ELISA

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M.Prasad NaiduMSc Medical Biochemistry, Ph.D,.
Enzyme-Linked Immunosorbent Assay
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INTRODUCTION

• Is a biochemical technique used mainly in
  immunology to detect the presence of an antibody
  or an antigen in a sample.
• ELISA is so named because the technique involves
  the use of an immunosorbent, an absorbing
  material specific for one of the components of
  the reaction, the antigen or antibody.
• ELISA is usually done using 96-well microtitre
  plate suitable for automation

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What is ELISA?

• ? Technique used to detect (assay) specific
  molecules (e.g. proteins carbohydrates) in
  samples.
• ? Immunological technique uses antibodies.
• ? Quantitative.
• ? Very sensitive.
• ? Commonly used in medicine and scientific
  research.

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PRINCIPLE
Substrate
Different antigens in sample
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• The technique is divided into
• 1- Competitive ELISA
• 2- Sandwich ELISA (also called direct ELISA)
• 3- Indirect ELISA

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COMPETITIVE ELISA

• The labelled antigen competes for primary
  antibody binding sites with the sample antigen
  (unlabeled).
• The more antigen in the sample, the less labelled
  antigen is retained in the well and the weaker
  the signal.
• Advantage
• The ability to use crude or impure samples and
  still selectively bind any antigen that may be
  present.
• Application
• Used for the detection of human T-cell
  leukemia-lymphoma virus type III (HTLV-III). 

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INDIRECT ELISA
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COLOR OF SOLUTION COLOR OF SOLUTION ARE
COMPLEMENTARY
COLOR OF FILTER WAVELENGTH COLOR OF SOLUTION
VIOLET 420 BROWN
BLUE 470 YELLOWISH BROWN
GREEN 520 PINK
YELLOW 580 PURPLE
RED 680 GREEN/BLUE
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INDIRECT METHOD

• This method is largely used to measure antibodies
  in almost all human infections
• Eg HIV antibody detection
• In patients with AIDS, the human immuno
  deficiency virus(HIV) produces specific antibody.
• To detect the HIV antibody, indirect ELISA method
  is used

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SANDWICH METHOD
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Eg Assay of thyroid hormone (T4)
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Reaction

• HRP
• H2O2 H2O O
• Diamino
  oxidized
• Benzidine
  DAB
• (colorless)
  (brown color)
• 
  

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MULTIPLE PORTABLE ELISA

• This new technique uses a solid phase made up of
  an immunosorbent polystyrene rod with 8-12
  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.

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• The advantages of this technique are as follows
• The ogives can each be sensitized to a different
  reagent, allowing the simultaneous detection of
  different antibodies and/or 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

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APPLICATIONS OF ELISA

• Serum Antibody Concentrations
• Detecting potential food allergens
• (milk, peanuts, walnuts, almonds and eggs)
• Disease outbreaks- tracking the spread
• of disease
• e.g. HIV, bird flu, common, colds,
  cholera,
• STD etc

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• Detections of antigens
• e.g. pregnancy hormones, drug allergen,
• , mad cow disease

• human chorionic gonadotropin (HCG), the commonly
  measured protein which indicates pregnancy.
• A mixture of purified HCG linked (coupled) to an
  enzyme and the test sample (blood, urine, etc)
  are added to the test system.
• If no HCG is present in the test sample, then
  only HCG with linked enzyme will bind.

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• The more HCG which is present in the test sample,
  the less enzyme linked HCG will bind.
• The substrate the enzyme acts on is then added,
  and the amount of product measured, such as a
  change in color of the solution.

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• Detection of antibodies in blood sample for past
  exposure to disease
• e.g. Lyme Disease, trichinosis, HIV, bird
  flu
• ELISA can also be used in toxicology as a rapid
  presumptive screen for certain classes of drugs.

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• To monitor diabetes, glucose concentrations will
  be checked.
• Bacterial left-over in milk can be determined
  with an ELISA test.
• ELISA assays are as well employed in foodstuff
  protection through signifying the occurrence of
  salmonella
• ELISA assays can also be utilised to diagnose
  several cancers (eg bladder cancer)

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ADVANTAGES OF ELISA

• ELISA tests are generally relatively accurate
  tests.
• Highly sensitive and specific
• Antigens of very low or unknown concentration can
  be detected since capture antibody only grabs
  specific antigen
• Generally safe do not require radioactive
  substances, contains diluted sulfuric acid
• Used in wide variety of tests

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DISADVANTAGES

• Only monoclonal antibodies can be used as matched
  pairs
• Monoclonal antibodies can cost more than
  polyclonal antibodies
• Monoclonal antibodies are more difficult to find
• Negative controls may indicate positive results
  if blocking solution is ineffective secondary Ab
  or antigen can bind to open sites in well
• Enzyme/substrate reaction is short term so
  microwells must be read as soon as possible

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