, albumin, fibronectin, etc.), comprehensive protocols to reproducibly immobilize different types of collagens in order to produce stable coatings are not readily available. Immobilizing collagen is surprisingly challenging because multiple experimental conditions may affect the efficiency of immobilization, including the type of collagen, the pH, the temperature, and the type of microplate used. Here, a detailed protocol to reproducibly immobilize and quantify type I and III collagens resulting in stable and reproducible gels/films is provided. Furthermore, this work demonstrates how to perform, analyze, and interpret in vitro time-resolved fluorescence binding studies to investigate the interactions between collagens and candidate collagen-binding compounds (e.g., a peptide conjugated to a metal chelate carrying, for example, europium [Eu(III)]). Such an approach can be universally applied to various biomedical applications, including the field of molecular imaging to develop targeted imaging probes, drug development, cell toxicity studies, cell proliferation studies, and immunoassays.

Introduction

The accumulation of fibrous connective tissue as part of the natural wound-healing process following tissue injury is known as fibrosis. However, if the deposition of fibrous tissue fails to terminate and continues beyond what is needed for tissue repair, then fibrosis becomes excessive^1,2. Excessive fibrosis impairs organ physiology and function and could lead to organ damage and potentially organ failure^3,4,5. Two main drivers of fibrosis are the extracellular matrix (ECM) proteins collagen type I and type III

Protocol

NOTE: All product information used for this work is presented in the Table of Materials.

1. Collagen immobilization

NOTE: Ensure each well in the microplate used during the binding assay has adjacent wells free to avoid cross-fluorescence. Carry out this part of the protocol on ice because collagen self-assembles at rising temperatures and pH levels. Perform this procedure in a tissue culture hood and under sterile conditions because .......

Discussion

This work presents a reproducible method for immobilizing type I and type III collagen. It also demonstrates a protocol for acquiring, analyzing, and interpreting in vitro Eu(III) TRF binding data to characterize the binding properties of a candidate ligand toward type I and III collagen. The protocols for immobilizing type I and type III collagen presented here were developed and optimized considering previously published work on type I and type III collagen fibrillogenesis in vitro^13.......

Materials

Name	Company	Catalog Number	Comments
10x PBS	Gibco	14200075	Use this to make 1x PBS by diluting in water (1:10)
2M HCL			Made in house and details are in the supporting document
2M Sodium hydroxide +2M Glycine			Made in house and details are in the supporting document
Cell-star 96 well  microplate	Greiner Bio-One	655 160
DELFIA enhacement solution	Perkin Elmer	1244-104
Ice
Infinite 200 PRO NanoQuant microplate reader	TECAN
Non-binding (NBS) 96 well microplates	Corning	3641
pH electrode Inlab Routine	Mettler Toledo	51343050
pH meter (sevenCompact)	Mettler Toledo
Pierce BCA protein assay kit	Thermofisher	23227
Tissue culture incubator (37 °C, 5% CO2)
Type I bovine collagen, 3 mg/mL	Corning	354231
Type III human placenta collagen, 0.99 mg/mL	Advanced Biomatrix	5021