Antibody applications and techniques

Antibodies are powerful research tools used in a variety of laboratory techniques. Here, we will provide a brief overview of the most popular laboratory techniques, focusing on how they use antibodies.

Enzyme-linked immunosorbent assay (ELISA)

ELISA, a plate-based technique, facilitates detection of antigens in biological samples. ELISA utilizes antibodies to detect target antigens with highly specific antibody-antigen interactions, making it one of the many immunoassays. ELISA allows for quantification and characterization of analytes and molecular interactions.

In an ELISA, the antigen is attached to a solid surface either directly or more often using a capture antibody, which is itself attached to the surface(Figure 1). The surface is then washed and incubated with detection antibodies conjugated to molecules such as enzymes or florophores.

In the presence of the antigen, these detection antibodies will stay attached to the plate, producing a signal. The intensity of this signal aligns with the concentration of the antigen within the sample.

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

ELISA are typically executed using a multi-well plate(96 or 384 wells), with the analytes immobilized to enable the separation of the antigen from the remaining sample components. Due to these traits, ELISA is one of the simplest assays to accomplish on numerous samples concurrently.

There are four primary types of ELISA: direct, indirect, sandwich and competitive, each having its own distinct benefits, drawbacks and appropriateness. The most suitable ELISA structure for any given experiment will depend on several factors, including desired sensitivity, specificity and assay duration.

Enzyme-linked immunospot (ELISPOT)

Enzyme-linked immunospot(ELISPOT) detects proteins secreted by cells, such as cytokines and growth factors. This technique allows for quantification and comparison of immune responses to different stimuli.

Cells are cultivated in 96-well plates with PVDF or nitrocellulose membranes coated in antibodies. The proteins of interest secreted by the cells are detected using primary and conjugated secondary antibodies. The cells that secrete the protein of interest will manifest as color or fluorescence spots. The membranes are examined and analyzed to measure the amount or percentage of cells that secrete the protein.

Western blot (WB)

Western blot technique is extensively used in research for the separation and identification of proteins. It enables the detection of proteins, determination of relative protein levels between samples and estimation of the target protein’s molecular weight, thereby providing insight into its post-translational processing.

The Western blot comprises three main steps:(1) separating proteins according to size, (2) transferring them to a membrane and (3) visualizing the target protein through primary and secondary antibodies (Fig. 2).

Initially, the proteins are loaded onto a gel and separated by size using gel electrophoresis. Subsequently, protein bands are transferred to a membrane through an electrical current. Protein transfer to the membrane is indispensable since gels utilised for electrophoresis are of inadequate quality for subsequent immunostaining, meaning that proteins on the gel aren’t sufficiently adhesive for antibodies.

Finally, the membrane can undergo further immunostaining with antibodies specific to the target of interest before being visualised using secondary antibodies and detection reagents.

Figure 2. A simplified diagram of western blotting.

Immunoprecipitation (IP) and Chromatin immunoprecipitation (ChIP)

Immunoprecipitation (IP) is a flexible method for isolating and purifying both individual and complexed proteins. This technique involves immobilizing antibodies on solid-phase substrates(e.g, magnetic or agarose beads) to capture antigens from complex solutions.

Chromatin immunoprecipitation (ChIP) is a technique used to determine if a specific protein binds to particular DNA sequence in living cells. This allows scientists to pinpoint particular genes and sequences where a protein of interest binds throughout the entire genome, providing valuable insights into their regulatory functions and mechanisms.

The ChIP method (Fig. 3) uses an antibody to isolate a particular protein, such as a transcription factor, and its associated DNA. Afterward, the recovered DNA undergoes analysis through PCR, micro-array or sequencing to locate and identify the genomic sequence where the protein attaches.

Figure 3. A step-by-step guide to conducting a ChIP experiment, which involves the following five stages: 1. cross-linking, 2. chromatin fragmentation, 3. immunoprecipitation, 4. recovery and purification of DNA and 5. analysis of DNA.

Immunohistochemistry (IHC)

Immunohistochemistry (IHC) is a technique that examines the distribution and location of antigens within tissue sections through antibody-antigen interactions. While not as quantitative as Western blot or ELISA, IHC allows protein expression to be characterized in the context of intact tissue, making it advantageous.

