elisa seminar final2
DESCRIPTION
Slides on ELISA - Definition, Procedure etc.Immunology.Heart of various diagnostic proceduresTRANSCRIPT
…By Dr. Deshkar DW
1798 - First demonstration of vaccination smallpox vaccination (Edward Jenner)
1890 - Demonstration of antibody activity against diphtheria and tetanus toxins.
1890 - Beginning of humoral theory of immunity. (Emil von Behring & Shibasaburo Kitasato)
1900 - Antibody formation theory (Paul Ehrlich)
1938 - Antigen-Antibody binding hypothesis (John Marrack)
1948 - antibody production in plasma B cells
1959 -1962 - Discovery of antibody structure
Enzyme Linked ImmunoSorbent Assay{ELISA}
History
• 1971 - Peter Perlmann and Eva Engvall at Stockholm University invented ELISA
• 1975 - Generation of the first monoclonal antibodies (George Kohler and Cesar Milstein)
• Prior to the development of the EIA/ELISA, the only option for conducting an immunoassay was radioimmunoassay, a technique using radioactively-labeled antigens or antibodies.
• Radioimmunoassay was first described in a paper by Rosalyn Sussman Yalow and Solomon Berson published in 1960.
Enzyme Linked ImmunoSorbent Assay{ELISA}
History
• ELISA – an immunological test, using an enzyme as a label to determine presence of target protein.
• The enzyme linkage or labeling allows you to follow your target protein and if present (qualify) and at what amounts (quantify).
• An enzyme conjugate is an enzyme bound or joined with an antibody which binds with your target protein.
• This enzyme labeling is a safe and effective way to track your antibody.
Enzyme Linked ImmunoSorbent Assay{ELISA}
Components
Antigen
• The antigen is your target protein which comes from your sample extract.
• The antigen binds to the antibody.
• Any substance that stimulates an immune response.
Antibody
• An antibody is a protein made in response to an antigen.
• Each antibody binds only to its antigen.
Enzyme Conjugate
• An enzyme conjugate (EC) is an antibody joined with an enzyme.
• Enzyme labeling allows the researcher to follow the antibody.
• This joining of the enzyme to antibody is often called conjugation.
• Horse radish peroxidase (HRP) and Alkaline phosphatase (ALP) are the two most widely used enzymes employed in ELISA assay.
Components
Enzyme Function
• Enzymes are proteins that speed up the rate of a chemical reaction without being used up and usually react only to particular substrates.
• The rate of this reaction is proportional to the amount of enzyme present. In the case of non-competitive ELISA, the more binding you have of the enzyme conjugate to the antigen, the stronger your color development will be.
Components
Substrate
• ALP substrate - For most applications pNPP (p-Nitrophenyl-phosphate) is the
most widely used substrate.
• HRP substrates– Hydrogen peroxide. TMB (3,3’,5,5’-tetramethylbenzidine) OPD (o-phenylenediamine dihydrochloride)
ABTS (2,2’-azino-di-[3-ethyl-benzothiazoline-6 sulfonic acid] diammonium salt
Substrate Function• A substrate is a compound or substance that undergoes change. Substrates
bind to active sites on the surface of enzymes and are converted or changed. In ELISA the specific substrate used changes color.
Components
Four major advantages of ELISA
Data analysedstatistically
ELISA Enzyme Linked Immunosorbent Assay
Why ELISA?
Versatile Simple Sensitive Quantifiable
Many systems using different
combinations of reagents are
possible
Uses micro-titre plates and
allied equipment
Enzyme catalyst
amplification
Colour
Passive attachment to solid phase
High Capacity Ideal range
of sensitivity
for diagnosis
Read by eye
Easy separation of
bound and unbound
reactants by washing step
Rapid
Cheap
Kits feasible
Multi-channelspectrophotometers
Data can be stored
ELISA : Pros and Cons
Pros: 1.No radioactivity needed : safe and inexpensive
2.Less expensive equipment
3.Huge capacity through standard 96-microwell pattern: miniaturization and automation.
