Antibody methods and techniques

​Contents

• Enzyme-linked immunosorbent assay (ELISA)
• Enzyme-linked immunospot (ELISPOT)
• Western blot (WB)
• Immunoprecipitation (IP) and Chromatin Immunoprecipitation (ChIP)
• Immunohistochemistry (IHC)
• Immunocytochemistry (ICC)
• Flow cytometry and FACS

Enzyme-linked immunosorbent assay (ELISA)

ELISA is a plate-based technique enabling the detection of antigens in biological samples. Like other immunoassays, ELISA relies on antibodies to detect a target antigen using highly specific antibody-antigen interactions. ELISA enables the quantification and characterization of analytes and molecular interactions. 

In an ELISA, the antigen is immobilized to a solid surface either directly or more commonly via a capture antibody, itself immobilized to the surface (Fig. 1). The surface is washed, then incubated with detection antibodies conjugated to molecules such as enzymes or fluorophores. 

In the antigen's presence, these detection antibodies will remain bound to the plate, providing a signal. The strength of this signal corresponds to antigen concentration within the sample. 

Figure 1. Sandwich ELISA setup. A capture antibody on a multi-well plate will immobilize the antigen of interest. This antigen will be recognized and bound by a detection antibody conjugated to biotin and streptavidin-HRP. 

An ELISA is typically performed in a multi-well plate (96- or 384-wells), and the analytes' immobilization facilitates the separation of the antigen from the rest of the sample components. These characteristics make ELISA one of the easiest assays to perform on multiple samples simultaneously.

There are four main types of ELISA: direct, indirect, sandwich, and competitive – each with unique advantages, disadvantages, and suitability. The most appropriate ELISA format for each experiment will depend on many factors, including desired sensitivity, specificity, and assay time. See more information here to help you choose the right type of ELISA.

For the same cost as a standard ELISA, our SimpleStep ELISA™ kits can halve assay time without compromising sensitivity or reliability.

Introduction to ELISA and troubleshooting webinar
ELISA resources
ELISA kits
SimpleStep ELISA™ kits
Benefits of SimpleStep ELISA™
ELISA troubleshooting tips
Introduction to In-Cell ELISA webinar

Enzyme-linked immunospot (ELISPOT)

Enzyme-linked immunospot (ELISPOT) is used to detect proteins secreted by cells, such as cytokines and growth factors. The technique enables quantification and comparison of immune responses to various stimuli.

Cells are grown in 96-well plates with antibody-coated PVDF or nitrocellulose membranes. The secreted proteins of interest are detected using primary and conjugated secondary antibodies. Cells secreting the protein of interest will appear as a spot of color or fluorescence. Membranes are scanned and analyzed to quantify the number or proportion of cells secreting the protein.

ELISPOT and FLUOROSPOT assays
ELISPOT protocol
ELISPOT troubleshooting tips
​ELISPOT products

Western blot (WB)

Western blot is widely used in research to separate and identify proteins. Western blot allows us to detect proteins, determine the relative protein levels between samples, and establish the target's molecular weight, providing insight into its post-translational processing. 

Western blot involves three main steps: (1) separation of proteins by size, (2) transfer of proteins to a membrane, and (3) visualizing the target protein using primary and secondary antibodies (Fig. 2). 

In the first step, the proteins are loaded onto a gel and separated based on size by gel electrophoresis. Protein bands are then migrated to a membrane using an electrical current. Protein transfer to the membrane is essential because gels used for electrophoresis provide an inferior surface for subsequent immunostaining, ie, antibodies don't stick to the gel's proteins. 

Finally, the membrane can be further immunostained with antibodies specific to the target of interest and visualized using secondary antibodies and detection reagents. 

Western blot resources
Western blot troubleshooting tips
Western blot training
​HRP detection kits
HRP conjugates

Immunoprecipitation (IP) and Chromatin immunoprecipitation (ChIP)

Immunoprecipitation (IP) is a versatile technique that isolates and purifies individual and complexed proteins. In this technique, antibodies are immobilized on solid-phase substrates (eg, magnetic/agarose beads), capturing antigens from complex solutions.

Chromatin immunoprecipitation (ChIP) is used to determine whether a given protein binds to a specific DNA sequence in vivo. ChIP allows researchers to identify specific genes and sequences where a protein of interest binds across the entire genome, providing critical clues to their regulatory functions and mechanisms.

The ChIP procedure (Fig. 3) utilizes an antibody to immunoprecipitate a protein of interest, such as a transcription factor, along with its associated DNA. The associated DNA is then recovered and analyzed by PCR, microarray or sequencing to determine the genomic sequence and location where the protein was bound.

Figure 3.  ChIP protocol workflow. A step-by-step approach to carrying out a ChIP experiment, where 1 - cross-linking, 2 – chromatin fragmentation, 3 – immunoprecipitation, 4 – DNA recovery and purification, 5 – DNA analysis.

Immunoprecipitation protocol
Webinar: Introduction to IP
Webinar: Advanced IP
Troubleshooting tips for IP
Chromatin immunoprecipitation: ChIP step-by-step webinar
ChIP guide
​ChIP training

Immunohistochemistry (IHC)

Immunohistochemistry (IHC) is a method to access the distribution and localization of antigens in tissue sections using antibody-antigen interactions. Although less quantitative than western blot or ELISA, IHC offers the advantage of characterizing protein expression in the context of intact tissue.

IHC is often used to diagnose tissue abnormalities in diseases such as cancer. IHC provides valuable perspective and support that can contextualize data obtained from other methods.

IHC staining relies on antibodies that recognize the target antigen. You can use chromogenic or fluorescent-based detection systems to visualize this antibody-antigen interaction. In chromogenic detection, an antibody is conjugated to an enzyme that produces a colored precipitate when exposed to a chromogen. In fluorescent detection, an antibody is conjugated to a fluorophore. There are various techniques for sample preparation and visualization, and the method used should be tailored to your type of specimen and the degree of sensitivity required.

Highly validated antibody conjugates
Understanding IHC basics webinar 
Immunohistochemistry training
The complete guide to immunohistochemistry
IHC protocols

Immunocytochemistry (ICC)

Immunocytochemistry (ICC) is used to study the subcellular distribution of proteins using labeled antibodies. In contrast to IHC, this technique focuses on samples of cells rather than blocks of tissues. 

In ICC staining, antibodies raised against a protein of interest are applied to cell culture samples that have been fixed and permeabilized. There are two types of ICC: direct and indirect. Direct ICC uses conjugated primary antibodies, whereas indirect ICC involves an unconjugated primary antibody, which will then be detected by a conjugated secondary antibody. For most ICC experiments, antibodies are labeled with fluorophores which is ideal for co-localization studies. Various imaging techniques, such as widefield, confocal or spinning disc microscopy, can be used to detect the signal. 

ICC/IF Immunocytochemistry and immunofluorescence protocol

Flow cytometry and FACS

Flow cytometry is a popular laser-based technology used to analyze the characteristics of cells or particles. The technique measures fluorescence emitted by labeled antibodies bound to individual cells in a mixed population. Also, the scattering of light by different cells is used to determine their size and properties.

Flow cytometry enables you to analyze the expression of cell surface and intracellular molecules, characterize and define different cell types in a heterogeneous cell population, assess the purity of isolated subpopulations, and analyze cell size and volume. It allows simultaneous multi-parameter analysis of single cells.

Fluorescence-activated cell sorting (FACS) is a derivative of flow cytometry that physically separates a population of cells into subpopulations based on fluorescent labeling. 

Highly validated antibody conjugates
​​Introduction to flow cytometry
Flow cytometry multicolor selector
Flow cytometry training

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