IHC is a common diagnostic tool for detecting tissue abnormalities in diseases such as cancer. It provides valuable perspective and support which can be used to contextualise data from other methods.

IHC staining depends on antibodies that detect the target antigen. Visualize this interaction by using chromogenic or fluorescent-based detection systems. In chromogenic detection, an enzyme-conjugated antibody releases a color precipitate upon exposure to a chromogen. In fluorophore detection, a fluorophore-conjugated antibody is used instead. Various techniques exist for sample preparation and visualization, and the selected method should be customized to your specimen type and desired level of sensitivity.

Figure 4. The left panel shows fluorescent multiplex IHC staining of normal human tonsil tissue (formalin fixed paraffin embedded sections). merged staining of anti-PD1(orange), anti-PDL1(green), anti-CD68(yellow), anti-CD3(red), anti-Ki67(light blue) and anti-PanCK(grey). the figure on the right shows IHC staining of formalin-fixed paraffin-embedded normal human tonsil sections with anti-Ki67 antibody.

Immunocytochemistry (ICC)

Immunocytochemistry (ICC) is utilized for tudying the sub-cellular location of proteins through labelled antibodies. This technique focuses on samples of cells unlike IHC, which centers on blocks of tissues.

Antibodies specific to a protein of interest are applied to fixed and permeabilized cell culture samples in IDD staining. Two ICC staining techniques are available: direct and indirect. Direct ICC employs conjugated primary antibodies, while indirect ICC employs unconjugated primary antibodies. The unconjugated primary antibodies are detected by a conjugated secondary antibody. Fluorophores are commonly used to label antibodies in most ICC experiments, which is highly desirable for co-localization studies. Multiple imaging techniques, including widefield, confocal and spinning disc microscopy are available for detecting the signal.

Figure 5. The left panel shows indirect ICC staining of SH-SY5Y cells with anti-PDGFRA antibody (detected with Alexa Flour®488 secondary antibody-green) and anti-microtubulin antibody (detected with Alexa Fluor®594 secondary antibody-red). the right panel shows direct ICC detection with conjugated anti-KRT14 antibody (Alexa Fluor®647-red) and conjugated anti-microtubulin antibody (Alexa Fluor®488-green). cell nuclei were stained with DAPI(blue). the top image shows relevant signals in wild-type cells and the bottom image shows specific signals not present in the knockout. Images were taken by confocal microscopy.

Flow cytometry and FACS

Flow cytometry is a common laser-based method employed in the analysis of cell or particle properties. The technique entails measuring fluorescence emitted by labelde antibodies bound to individual cells within a mixed population. Additionally, the light scattering of distinct cells is used to determine their sizes and properties.

Figure 6:Overview of flow cytometry.

Flow cytometry allows you to analyse the expression of cell surface and intracellular molecules, characterise different cell types in heterogeneous cell populations, assess the purity of isolated sub-populations, and analyse cell size and volume. It allows simultaneous multi-parameter analysis of individual cells.

Fluorescence-activated cell sorting (FACS) is a flow cytometry-derived technique that physically separates cell populations into sub-populations based on fluorescent labeling.

Reference

1 Bonifacino, J.S., and Dell'Angelica, E.C. (2001). Immunoprecipitation. Curr. Protoc. Cell Biol. Chapter 7, Unit 7.2.

2 Harlow, E., and Lane, D. (1988). Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press.

3 Gundry, R.L., and White, M.Y. (2010). Immunoprecipitation. In: Protein Chromatography: Methods and Protocols, eds. R. Gundry and M.Y. White, pp. 189-203. Humana Press.

4 Towbin H, Staehelin T, Gordon J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 76(9):4350-4.

5 Taylor, C. R., & Levenson, R. M. (2006). Quantification of immunohistochemistry—issues concerning methods, utility and semiquantitative assessment II. Histopathology, 49(4), 411-424.

6 Van Weemen, B. K., & Schuurs, A. H. (1971). Immunoassay using antigen-enzyme conjugates. FEBS letters, 15(3), 232-236.