Different Types of Immunological Techniques With Examples

Immunological techniques is the set of laboratory methods that is based on the specific antigen–antibody reaction, and it is used to detect, identify or measure different biological substances in a sample. It is the process where an antibody binds with its corresponding antigen and this binding is used for locating proteins, hormones, pathogens or other molecules present in blood, serum or tissue.

These techniques are used in tests like immunodiffusion, ELISA, and radioimmunoassay, where the antigen–antibody reaction is visualised either as a visible band or by using a labelled enzyme or radioactive marker.

It is an important group of methods in diagnosis of infections and in studying immune responses, because the reactions formed are highly specific and give reliable indication of the presence of a particular antigen or antibody in the sample.

Different Types of Immunological Techniques

A. Classical Techniques

a. Precipitation Reactions

• It is the reaction where a soluble antigen reacts with a soluble antibody to form an insoluble precipitate.
• It is the process in which a lattice structure is formed when both antigen and antibody are present in proper proportion (zone of equivalence).
• It is required that antibody molecules are bivalent and antigens are bivalent or polyvalent for lattice formation.
• It is different from agglutination because precipitation involves soluble molecules whereas agglutination involves particulate antigens.
• It is observed either in liquid or in gel medium depending on the method used.
• Some of the main techniques are– the precipitin ring test, flocculation test, Ouchterlony double diffusion, and radial immunodiffusion.

Some of the main types are–

Precipitin Ring Test

It is the method where antigen is layered over antiserum and a ring is formed at the junction.

• It is the simple precipitation test used to detect the presence of antigen or antibody in liquid medium.
• It is performed in a narrow test tube where antigen solution is carefully layered over antiserum solution.
• It is the process where both solutions diffuse slowly at the junction without mixing immediately.
• A visible white ring is formed at the interface if antigen and antibody are in proper proportion (zone of equivalence).
• The white ring indicates a positive reaction for the specific antigen–antibody pair.
• Some common uses are– C-reactive protein (CRP) test, Lancefield grouping of β-haemolytic streptococci, and Ascoli’s thermoprecipitin test.

Radial Immunodiffusion (RID)

Antigen diffuses through antibody containing gel and a precipitin ring develops whose diameter indicates concentration.

• It is the quantitative immunodiffusion method used to measure the exact concentration of a specific antigen.
• It is the process where antibody is mixed uniformly in agarose gel and small wells are made to load the antigen.
• Antigen diffuses radially from the well and meets antibody in the gel, forming a visible precipitin ring.
• The ring forms at the zone of equivalence where antigen and antibody are in proper proportion.
• The square of the diameter of the ring is directly proportional to the antigen concentration.
• A standard curve is used to calculate unknown antigen concentration by comparing ring sizes.
• Ring formation depends on proper well filling, correct antiserum concentration, and proper gel temperature.
• It is commonly used for measurement of serum immunoglobulins like IgG, IgM and IgA.

Ouchterlony Double Diffusion

Both antigen and antibody diffuse in agar and different line patterns are produced.

• It is the qualitative immunodiffusion method where both antigen and antibody diffuse through agar gel.
• It is called double diffusion because antigen and antibody move independently in two directions inside the gel.
• Wells are made in agar with antibody in the center well and different antigens in surrounding wells.
• Antigen and antibody diffuse outward and meet in the gel to form visible precipitin bands.
• Bands appear at the zone of equivalence where antigen and antibody are in proper proportion.
• It is used to compare different antigens and study their relationship.
• Three main patterns are seen – identity (smooth arc), non-identity (crossed bands), and partial identity (spur formation).

Immunoelectrophoresis

It is the process where electrophoresis and immunodiffusion are combined for separation and identification of serum proteins.

