Immunohistochemistry Techniques and Antibodies

Immunohistochemistry & Immunocytochemistry Techniques

• Immunohistochemistry and immunocytochemistry are techniques that rely on the specific binding between antigens and antibodies.
• Stereospecific interaction is characterized by the highest specificity between chemical compounds.
• Covalent bonds are not formed, but weak bonds cause molecular interaction.
• Antigenic-antibody binding involves hydrogen bonding, electrostatic interactions, and Van der Waals forces.
• Affinity histochemistry is a key histochemical technique leveraging high affinity bonding of antigens an antibodies.
• Immunohistochemistry (IHC) and hybrid cytochemistry are examples of affinity histochemistry.

Introduction

• Antibodies are characterized by their highest specificity for binding to antigens.
• This specificity results from the spatial matching (complementarity) of bonding molecules.
• Antibody-antigen binding, or purine-pyrimidine bonding in nucleic acids, occurs via weak multiple bonds from atom proximity.

Immuno... Reactions

• IHC methods rely on antigen-antibody reactions to detect and localize cells and tissues.
• These methods allow for the detection of substances with specific antigenic characteristics.
• Routinely used in cell type identification and cancer marker detection.

Cancer Type/Useful Markers

• Useful cancer markers are provided for various cancer types.

Antigen

• Antigens are multi-component substances recognized by the immune system— eliciting an immune response with specific antibody production.
• Immunogenicity refers to the antigen's ability to generate an immune response.
• Antigenicity describes an antigen's ability to bind to antibodies or receptors.
• Immunogenes possess both immunogenicity and antigenicity whereas haptens exhibit only antigenicity.

Antigen cont.

• An epitope is a part of an antigen that the immune system recognizes, usually via antibodies, B-cells, or T-cells.
• Antigens can be monovalent (possessing one epitope) or polyvalent (possessing multiple epitopes).

Antibody

• Antibodies (immunoglobulins) are glycoproteins produced by differentiated B lymphocytes (plasma cells).
• They bind specifically to antigens via variable regions (V) and constant regions (C).
• Five major immunoglobulin classes are: IgA, IgD, IgE, IgG, and IgM; IgG is most often used in immunocyto/histo/chemistry.
• Antibody variable regions contain specific antigen-binding sites (paratopes), which determine specificity.

Types of antibodies used in IHC

• Monoclonal antibodies: highly specific, recognize one antigen determinant.
  • Advantages: highly specific; enabling differentiation of minimal variations in antigens; reduce background effects when compared to polyclonal antibodies.
  • Disadvantages: can be expensive; time consuming; can struggle to bind to an antigen if its structure is modified, for example, due to denaturation as an epitope change.
• Polyclonal antibodies: less specific; recognize multiple epitopes, and generated from several different B cells clones.
  • Advantages: react with various epitopes; high binding affinity, fewer chances of false negative results; potentially less expensive
  • Disadvantages: high degree of non-specificity or cross reactivity; more background effects compared to monoclonal antibodies.

Markers used in IHC

• Markers can be directly observed using fluorochromes or indirectly through enzymes or metals.
• Fluorochromes: fluorescent dyes; visual under fluorescent microscope.
• Enzymes: require detection of enzyme activity, creating a visible marker; observed by light or electron microscope.
• Metals: observed under the electron microscope (e.g., gold); providing high contrast.

Markers used in immuno... methods

• Fluorochromes are substances that emit light after radiation.
• Commonly used fluorochromes include fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC) and Texas Red.

Markers used in IHC

• Enzymes are used for indirect detection methods.
• Examples include horseradish peroxidase, alkaline phosphatase, and glucose oxidase.

Heavy metals

• Heavy metals, such as ferritin and colloidal gold, are used for electron microscopy.
• Colloidal gold produces good contrast due to the binding of tissue with gold, and reduction of silver salt which forms a colorful precipitate.

Preparing histopathological material for tests

• Preparation involves steps like tissue fixation, processing, embedding in paraffin, slicing, deparaffinization, rehydration, staining, dehydration and clearing, concluding with slide mounting.

What's important during IHC reaction?

• Key steps include fixing, embedding, and cutting paraffin blocks, antigen retrieval, background busting, antibody selection, control reactions, and the direct or indirect method, in addition to examination.
• Each of the preceding process steps has a future influence on the final IHC reaction result.

What kind of material is used to perform IHC reaction?

• Formalin-fixed, paraffin-embedded (FFPE) tissue sections, typically 3-4 μm thick are commonly used.
• Smears can, in some circumstances, also be used.

Paraffin section preparation

• Tissue sections are deparaffinized using xylene at 56-60°C and subsequently at room temperature.
• Rehydration involves using decreasing concentrations of alcohols.

Utrwalenie materiału do IHC

• Buffered formalin (10%, pH 7.2) is a common tissue fixative.
• Aldehydes (formaldehyde, paraformaldehyde, glutaraldehyde) bind different functional groups on proteins creating crosslinks during fixation.

