Unit 3: Antigen-antibody interactions Theory: Precipitation, agglutination and complement

mediated immune reactions; advanced immunological techniques: RIA, ELISA, Western

blotting, ELISPOT assay, immunofluorescence microscopy, flow cytometry and
immunoelectron microscopy; surface plasmon resonance, biosensor assays for assessing
ligand–receptor interaction; CMI techniques: lymphoproliferation assay, mixed lymphocyte
reaction, cell cytotoxicity assays, apoptosis, microarrays, transgenic mice, gene knockouts
Precipitation, agglutination and complement mediated immune reactions

Precipitation

Precipitation reaction can define as the immunological reaction where visible precipitate forms
as a result of cross-linkage between antigen and antibody. The resulted compound in
precipitation reaction refers as “Precipitin” which is a precipitate formed by the antibody on
reaction with the antigen.

Agglutination

Agglutination reactions are surface reactions, and thus the surface of the antigens must be
exposed for the antibody to bind and form visible clumps. The concentration of
antigen and antibody should be equal. Any change in this equivalence prevents the formation of
precipitins. Agglutination reactions might require minutes to hours for completion.
Complement mediated immune reactions

Complement, in
immunology, a complex
system of more than 30
proteins that
act in concert to help
eliminate infectious
microorganisms.
Specifically, the
complement system
causes the lysis (bursting)
of foreign and infected
cells, the phagocytosis
(ingestion) of foreign
particles and cell debris,
and the inflammation of
surrounding tissue .
Advanced immunological techniques:

RIA
ELISA
Western blotting
 ELISPOT assay
Schematic illustration of the
principle of the ELISpot
assay. The enzyme-linked
immunospot.
(ELISpot) assay is a highly
sensitive immunoassay that
measures the frequency of
cytokine-secreting cells at
the single-cell level.
Immunofluorescence microscopy
Applications

Scientists can use the immunofluorescence technique to perform a variety of lab tests and
observations, each of which provides a fresh perspective for the sample currently undergoing
analysis.
For instance, a researcher may opt to use this testing method to examine tissue samples and
beads, detect particular proteins using microarrays, or they might choose to evaluate cultured
cells, as well as those in suspension.
An observer can apply this technique to samples that are fixed or fresh, thus providing more
opportunity for diverse analysis and accurate results.
A researcher may also choose to apply this technique when studying DNA sequences
on chromosomes, as these particles are extremely small and hard to detect. Obtaining spatial
data concerning tissue or cell genetics is another way to use immunofluorescence, as is the
observation of parasites and bacteria.
The deeper layers of a cell or tissue, along with the antigens that infect these layers, is another
area that scientists can study using this staining method.
Flow cytometry

Specifically, flow cytometry is used in research for a number of purposes, including:

Cell counting.
Cell sorting.
Determining cell function.
Determining cell characteristics.
Detecting microorganisms, such as bacteria, fungus or yeast.
Finding biomarkers (characteristics that indicate normal function).
Diagnosis and potential treatment of blood and bone marrow cancers.
Immunoelectron microscopy

Immune electron microscopy , sometimes called immunoelectron microscopy is a method

used in electron microscopy for diagnosis of viral infections.
The technique was first described in the 1940s using tobacco mosaic virus.