• It is the qualitative technique where electrophoresis and immunodiffusion are combined to separate and identify proteins.
• It is the process where proteins are first separated in agar gel according to charge and size by applying electric current.
• After separation, a trough is cut in the gel and specific antiserum is added for diffusion.
• Antigens diffuse from their separated positions and antibodies diffuse from the trough until they meet.
• Visible precipitin arcs are formed at the zone of equivalence showing antigen–antibody reaction.
• Rocket immunoelectrophoresis is the variation where antibody is mixed in the gel and antigen moves under electric field to form rocket-shaped precipitate.
• It is used to identify serum proteins and detect abnormalities like multiple myeloma or hypogammaglobulinemia.

b. Agglutination Reactions

• It is the reaction where particulate antigens like bacteria, RBCs or latex beads react with specific antibodies to form visible clumps.
• It is the process in which antibodies bind to antigens present on the particle surface and form cross-links causing aggregation.
• It is different from precipitation because agglutination involves insoluble particles whereas precipitation involves soluble molecules.
• It is observed clearly when antigen and antibody are present in proper concentration, so clumps become visible.
• Direct agglutination is the method where antibody reacts with natural surface antigens of the particles.
• Passive agglutination is done when soluble antigens are coated on carrier particles and then react with antibody.
• Hemagglutination is the process where RBCs clump with specific antibodies and it is used for blood grouping.
• It is used in ABO and Rh typing, microbial identification and determination of immune status.

Some of the important are–

• Hemagglutination – Used in blood grouping where RBCs agglutinate in presence of specific antibody.
  • It is the agglutination test where red blood cells (RBCs) react with specific antibodies to form visible clumps.
  • It is the process in which multivalent antibodies bind to antigens present on the surface of several RBCs and form a lattice.
  • The clumping of RBCs becomes visible to the naked eye because the cells are large.
  • It is mainly used in ABO and Rh blood grouping.
  • A positive reaction occurs when RBCs have the specific antigen and the reagent antibody binds, producing clear clumps.
  • A negative reaction occurs when the antigen is absent and no clumping is seen.
  • It is used because it gives quick and clear results needed for transfusion and transplantation procedures.
• Flocculation – It is used in tests like VDRL where fine clumps are formed in suspension.
  • It is the precipitation reaction that occurs in liquid medium where soluble antigen reacts with soluble antibody to form visible floccules.
  • It is the process in which small clumps appear instead of a continuous ring or lattice.
  • Slide flocculation test is done by mixing antigen and serum on a slide to observe clumps directly.
  • VDRL test is the common slide flocculation test used for detecting reaginic antibodies in syphilis.
  • Tube flocculation test is performed in a test tube and clumps are seen after gentle mixing.
  • Kahn test and some toxin–toxoids standardization tests are examples of tube flocculation methods.

B. Labeled Immunoassays

It is the method where antibodies or antigens are attached with labels (enzyme or fluorescent molecule) and the reaction is detected by signal production.

• It is the group of immunological methods where a label is attached with antigen or antibody to produce a measurable signal.
• It is the process that detects analytes by enzyme, fluorescent, chemiluminescent or radioactive signals instead of visible clumps or precipitates.
• These assays are more sensitive because the signal is measured even at very low antigen or antibody concentration.
• ELISA is the common labeled assay using enzyme label for color formation.
• RIA uses radioactive label but is less used now due to safety issues.
• CLIA uses chemical labels that emit light and it gives high sensitivity.
• Lateral flow immunoassay uses colored particles on membrane for rapid visual detection.
• Digital ELISA isolates single molecules for extremely sensitive measurement.
• Competitive assays use labeled analog competing with sample antigen, giving inverse signal.
• Sandwich assays use excess antibody to capture antigen and signal becomes directly proportional to antigen concentration.

Some of the common types are–

• ELISA (Enzyme Linked Immunosorbent Assay) – It is used for detection and quantification of antigen or antibody.
  • Direct ELISA
  • Indirect ELISA
  • Sandwich ELISA
  • Competitive ELISA
• Chemiluminescence Immunoassay – Light emission is measured as the indicator.
• Lateral Flow Immunoassay – It is used in rapid test kits.
• Radioimmunoassay (RIA) – Uses radioactive label but is less used due to hazards.

C. Separation-Based Immunological Techniques

These techniques separate components first and then apply antigen–antibody reactions.

