Immuno chemical techniques are protein assay techniques of high sensitivity & specificity and utilize the high specific affinity of an antibody for its antigen.
It is used to identify, localize, purify, and quantify the protein in a given medium, tissue or cell
Immuno assays are based on the principle of labling antigens and antibodies Immunolabeling is a biochemical process that enables the detection and localization of an antigen/antibody/protein to a particular site within a cell, tissue, or organ by tagging with a fluorescent compound, gold beads or an enzyme
ANTIGEN,ANTIBODY AND ANALYTE
An immunoassay is a test that uses antibody and antigen complexes (immune complex)
• An antibody is a glycoprotein (immunoglobulin) produced by the immune system- B- lymphocytes in response to an “invading” (foreign) substance or antigen
• An antigen is a substance when enters in to the body elicits immune response (production of anti bodies and formation of immune complex)
• Analyte is - a chemical substance that is the subject of analysis
Based on the formation of immune complex these test can detect the presence / absence of antigen qualitatively and quantitatively.
Types of Immuno Assays :
1. ELISA - ENZYME LINKED IMMUNO SORBENT ASSAY
2. RIA -RADIO IMMNUNO ASSAY
3. IFT- IMMUNO FLUORESENCE TECHNIQUE These are Sensitive, specific, simple and speed to carry out, analyses - multiple samples and easy for standardization.
ENZYME LINKED IMMUNO SORBENT ASSAY
➢ It is a plate based assay designed for detecting and quantifying antigens and antibodies in a solution
➢ An enzyme linked with an antibody (binds to antigen-immuno) adsorbed to poly styrene surface (sorbent) react with colourless substrate (chromogenic ) to generate colourful product.
➢ ELISA is used as a diagnostic tool in medicine, plantpathology,biotechnology and as a quality control check in various industries.
This technique is carried out in four different methods
1. DIRECT ELISA
2. INDIRECT ELISA
3. SANDWICH ELISA
4. COMPETITIVE ELISA
• In Direct ELSA Primary antibody is labelled with enzyme
• In indirect ELISA Secondary antibody is labelled
ENZYMES EMPLOYED FOR ELISA
➢ 1. ALKALINE PHOSPHATASE
SUBSTRATE; PNPP (p-Nitrophenyl Phosphate Disodium Salt) to p-Nitrophenol (405 nm (yellow colour)
➢ 2. HORSE RADISH PEROXIDASE
SUBSTRATE: OPD (o-phenylenediamine dihydrochloride)
TMB (3,3',5,5'-tetramethylbenzidine)
➢ 3. BETA GALACTOSIDASE
SUBSTRATE: ONPG (o-nitrophenyl-β-D- galactopyranoside
ELISA is carried out on Polystyrene Microtiter Plate
DIRECT ELISA
Step 1: A buffered solution of antigen is added to each well of a microtiter plate which is coated with polystyrene to adhere to the plastic through charge interactions and washed to remove the unbounded protein(antigen)
Step 2: A solution of non reacting protein, such as bovine serum albumin or casein is added to each well to cover any plastic surface in the well which remains uncoated by the antigen.
Step 3: The primary antibody with an attached (conjugated) enzyme is added, which binds specifically to the test antigen coating the well.
Step 4: The wells are washed for unbounded enzyme
Step 5: A chromogenic substrate for this enzyme is then added..
Step 6: colour change is quantitatively measured by spectrometer (Elisa Reader)
Advantages: Simple, easy, Less time is required
Disadvantages: less sensitive, no signal amplification
INDIRECT ELISA
1. Micro-well plates are incubated with antigens, washed up and blocked with BSA.
2. Samples with antibodies are added and washed
3. Enzyme linked secondary antibodies are added and washed 4. Chromogenic substrate is added. Colour change is measured by ELISA Reader.
SANDWICH ELISA Sandwich of Ab-Ag-Ab/ enzyme bound to microwellSandwich ELISA is named so as antigen is sandwiched between two antibodies. The sandwich assay uses two different antibodies that are reactive with different epitopes on the same antigen
• It has high specificity since two antibodies are used and antigen/analyte is specifically captured and detected.
• It is suitable for complex samples as antigen does not require purification prior to measurement.
• Has good flexibility and sensitivity, since both direct and indirect detection methods can be used.
COMPETITIVE ELISA
• Used to determine small molecules like T₃ , T₄ & Progesterone
• Steps: 1.Unlabeled antibody is incubated in the presence of its antigen (sample).
• These bound antibody/antigen complexes are then added to an antigen-coated well. • The plate is washed, so unbound antibodies are removed. (The more antigen in the sample, the more Ag-Ab complexes are formed )
• there are less unbound antibodies available to bind to the antigen in the well, hence "competition".
• The secondary antibody, specific to the primary antibody, is added. This second antibody is coupled to the enzyme.
• A substrate is added, and remaining enzymes elicit a chromogenic or fluorescent signal.
• The reaction is stopped to prevent eventual saturation of the signal.
Applications of ELISA: ELISA is used for the
Diagnostics in Medical laboratories
Detection of dengue, malaria diseases Detection of Mycobacterium antibodies in tuberculosis
Detection of rotavirus in feces
Detection of hepatitis B markers in serum
Detection of hepatitis C markers in serum
Detection of enterotoxin of E. coli in feces
Detection of HIV antibodies in blood samples and many more detections.
It has also found applications in the food industry in detecting potential food allergens such as milk, peanuts, walnuts, almonds and eggs .
Used in serological blood test for coeliac disease
ELISA can also be used in toxicology as a rapid presumptive screen for certain classes of drugs.
