Transcript
(EnzymelinkedImmunoAbsorbant Assay)
Objectives
• To understand the steps involved in performing an ELISA and how it is used as a diagnostic tool
• To understand the disease-causing agent and transmission patterns of certain infectious diseases
Quick Recall:
1. The body possesses several lines of defense against infection by pathogenic organisms.
2. The first two defense modes are nonspecific.1. body’s physical barriers2. nonspecific immune system
3. Specific immune responses are tailored to the type of invading pathogen.
Specific Immune Response
• The specific immune system is complex and involves several organs and tissues, including the thymus, spleen, lymph nodes, bone marrow, and white blood cells.
• Specific immune responses are triggered by antigen molecules. Antigens include proteins and other molecules produced by pathogens.
• The key players in the specific immune defense are dendritic cells, macrophages, and small white blood cells called B lymphocytes (B cells) and T lymphocytes (T cells).
• Phagocytic macrophages and dendritic cells break down pathogens and display antigenic fragments from the pathogens on the surface of their cell membranes.
http://www.youtube.com/watch?v=BDr44vLNnPY
• B and T lymphocytes circulate through the body in the blood and lymph.
• When T cells see displayed antigenic fragments, they stimulate specific B cells to reproduce and generate antibodies designed against the specific structure of the antigen encountered. Thus, the word antigen is derived from the term “antibody generator.”
• Antibodies are a group of serum proteins (also referred to as immunoglobulins) that are found in the bloodstream or bound to cell membranes.
• These proteins all have the same basic Y-shaped structure, but have different antigen binding sites at their ends.
• Antigen binding sites are designed to fit the shape of specific antigens. Antibodies bind to antigens like a lock and key, forming antigen-antibody complexes
• When an antibody forms an antigen-antibody complex, generally it marks the invading organism/antigen for destruction or for clearance from the bloodstream by phagocytic cells.
• This removal is designed to prevent the organism/antigen from infecting the cell. Antigen-antibody complexes also stimulate additional immune responses to aid the body in clearing an infection.
ELISA
Assay for detecting infection by specific organisms
• ELISA is commonly used to test blood serum for the presence of antibodies against disease-causing pathogens such as viruses and bacteria.
• In this way, the assay indirectly detects infection by particular disease-causing agents.
• Based on the principle that antibodies produced in response to pathogens attach to their antigen targets with great specificity to form antigen-antibody complexes.
• ELISAs can be used to test for infection by HIV, influenza virus, the bacterium that causes Lyme Disease, smallpox virus, SARS coronavirus, West Nile virus, and other disease agents.
Types of ELISA
• Indirect ELISA• Direct ELISA• Sandwhich ELISA
• COMPETETIVE ELISA
NON -COMPETETIVE ELISA
MATERIALS NEEDED FOR ELISA KIT
ELISA Plate
Positive control
Negative control
Dilution Buffer
Conjugate
TMB Substrate
Stop Solution
Indirect ELISA
• Indirect ELISA is used to detect infection by testing patients’ blood for the presence or absence of ANTIBODIES against a particular pathogen.
• The presence of such antibodies indicates that the individual has been infected and that their body has launched an immune response against the disease-causing agent.
INDIRECT ELISA
Antigen is added to plate.
Added Blocking buffer.
Suitable primary antibody is added.
Secondary antibody- HRPO is then added which recognizes and binds to primary antibody.
TMB substrate is added, is converted to detectable form.
Under standard condition ,the enzyme activity measured is proportional to amount of specific antibody in the original serum.
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.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.
Direct ELISA
• assays for the presence or absence of certain ANTIGENS in patients’ blood.
DIRECT ELISA
1. Apply a sample of known antigen to a surface.
2. Enzyme linked primary antibody is applied to the plate.
3. Washed, After this wash, only the antibody-antigen complexes remain attached.
4. Apply a substrate which is converted by the enzyme to elicit a chromogenic signal.
Under standard condition ,the enzyme activity measured is proportional to amount of specific antibody in the original serum.
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.
SANDWICH ELISA
1. a. Plate is coated with suitable antibody.b. Blocking buffer is added.
2. Sample is added to plate so antigen is bounded by capture antibody.3. A suitable biotin labeled detection antibody is added to plate.4. Enzyme HRPO is added and binds the biotin labeled detection
antibody.5. TMB substrate is added and converted by HRPO to colored product.
Under standard condition ,the enzyme activity measured is proportional to the Amount of specific antigen in the original serum.
Wash
3X
Wash
4X
Wash
4X
Wash
4X
A EDCB
Alkaline phosphatase
substrate is added and developed
colour is read at 405 nm
wavelength to measure plasma
cencentration
Wells are coated with 0.2
μg primary antibody
Diluted plasma
is added to coated wells,
which bind to
antibodies
0.1 μg of biotinylated (biotin =
– ) antihuman secondary antibody
Incubated overnight at
4˚C
Incubated at room temperature (24˚C)
2h 2h 1h
PROCEDURE OF ELISA
Add 1.2000 dilution of streptavidi
n conjugate to alkaline phosphatas
e ( E)
FINAL PLATE OF ELISA
COMPETETIVE ELISACOMPETETIVE ELISA
COMPETETIVE ELISA
Solid phase coated with antibody
Add unknown amount of unlabeled antigen and known amount of labeled antigen
Free and labeled antigen are captured
Color formation by oxidation of substrate into a colored compound
Under standard condition ,the enzyme activity measured is proportional to the proportion of labeled antigen in the mixture of labeled and unlabled antigen.
ADVANTAGES:
Suitable for complex (crude or impure) samples, since the antigen does not require purification prior to measurement.
DISADVANTAGES
Each antigen may require a different method to couple it to the enzyme.
COMPARISON BETWEEN VARIOUS TYPES OF ELISA
Direct ELISA Indirect ELISA Sandwich ELISA Competitive ELISA
SENSITIVITY
ELISAs are one of the most sensitive immunoassays available. The typical detection range for an ELISA is 0.1 to 1 fmole or 0.01 ng to 0.1 ng, with sensitivity dependent upon the particular characteristics of the antibody – antigen interaction.
In addition, some substrates such as those yielding enhanced chemiluminescent or fluorescent signal, can be used to improve results.
As mentioned earlier, indirect detection will produce higher levels of signal and should therefore be more sensitive. However, it can also cause higher background signal thus reducing net specific signal levels.
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)
Detecting infections
Sexually-transmitted agents like HIV, syphilis and chlamydia
Hepatitis B and C Toxoplasma gondii
Detecting illicit drugs.
Detecting allergens in food and house dust
THANK YOU
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