• It is the group of methods where a physical separation step is done before applying antigen–antibody reaction.
• It is the process that separates molecules by size or charge so that antibody reacts only with the specific component.
• These techniques increase specificity because proteins are resolved first and then detected.
• Western blot is used where proteins are separated by electrophoresis, transferred to membrane and detected by antibodies.
• It confirms presence of specific proteins and is used after screening tests like ELISA.
• Immunoprecipitation is done when antibody is attached on beads to pull out a specific protein from a mixture.
• Co-immunoprecipitation is used to study protein–protein interaction by isolating whole protein complexes.
• Immunoelectrophoresis combines electrophoresis and immunodiffusion to form precipitin arcs after separation.
• It is used to identify serum proteins and to detect abnormalities like monoclonal or polyclonal gammopathies.

Some of the important are–

• Western Blot – Proteins are separated by electrophoresis and then detected by antibodies.
• Immunoprecipitation – Specific proteins are isolated from mixtures using antibody bound to beads.

D. Cytometry and Single-Cell Techniques

These techniques study cells individually and identify surface markers.

• It is the group of advanced methods that study individual cells instead of the whole cell population.
• It is the process that detects cell size, markers, and functions at the single-cell level showing cell-to-cell differences.
• Flow cytometry is used where cells pass through a laser beam and light scatter and fluorescence are measured.
• It is used for immunophenotyping of blood cells and diagnosis of leukemia and lymphoma.
• FACS is the sorting form of flow cytometry where labeled cells are separated into different containers.
• Mass cytometry uses heavy metal tagged antibodies and detects many markers because there is no spectral overlap.
• It is suitable for deep immune profiling but cells are destroyed during the process.
• ELISpot detects cytokine secreting cells and each spot indicates one active cell.
• FluoroSpot detects more than one cytokine from the same cell using fluorescent labels.
• Single-cell RNA sequencing studies gene expression of each cell and identifies new cell types.
• TCR and BCR sequencing analyzes immune receptor genes to study clonality and immune diversity.

Some of the main are–

• Flow Cytometry – Cells pass through laser beam and fluorescence is measured. It is used for immunophenotyping.
• FACS (Fluorescence Activated Cell Sorting) – It sorts cells based on markers.
• Mass Cytometry (CyTOF) – Uses metal tagged antibodies and detects multiple markers.

E. Functional Immunological Assays

• It is the group of assays that measure the actual biological activity of immune cells instead of only measuring protein amount.
• It is the process that studies how cells secrete cytokines, proliferate or kill pathogens at functional level.
• ELISpot is used where cytokine secreting cells are captured on membrane and each colored spot shows one active cell.
• It is highly sensitive and detects rare antigen-specific T or B cells important in vaccine studies.
• FluoroSpot is the advanced form where fluorescent labels detect more than one cytokine from the same cell.
• It helps in identifying polyfunctional T cells.
• Flow-cytometry functional assays are used for intracellular cytokine staining, proliferation tracking, calcium flux and phagocytosis study.
• Neutralization assays measure how antibodies block viral infection.
• Cytotoxicity assays detect T-cell killing ability such as CD107a expression.

Examples of Functional Immunological Assays

1. Enzyme-Linked ImmunoSpot (ELISpot)

ELISpot is considered the sensitive assay that detects functional immune responses at the single-cell level. It is the process where cells such as PBMCs are placed on a membrane coated with a capture antibody like anti–IFN-γ. When antigen stimulation occurs, the activated cells secrete cytokines which bind immediately to the antibody present beneath the cell. Later detection steps develop a visible colored spot.

Each spot represents one actively secreting cell. This is the point that makes ELISpot different from ELISA because ELISA measures total cytokine amount in the solution but cannot tell how many cells produced it or whether a small number of cells produced high amount. ELISpot is highly sensitive and can detect rare antigen-specific T cells and B cells. Its sensitivity is reported to be several folds higher than the conventional ELISA method.

Some of the applications are–

• It is used in vaccine development to measure antigen-specific memory T and B cells.
• It is essential in immuno-oncology studies where functional activity of immune cells are monitored.
• It is used in infectious diseases like tuberculosis and COVID-19 to detect antigen-responsive cells.

