Enzyme-Linked Immunosorbent Assay

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Introduction

• ELISA• Types• Applications• Principles

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What is ELISA?• Biochemical technique

used mainly in immunology.

• first and most basic test to determine if an individual is positive for a selected pathogen, such as HIV.

• 8 cm x 12 cm plastic plate which contains an 8 x 12 matrix of 96 wells, each of which are about 1 cm high and 0.7 cm in diameter.

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Types of ELISA

• Qualitative ELISA– Postive or Negative results

• Quantitative ELISA– optical density or fluorescent units of the sample is

interpolated into a standard curve, which is typically a serial dilution of the target.

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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• Detections of antigens• e.g. pregnancy hormones, drug allergen, • GMO, mad cow disease• Detection of antibodies in blood sample for • past exposure to disease• e.g. Lyme Disease, trichinosis, HIV, bird flu

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Basic principles of ELISA

• the antibodies fixed to a solid surface, such as the inner surface of a test tube;

• a preparation of the same antibodies coupled to an enzyme. This is one (e.g., β-galactosidase) that produces a colored product from a colorless substrate.

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Objectives

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• To be able to determine if a serum sample is positive or negative which may help in the diagnosis and treatment of a particular disease like HIV and leptospirosis and alike.

• To be able to apply the proper methods in achieving an accurate results.

• To be able to know the different reagents, their properties and functions.

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Methodology

ELISA for Tracking Disease Outbreaks

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Methods

• A yellow tube and plastic transfer was labeled.

• The “bodily fluid” was then transferred into the tube of another group. The samples were then mixed and after which, the half of the shared sample (750μl) was placed in the group’s tube.

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Methods

• The sharing protocol was repeated twice.

• The 12-well strip was then labeled. On the first 3 wells, it was labeled as “+” while for the next 3 it was labeled as “-”.

• The remaining wells were labeled with two of the member’s initials.

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Methods

• A fresh pipette tip was used to transfer 50μl of the positive control into the “+” wells (the same was done for the “-” wells).

• 50μl of the group’s sample was transferred into the the appropriately initialed three wells.

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Methods

• There was a 5 minute waiting period so as to allow the proteins in the samples to bind to the plastic wells.

• The microplate strip was then tapped onto the paper towels provided.

• The well was then filled with wash buffer.

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Methods

• A 50μl of primary antibody was then transferred into the 12 wells of the microplate strip.

• The fluids was then allowed to stand for another five minutes so as to allow the antibody to bind.

• Washing of the wells was again performed.

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Methods

• A 50μl of secondary antibody was then transferred into the 12 wells of the microplate strip.

• A 5 minute waiting period and washing of the microplate wells was again performed (2x).

• A 50μl of enzyme substrate was then transferred into the 12 wells of the microplate strip.

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Methods

• The enzyme substrate was let to stand for five minutes.

• The results were then observed and recorded.

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Results and discussion

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