Antigen sites retrieval

• Fixation and embedding can modify antigen molecules, blocking antibody recognition.
• Retrieval methods restore original antigen characteristics to permit antibody binding.
• Techniques include proteolytic enzymes (e.g., trypsin, chymotrypsin) and microwaves.

Enzyme-induced epitope retrieval (EIER)

• Proteolytic enzymes are used to digest the tissue sections.
• Common proteolytic enzymes are K proteinase, E proteinase, and pronase, trypsin, or chymotrypsin in PBS buffer (pH 7.2).
• The digestion process occurs at room temperature or 37°C.

Heat-induced epitope retrieval (HIER)

• Tissue sections are boiled in a buffer, such as citrate buffer (pH 6.0), or EDTA (pH 9.0) at 95°C for 15–20 minutes, followed by cooling.
• This process can restore or expose hidden epitopes.

Inhibiting endogenous enzymatic activity

• Endogenous enzyme activity is neutralized to prevent unwanted background staining.
• This is done by incubating sections in 3% hydrogen peroxide solution for peroxidase.
• Specific inhibitors like levamisole can block alkaline phosphatase activity.

Immunoglobulins' unspecific binding sites blocking

• Blocking nonspecific sites on the tissues is crucial to prevent them from binding unrelated proteins, which can cause unwanted staining.
• Unspecific binding proteins are therefore blocked with solutions containing proteins from another species (e.g. bovine serum albumin (BSA) or non-immunized animal serum).

IHC reactions

• IHC reactions can either be direct (using a primary antibody that is marked) or indirect (using a primary antibody followed by a secondary, marked antibody).
• Direct methods bind a marked antibody directly with the target antigen, while indirect methods use a secondary antibody.
• Indirect methods are generally considered to be more sensitive to signal the reaction.

Direct reaction

• This involves incubating a tissue sample with a marked primary antibody that directly binds to the target antigen.

Indirect reaction

• This involves incubating a tissue sample with an unmarked primary antibody, followed by marked secondary antibody that binds to the primary antibody.

Detection of marking enzymes

• Detection of enzymes is the final step of immunoenzyme processes.
• A reaction detecting enzyme activity results in a colourful insoluble product, marking the antigen-antibody complex location.

Detection of Peroxidase activity

• Peroxidase activity is detected by a reaction involving diaminobenzidine (DAB) which oxidizes into a brown/dark brown product.
• The use of DAB produces a deposition product which is then visualized.

Detection of alkaline phosphatase activity

• Alkaline phosphatase activity can be detected via reactions using BCIP in conjunction with NBT.
• This yields an indigo visible product.

Staining basophilic structures

• Hematoxylin, according to Mayer, stains basophilic structures, like nuclei, dark purple/dark blue, highlighting important morphological features.

Final stages of preparing slides

• Final stages include steps like dehydration using increasing alcohol concentrations, clearing the slides with xylene I-IV, applying mounting medium, and covering with a cover slip.

Closing glass slides in medium

• Mounting medium is used to close glass slides.
• This helps improve microscopic image quality/clarity, protects the staining from physical damage/environmental factors during microscopic imaging.

Control reactions

• Control reactions are a crucial aspect of IHC testing.
• They help control results credibility, confirm methodology specificity, and highlight any artefacts.
• Positive controls reveal appropriate staining of antigens in a tissue/process; negative controls are the samples which should not stain.

Positive control reaction

• A positive control reaction uses a known positive tissue sample to confirm whether antibody-related procedures are correctly executed and will result in a positive outcome.

Negative control reactions

• Negative controls are samples that should not show staining under specific circumstances to confirm methodology accuracy and the absence of cross-reactions.

Stages of IHC reaction - Reagents

• Reagents for the different stages of IHC are detailed. This includes the processing stages such as deparaffinization and hydration, antigen retrieval, blocking endogenous peroxidase activity, background elimination, antibody incubation, and visualization and staining of basophilic structures

Detecting markers

• Markers like DAB and NBT/BCIP, used for peroxidase and alkaline phosphatase respectively, are mentioned and how their detection is accomplished.

Localization of the Ag

• Localization of target antigens is described in relation to different cellular structures.

Panels IHC

• Panels are used to examine multiple antigens using IHC, improving the evaluation of specific cancer cases.

Other markers

• List of other markers utilized for tumor detection.

Metastasis

• Information about biomarkers relevant to metastasis in various cancer types

Histology

• Examples of markers for detecting various cancer types.

Why is IHC testing done?

• IHC testing can provide a more specific diagnosis after other standard tests have been completed.
• Pathologists may use IHC tests for specific cancer types when other processes don't yield enough information.
• Individual cases may require or not require IHC testing.

Benefits and Risks of IHC testing

• IHC benefits in improving the diagnosis and treatment of various cancers.
• IHC accuracy and risks are associated with several factors.

IHC test kits

• Procedures and regulations affecting IHC tests are highlighted.

More information

• Useful web links regarding immunohistochemistry are provided.

Immunohistochemistry

• Illustrative diagram showing the general concept of an IHC technique.