2. FluoroSpot

FluoroSpot is the modified form of ELISpot where fluorescent labels are used instead of enzyme-based color reactions. It is the process suitable for detecting more than one analyte in the same assay.

One of the important features is that multiple cytokines secreted by the same cell can be detected and separated by their fluorescent signals. This identifies polyfunctional T cells which produce more than one cytokine like IFN-γ and IL-2. These cells indicate the quality and type of immune response and help in distinguishing patterns like Th1 and Th2.

F. Tissue and Spatial Immunological Techniques

• It is the group of techniques used to detect antigens directly inside preserved tissue sections.
• It is the process where tissue structure is kept intact so the exact location of proteins can be seen.
• Immunohistochemistry (IHC) uses enzyme-linked antibodies to produce a colored stain at antigen sites.
• It preserves tissue morphology and gives permanent slides used in tumor staging and diagnosis.
• Immunofluorescence (IF) uses fluorescent-labeled antibodies and requires fluorescence microscope.
• Direct IF uses labeled primary antibody while indirect IF uses labeled secondary antibody for stronger signal.
• It allows multi-color imaging and detects low-abundance proteins but signals fade with time.
• Tissue microarray (TMA) places many small tissues on one slide for high-throughput analysis.
• In vivo imaging uses antibody-linked contrast agents to visualize markers inside living organisms.
• Antigen mapping uses IF to detect exact level of skin splitting in hereditary blistering diseases.

Examples of Tissue and Spatial Immunological Techniques

1. Immunohistochemistry (IHC)

Immunohistochemistry is the technique used for visualizing proteins in fixed tissue sections by producing a colored reaction at the antigen site. It is carried out with antibodies that are linked to enzymes such as horseradish peroxidase or alkaline phosphatase. When the substrate (chromogen) is added, the reaction forms an insoluble colored precipitate at the place where the antigen–antibody binding occurs. The stained section is viewed under a brightfield microscope.

It is important because the tissue morphology is preserved, so the distribution of the protein can be seen in relation to cells and structures. The stained sections also remain permanent which makes the slides useful for long-term record and re-examination later. It is also comparatively cost-effective in diagnostic laboratories.

Some of the applications are–

• It is required in tumor staging and for predicting treatment response.
• In breast cancer, it is used to detect hormone receptor status (estrogen and progesterone) and HER2 expression.
• In lymphomas, it helps in differentiating infection from cancer and in identifying the particular lymphoma subtype.
• It is also used in prostate cancer diagnosis where routine tests sometimes do not give clarity.

2. Immunofluorescence (IF)

Immunofluorescence is the method where fluorophores are used instead of enzymes. These fluorophores emit light when excited with a specific wavelength, so a fluorescence microscope is required to visualize the reaction.

There are two common methods. In Direct IF, a primary antibody that is already labeled with a fluorophore binds directly to the antigen. In Indirect IF, an unlabeled primary antibody binds first and then a fluorophore-linked secondary antibody binds to it. It is the process that gives amplification of signal and higher sensitivity.

Some of the important features are–

• It supports multi-color imaging, so multiple proteins can be detected on the same tissue.
• It is more sensitive in detecting low-abundance proteins compared to brightfield IHC.
• The main limitation is that fluorescence fades over time (photobleaching), so the slides are not permanent.

Applications include–

• In dermatology, Direct IF is the standard method to diagnose autoimmune blistering disorders like Pemphigus and Bullous Pemphigoid by showing IgG, IgA, or C3 deposits.
• In renal pathology, it identifies immunoglobulin and complement deposition in kidney biopsies.

3. Advanced and High-Throughput Spatial Methods

Several modern techniques are available to examine spatial arrangement and molecular patterns in tissues.