4.Equal or even better sensitivity than RIA
5.Versatility : same principle allows the use of different labels, incl. enzymes (colored end product or light) and fluorochromes.
Cons: Somewhat more difficult to develop, control and standardize than RIA.
General Procedure Of ELISA
Coating with Capture antibody
1. Coat the wells of a PVC micro-titer plate with the capture antibody2. Cover the plate with an adhesive plastic and incubate overnight at
4°C.3. Remove the coating solution and wash the plate twice by filling the
wells with 200 μl PBS.
Adding Samples4. Add 100 μl of appropriately diluted samples to each well, & Incubate
for 90 min at 37°C.5. Remove the samples and wash the plate twice by filling the wells with
200 μl PBS.
Incubation with Detection antibody6. Add 100 μl of detection antibody conjugated, diluted at the optimal
concentration in blocking buffer immediately before use.7. Cover the plate with an adhesive plastic and incubate for 1-2 h at
room temperature.8. Wash the plate four times with PBS.
Detection9. Dispense 100 μl (or 50 μl) of the substrate solution per well with a multi-
channel pipette or a multi-pipette.
A Microtiter plate (96-well) used for ELISA
General Procedure Of ELISA
Reading plates
•Select proper wave-length on machine.
• Carefully wipe bottom of plate to removeexcess moisture. Plates that are wet maydiffuse your light source, giving inaccuratereadings.
•Mix the plate. This will distribute colorevenly. Some machines have a ‘mix’ setting.
General Procedure Of ELISA
There is no need for phase separation. It is less sensitive than heterogenous ELISA.
Uses:
It is suitable for determination of lowmolecular weight substances. e.g drug monitoring.
PRINCIPAL:
Enzyme labeled Ag & unknown Ag compete together for Ab-binding sites .The enzyme labeled Ag that remain free is enzymatically active & can be determined in the presence of the bound form.
HOMOGENOUS ELISA
HETEROGENOUS ELISA
PRINCIPAL:
• An essential component of these assay is the separation of the bound enzyme labeled component from free labeled one after the immunological incubation.
• The assay can be :a) Competitive.b) Non Competitive
TYPES OF ELISA
• There are three main methods that form the basis to all ELISAs. 1. DIRECT ELISA 2. INDIRECT ELISA 3. SANDWICH ELISA
All these systems (1-3) can be used in assays called:4. COMPETITION OR INHIBITION ELISA
DIRECT ELISA
The direct ELISA uses the method of directly labeling the
antibody itself. Microwell plates are coated with a sample
containing the target antigen, and the binding of labeled
antibody is quantitated by a colorimetric,
chemiluminescent, or fluorescent end-point.
DIRECT ELISA
DIRECT ELISA
DIRECT ELISA
Advantages of Direct Detection
• Quick methodology since only one antibody is used. • Cross-reactivity of secondary antibody is
eliminated.
Disadvantages of Direct Detection
• Immunoreactivity of the primary antibody may be reduced as a result of labeling.
• Labeling of every primary antibody is time-consuming and expensive.
• No flexibility in choice of primary antibody label from one experiment to another.
• Little signal amplification.
DIRECT ELISA
The indirect ELISA utilizes an unlabeled primary antibody in conjunction with a labeled secondary antibody.
Since the labeled secondary antibody is directed against
all antibodies of a given species (e.g.anti-mouse), it can be
used with a wide variety of primary antibodies (e.g. all
mouse monoclonal antibodies).
INDIRECT ELISA
INDIRECT ELISA
INDIRECT ELISA
Advantages of indirect detection
• Wide variety of labeled secondary antibodies are available commercially.
• Versatile, since many primary antibodies can be made in one species and the same labeled secondary antibody can be used for detection.
• Immunoreactivity of the primary antibody is not affected by labeling.
• Sensitivity is increased because each primary antibody contains several epitopes that can be bound by the labeled secondary antibody, allowing for signal amplification.
INDIRECT ELISA
Disadvantages of indirect detection
• Cross-reactivity may occur with the secondary antibody, resulting in nonspecific signal.
• An extra incubation step is required in the procedure.