ELISA FOR DETECTING HIV
• It is a useful tool for determining serum antibody concentrations of HIV virus
• ELISA was the first screening test widely used for HIV because of its high sensitivity.
• In an ELISA, a person's serum is diluted 400 times and applied to a plate to which HIV antigens are attached.
• If antibodies to HIV are present in the serum, they may bind to these HIV antigens..
• ELISA results are reported as a number
• The most controversial aspect of this test is determining the "cut-off" point between a positive and a negative result.
• A cut-off point may be determined by comparing it with a known standar
RADIO IMMUNO ASSAY
Radioactivity : It Is the property of emitting energy and subatomic particles by radio isotope molecules to get stability
FIRST INVITRO ASSAY TO DETECT HORMONE
• Developed in 1959 by Rosalyn Yalow and Solomon Berson for measurement of insulin in plasma.
• In 1977 Yalow received the Nobel Prize for her and Berson, for development of RIA
PRINCIPLE OF RIA • The competitive binding of radiolabelled antigen and unlabelled antigen to a high affinity antibody.
• The labelled antigen is mixed with the antibody at a concentration that saturates the antigen – binding sites of the antibody.
• With the increase in the concentration of the unlabelled antigen more labelled antigen will be replaced from the binding sites of antibody.
• The decrease in the amount of radiolabelled antigen bound specific antibody in the presence of the test samples is measured to determine the amount of antigen Present in the test sample.
REAGENTS USED IN RIA
1.A tracer i.e. a labelled ligand
Radioisotopes used in RIA technique:
Ex: Beta emitters-tritium and C-14
Gamma emitters I- 125
2. A binder (Antibody) which is the specific antiserum.
3. A separation system to separate the ‘bound’ and ‘free’ phases.
4. A standard curve
PROCEDURE FOR RIA
PROCEDURE FOR RIA Radioactive antigen
Radiolabeled antigen + known amount of antibody
Sample /Serum from a patient containing an unknown quantity of that same antigen is added
Competition between unlabelled antigen from the serum with the radiolabeled antigen for antibody binding sites As the concentration of unlabelled antigen increase, more of it binds to the antibody.
• The bound antigens are then separated from the unbound ones the radioactivity of the free antigen remaining in the supernatant is measured
Advantages Of RIA
• Radio immuno assay is very sensitive technique
• Used to measure pico gram concentrations of antigen
• It is structurally specific as antigen: antibody reaction are highly specific.
• It is indirect method of analysis.
As It is a saturation analysis active reagent is added in smaller quantity than that of analyte
Disadvatages of RIA
➢ Prolonged reaction time (in days) as a consequence highly diluted reagent is used.
➢ Radioactive Iodine is not a cheap reagent.
➢ Possible health hazards due to handling of radioisotopes.
➢ Limited assay range.
➢ Lack of direct linear relationship between analyte concentration and signal response. ➢ Difficulty of automation.
➢ Lengthy counting time
Applications of RIA
• Used for drug detection
• Blood screening for the hepatitis virus
• Early cancer detection
• Measurement of growth hormone levels
• Diagnosis for leukemia virus
• Diagnosis and treatment of peptic ulcers
• Research with neurotransmitters
IMMUNOFLUORESENCE TECHNIQUE
Immunofluorescence :
Immunofluorescence is a technique allowing the visualization of a specific protein or antigen in tissue sections by binding a specific antibody chemically conjugated with Fluorochromes
• In 1944 Albert Coons was first person to apply the IF technique to demonstrate the microorganism in the infected tissue
Fluorochromes
Fluorochromes are compounds which when irradiated with a light of a shorter wavelength achieve an unstable higher energetic state. On returning to the ground state as a spontaneous process, they emit light of a longer wavelength which can be observed by fluorescent microscope
Fluorophores
Fluorescein asorbs blue light(490 nm) and emits yellow green (517nm) fluorescence
Rhodamine absorbs yellow green(515nm) and emits deep red colour (546nm) fluorescence
Phycoerythrin-brillint emitter of red fluorescence
Types of immunofluorescence:----
There are two classes of immunofluorescence
1.Direct immunofluorescence
A single antibody is used to target antigen of interest. The primary antibody is directly coupled to a fluorophore.
2.Indirect immunofluorescence
Two antibodies are used. Primary antibody in this case is unconjugated , the second fluorophore- conjugated antibody is used for acting against the primary antibody DIRECT AND INDIRECT IMMUNOFLUORESCENCE
DIRECT IMMUNOFLUORESCENCE
• Direct attachment of the antibody reduces the number of steps in the procedure
• Saves time
Direct immunofluorescence also requires the use of much more primary antibody ,which is extremely expensive
INDIRECT IMMUNOFLUORESCENCE
This provides signal amplification by increasing the number of fluorophore molecules per antigen. Increases the sensitivity.
• Immunofluorescence can be used on tissue sections, cultured cell lines or individual cells, and may be used to analyze the distribution of proteins glycans and small biological and non- biological molecules.
• Applied to identify subpopulations of lymphocytes.
• For detecting of bacterial species, detecting antigen and antibody complexes in immune diseases Flow Cytometry & Fluorescence
Flow cytometry (FCM) is a technique used to detect and measure physical and chemical characteristics of a population of cells or particles. In this process, a sample containing cells or particles is suspended in a fluid and injected into the flow cytometer instrument.
• A laser beam of light is used to detect single intact cells in suspension.
• Flourescently tagged antibody bound to cells are excited by laser and emit light that is recorded by detectors.
• Cell sorting based on type of the cells is also carried.