• Tissue Microarrays (TMA)– TMA is the technique where hundreds of small tissue cores from many donors are arranged together on a single slide. It allows the analysis of molecular markers in a large group of samples at the same time under identical laboratory conditions. It reduces experiment-to-experiment variation and saves reagents.
• In Vivo Imaging– This process uses contrast agents that combine antibodies with nanoparticles like superparamagnetic iron oxide. These agents help in tracking biological markers inside a living organism using MRI. It is mainly applied for imaging inflammation or other immune-related activity inside tissues without removing the sample.
• Antigen Mapping– Antigen mapping is a modified IF technique used in dermatology. It detects the level of skin splitting in hereditary epidermolysis bullosa (EB). Antibodies against specific basement membrane proteins are used to locate the exact layer where the separation occurs.

G. Molecular Immunological Techniques

These are advanced techniques to study immune receptor genes and cell diversity.

• It is the group of advanced methods that study immune system at genetic and transcript level.
• It is the process where gene expression, clonal diversity and molecular changes of immune cells are analyzed.
• qPCR is used to measure mRNA levels of immune-related genes and study transcriptional response.
• It detects expression of markers like interferon-gamma, TNF or TCR related genes but mRNA level may not always match protein activity.
• High-throughput sequencing (NGS) is used for TCR and BCR sequencing by analyzing the CDR3 region.
• It can be done using genomic DNA or mRNA to study clonal expansion and immune repertoire diversity.
• It is used in autoimmune diseases, vaccine studies and tracking immune clones during therapy.
• Single-cell RNA sequencing measures transcriptome of each individual cell to identify rare subsets.
• It shows cell heterogeneity and allows reconstruction of immune cell development pathways.
• Molecular diagnostics in transplantation uses expression profiling of HLA genes and gene-editing approaches to reduce rejection.

Examples of Molecular Immunological Techniques

1. Quantitative Polymerase Chain Reaction (qPCR)

qPCR is used to detect and quantify expression levels of immune-related genes. It is the process where fluorescence signals increase according to the amplified DNA and this increase is monitored during the reaction. This is referred to as real-time detection of nucleic acid amplification. It is mainly applied to measure mRNA transcripts of signaling pathways like T-cell receptor (TCR), interferon-gamma, and tumor necrosis factor.

Some of the main features are–

• It characterizes transcriptional response of immune cells during diseases or in experimental treatment.
• It is important to note that mRNA concentration is not always equal to the final protein quantity.

2. High-Throughput Sequencing (HTS) of Immune Repertoires

It is also known as Next-Generation Sequencing (NGS). It is the technique that sequences immune receptors deeply, especially TCR and BCR. The receptors contain Complementarity Determining Region 3 (CDR3) which is formed after V(D)J recombination, and this region acts as a unique tag for each clone lineage.

In this method sequencing is done either from genomic DNA or from mRNA. gDNA is generally used to count templates accurately for clonal expansion and tissue density, but mRNA may vary in expression levels in each cell, so the interpretation is sometimes affected.

Some of the important applications are–

• Tracking specific clonal expansion or contraction in immune responses.
• Identification of dominant clonotypes in diseases like severe aplastic anemia and autoimmune disorders like Multiple Sclerosis.
• It also helps in vaccine development by explaining receptor diversity and antibody formation.

3. Single-Cell RNA Sequencing (scRNA-seq)

scRNA-seq is the technique where transcriptome of individual cells is measured. It is the process that captures single cells (microdroplets or microwells), converts mRNA into cDNA, amplifies it and sequencing is then performed.

These are used to understand heterogeneity among immune cells because classification is based on transcriptome rather than surface markers. Rare cell subsets are also detected. It is also used to reconstruct developmental and differentiation pathways, forming a type of molecular atlas for immune cells.

Some advanced uses integrate RNA sequencing with TCR tracking, such as in STARTRAC, where functional state like exhaustion is connected with clonal identity.

4. Molecular Diagnostics in Transplantation

In transplantation research, molecular techniques are used to study immunocompatibility. RNA-based NGS is used to profile allele-specific HLA expression for selection of suitable donors.

Gene-editing tools like CRISPR/Cas9 are used to modify donor organs, such as silencing immunogenic HLA molecules. It is the process that helps in reducing rejection risk and improving graft acceptance.