INDIRECT ELISA
SANDWICH ELISA
1. Plate is coated with a capture antibody
2. Sample is added, and any antigen present binds to capture antibody
3. Detecting antibody is added, and binds to antigen
4. Enzyme-linked secondary antibody is added, and binds to detecting antibody
5. Substrate is added, and is converted by enzyme to detectable form.
SANDWICH ELISA
SANDWICH ELISA TYPES
– Sandwich Direct – Sandwich Indirect
Sandwich Direct
A. The detecting antibody is labelled (conjugate)
B. The capture and detecting antibodies can be from the same sample
C. The detecting antibody can be from different species
Sandwich Indirect
A. The detecting antibody is NOT labelled
B. The detecting antibody is not prepared in same species as capture antibody
C. The detecting antibody is detected using an anti-species conjugate
SANDWICH DIRECT
SANDWICH DIRECT
SANDWICH INDIRECT
SANDWICH INDIRECT
COMPETITIVE ELISA
In this Unlabeled antibody is incubated in the presence of its antigen.
• These bound antibody/antigen complexes are then added to an antigen coated well.
• The plate is washed unbound antibody is removed.
• 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.
• For competitive ELISA, the higher the original antigen concentration, the weaker the eventual signal.
COMPETITIVE ELISA
ELISA Reverse method & device (ELISA-R m&d)
A newer technique uses an solid phase made up of an
immunosorbent polystyrene rod with 4-12 protruding
ogives. The entire device is immersed in a test tube
containing the collected sample and the following steps (washing, incubation in conjugate and incubation in
chromogenous) are carried out by dipping the ogives in
microwells of standard microplates pre-filled with reagents.
ADVANTAGES
• The ogives can each be sensitized to a different reagent, allowing the simultaneous detection of different antibodies and different antigens for multi-target assays.
• The sample volume can be increased to improve the test sensitivity in clinical (saliva, urine), food (bulk milk, pooled eggs) and environmental (water) samples.
• One ogive is left unsensitized to measure the non-specific reactions of the sample.
• The use of laboratory supplies for dispensing sample aliquots, washing solution and reagents in microwells is not required, facilitating ready-to-use lab-kits and on-site kits.
PRECAUTIONS
Negative control with strong signal
The excessive background signal can be caused by
inadequate rinsing of plates, reagents not sufficiently
diluted, inadequate blocking of plates or non-specific
binding of enzyme conjugate. The appearance of color in
negative control wells may also indicate cross-reactivity of
secondary antibody with components in the antigen
sample.
PRECAUTIONS contd…..
• Negative control with strong signal
The excessive background signal can be caused by
inadequate rinsing of plates, reagents not sufficiently
diluted, inadequate blocking of plates or non-specific
binding of enzyme conjugate.
The appearance of color in negative control wells may also
indicate cross-reactivity of secondary antibody with
components in the antigen sample.
ELISA with weak signal
• Wash buffer not adequately drained after every wash step.
• Inadequate incubation times.
• Detection reagents too dilute. Perform checkerboard titrations.
• Enzyme conjugate defective or inhibited by contaminant.
• Substrate defective or contaminated.
• Microwell plates poorly coated.
• Loss of capture antibody during blocking/washing. Decrease or eliminate use of Tween-20.
PRECAUTIONS contd…..
APPLICATIONS
• Screening donated blood for evidence of viral contamination by – HIV-1 and HIV-2 (presence of anti-HIV
antibodies) – hepatitis C (presence of antibodies) – hepatitis B (testing for both antibodies and
a viral antigen)
• Measuring hormone levels – HCG (as a test for pregnancy) – LH (determining the time of ovulation) – TSH, T3 and T4 (for thyroid function)
APPLICATIONS contd….
• Detecting infections – sexually-transmitted agents like HIV, syphilis
and chlamydia – hepatitis B and C – Toxoplasma gondii
• Detecting allergens in food and house dust • Measuring "rheumatoid factors" and other
autoantibody in autoimmune diseases like lupus erythematosus
• Measuring toxins in contaminated food• • Detecting illicit drugs, e.g.,
– cocaine – opiates