This technique allows the investigator to identify antibody/antigen complexes that localize to a
particular subcellular organelle or compartment by using a preembedding streptavidin-biotin
technique with diaminobenzidine (DAB) as a chromagen which is silver enhanced. The tissue is
post-fixed in osmium tetroxide, dehydrated and embedded in EPON/Araldite resin.
Paraffin embedded tissue (same tissue) sections are cut and screened by light microscopy using
a streptavidin-biotin technique to determine the appropriate antibody concentration to be used
for the EM immunogold procedure. Ultrathin sections from the same block(s) are cut, incubated
with primary antibody and then later incubated with Protein A gold particles (size range is 5 nm
to 20 nm). The gold particles bind to the Fc portion of the antibody and are detected by EM. A
variation of the large block "pop-off" technique for immunoelectron microscopy is also
available.
Protein A gold labeling of a prostatic endocrine-paracrine cell demonstrating
localization of calcitonin (inset) to the neuroscretory granules.
Surface plasmon resonance
•Surface Plasmon Resonance (SPR) is an optical technique used to measure molecular
interactions in real time. SPR can occur when plane-polarized light hits a metal film under total
internal reflection conditions.
•SPR signal is directly dependent on the refractive index of the medium on the sensor chip. The
binding of biomolecules results in changes in the refractive index on the sensor surface.
•In an SPR experiment, one molecule (the Ligand) is immobilized on a sensor chip and binding to
a second molecule (the Analyte) is measured under flow.
•Response is measured in resonance units (RU) and is proportional to the mass on the surface,
and for any given interactant, the response is proportional to the number of molecules bound to
the surface.
•Response is recorded and displayed as a sensogram in real time. SPR experiments can be used
to measure kinetic binding constants (ka, kd) and equilibrium binding constants (affinity, Ka =
1/Kd).
Biosensor assays for assessing ligand–receptor interaction

Fluorescent labeled ligand binding assay is applied to detect its binding to a target.
These assays have a broad spectrum of wavelengths, therefore it can apply multiple colors.
CMI techniques: Where we take cell mediated immunity in concern.

Lympho proliferation assay

Lymphocyte proliferation assay (LPA) measures the ability of lymphocytes placed in short-term
tissue culture to undergo a clonal proliferation when stimulated in vitro by a foreign molecule,
antigen or mitogen.
CD4+ lymphocytes proliferate in response to antigenic peptides in association with class II major
histocompatibility complex (MHC) molecules on antigen-presenting cells (APCs).
This proliferative response of lymphocytes to antigen in vitro occurs only if the patient has been
immunized to that antigen, either by having recovered from an infection with the
microorganism containing that antigen, or by having been vaccinated.
Therefore, some normal individuals may not respond to a given antigen, but most people will
respond to at least one of several common microbial antigens.
The MTT assay is a colorimetric assay for assessing cell metabolic activity. NAD(P)H-dependent
cellular oxidoreductase enzymes may, under defined conditions, reflect the number of viable
cells present.
These enzymes are capable of reducing the tetrazolium dye MTT, which is chemically 3-(4,5-
dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide, to its insoluble formazan, which has a
purple color.
Other closely related tetrazolium dyes including XTT, MTS and the WSTs, are used in conjunction
with the intermediate electron acceptor, 1-methoxy phenazine methosulfate (PMS). With WST-
1, which is cell-impermeable, reduction occurs outside the cell via plasma membrane electron
transport.
However, this traditionally assumed explanation is currently contended as proof has also been
found of MTT reduction to formazan in lipidic cellular structures without apparent involvement
of oxidoreductases.
Mixed lymphocyte reaction
It’s an ex-vivo cellular immune assay that occurs between two
allogeneic lymphocyte populations (same species but genetically distinct).
MLR’s are performed to assess how T-cells react to external stimuli. T cells are a type of white
blood cell that scans for cellular abnormalities and infections. They are essential to human
immunity

Cytotoxicity assays

Cell cytotoxicity and proliferation assays are generally used for drug screening to detect
whether the test molecules have effects on cell proliferation or display direct cytotoxic effects.
Regardless of the type of cell-based assay being used, it is important to know how many viable
cells are remaining at the end of the experiment.
Apoptosis
Transgenic mice
Gene knockouts
A process of suppressing gene function by gene manipulation is called gene knockout.
ES: Embryonic Stem Cells.
Unit 5: Complement genes of the human major histocompatibility complex: implication for
linkage disequilibrium and disease associations, genetic studies of rheumatoid arthritis, systemic
lupus erythematosus and multiple sclerosis, immunogenetics of spontaneous control of HIV, KIR
complex.