UNIVERSITY OF CAPE COAST

COLLEGE OF HEALTH AND ALLIED HEALTH SCIENCES

SCHOOL OF ALLIED HEALTH SCIENCES
DEPARTMENT OF BIOMEDICAL SCIENCES
BMD 307:METHODS IN MOLECULAR BIOLOGY
ASSIGNMENT ONE

LECTURER’S NAME : DR. PAULINA

STUDENT’S NAME : ISAAC DARMEY
INDEX NUMBER: AH/BMS/21/0064
QUESTION
Write a protocol on ELISA.

Enzyme-Linked Immunosorbent Assay (ELISA) is a powerful immunological technique used for the
detection and quantification of various substances, such as proteins, antibodies, hormones, and antigens.
ELISA protocols generally include the following key steps:

Coating:
Microplates are coated with the antigen or antibody of interest.
This step ensures that the surface of the well is covered with the molecule to which the target will bind.
Blocking:
After coating, the plate is blocked with a blocking agent (e.g., BSA or milk) to prevent nonspecific binding
and reduce background noise.
Sample Incubation:
The sample, containing the analyte of interest, is added to the wells.
If the analyte is present, it binds to the coated surface.
Washing:
Unbound substances are removed through thorough washing steps.
Washing is crucial to minimize background signals and increase assay specificity.
Detection:
A secondary antibody or enzyme-linked conjugate is added.
This secondary component binds to the analyte or to the primary antibody, amplifying the signal.
Substrate Addition:
A substrate specific to the enzyme (e.g., HRP or AP) is added.
The enzyme catalyzes a reaction, resulting in a detectable color change or fluorescence.
Measurement:
The intensity of the generated signal is measured, often using a spectrophotometer or a plate reader.
The signal is proportional to the amount of analyte present in the sample.
Data Analysis:
Results are analyzed, and the concentration of the target analyte in the sample is determined based on a
standard curve.