ELISA : ELISA HISTORY : HISTORY 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. Continued : Continued Certain enzymes (such as peroxidase) react with appropriate substrates (such as 3,3’,5,5’-Tetramethylbenzidine), they can result in changes in color, which can be used as a signal.
This signal has to be associated with the presence of antibody or antigen
This linking process was independently developed by Stratis Avrameas and G.B. Pierce INTRODUCTION TO ELISA : INTRODUCTION TO ELISA ELISA, or enzyme-linked immunosorbent assay, is an immunoassay technique involving the reaction of antigen and antibody in vitro. ELISA is a sensitive and specific assay for the detection and quantitation of antigens or antibodies. ELISA tests are usually performed in microwell plates.
The ELISA test, or the enzyme immunoassay (EIA), was the first screening test commonly employed for HIV. It has a high sensitivity. Slide 5: A 96-WELL MICROTITER PLATE USED FOR ELISA COMPONENTS OF AN ELISA : COMPONENTS OF AN ELISA Antibody: IgG fraction of serum purified by affinity chromatography
Enzyme: Horse Radish Peroxidase (HRP) MW 44, 000, glycoprotein with 4 lysine residues
Substrate: TMB (3,3',5,5', tetramethylbenzidine) The enzyme acts as a catalyst to oxidize substrate in the presence of Hydrogen peroxide to produce a blue color. Reaction stopped with dilute acid to cause complex to turn yellow. PRINCIPLE OF ELISA : PRINCIPLE OF ELISA Antibody is immobilized on micro-plate wells
Competition between in sample and labeled enzyme for antibody binding sites
The unbound material is washed out
Chromogenic substrate added to develop color
Resulting color is read in a spectrophotometer Slide 8: Principle of ELISA TYPES OF ELISA : TYPES OF ELISA DIRECT ELISA
COMPETETIVE ELISA DIRECT 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 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. INDIRECT ELISA : INDIRECT 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 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. Slide 14: 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. SANDWICH ELISA : SANDWICH ELISA Plate is coated with a capture antibody
Sample is added, and any antigen present binds to capture antibody
Detecting antibody is added, and binds to antigen
Enzyme-linked secondary antibody is added, and binds to detecting antibody
Substrate is added, and is converted by enzyme to detectable form. COMPETITIVE ELISA : 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. ELISA Reverse method & device (ELISA-R m&d) : 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 : 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 : 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. Slide 22: Positive control with no signal
Microwell plates not coated properly.
Reagents applied in wrong order or step omitted.
Secondary antibody not matched to the species of primary antibody.
Enzyme conjugate defective or inhibited by contaminant.
Detector antibody not compatible with capture antibody (for sandwich assays). Slide 23: 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. APPLICATIONS : 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) Slide 25: Detecting infections
sexually-transmitted agents like HIV, syphilis and chlamydia
hepatitis B and C
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.,
opiates THANKS : THANKS