Introduction and Patient History-– Biology of Disease

Read through Introduction and Patient History
 Read procedure for estimating carcinoembryonic antigen (CEA)
concentration using Sandwich Enzyme Linked Immunoabsorbant Assay
 Complete the Immunology questions
BACKGROUND
 CA has undergone surgery, progress is monitored
 Serum samples collected-
 pre-operation- Serum 1
 three weeks later at follow-up – Serum 2
 eight weeks post-operation- Serum
 CEA is a type of tumor marker
 High level of CEA can be a sign of certain types of cancers
AIM
 Estimate the levels of Carcinoembryonic antigen (CEA) in
the three serum samples (Serum 1, Serum 2, Serum 3) using
Sandwich Enzyme Linked Immunoabsorbant Assay
 Read through sheets before starting
ENZYME-LINKED IMMUNOSORBENT ASSAY
(ELISA)
 A sensitive immunoassay of specific protein, antigen or antibodies
 Depends on the specific binding between the antibody and the
antigen
 Uses an enzyme linked to an antibody or antigen as a marker for
the detection
 Plate-based technique designed for detecting peptides, proteins,
antibodies and hormones
ELISA IS QUANTITATIVE
Detect using
 colour/chemiluminescence
 Fluorescence
 Radioactivity (125I)
Generate standard curve
Concentration
10 100 1000
0.08
0.13
0.18
0.23
0.28
Standard Curve
4-P Fit: y = (A – D)/( 1 + (x/C)^B ) + D: A B C D R^2
Std (Standards: Concentration vs MeanValue) 0.0905 1.5 6.33e+06 1.05e+05 0.998
__________
Weighting: Fixed
ELISA FORMATS
Direct ELISA
 Antigen coated to multi-well
 Antibody directly conjugated to an enzyme added
Or Reverse
Advantages
 Fast
 Less prone to error
Source: Thermofisher
Indirect ELISA
 Antigen coated on the polystyrene well
 First unlabelled primary antibody specific
for antigen is applied
 Second step- Enzyme-linked secondary
 antibody against the primary antibody is applied
Advantages
 Increased sensitivity (more than one ab bound to primary ab)
 Flexibility (different primary antibodies can be used)
 Cost saving
Source: www.chemgapedia.de
Sandwich ELISA
 Use of matched antibody pairs
 Capture antibody coated first on polystyrene plate
 Sample solution is added
 Second antibody layer of detection antibody added
 Detection antibody could be labelled or unlabelled
(direct or indirect)
Advantages
 Highly specificity
 Suitable for complex samples
 Flexibility and sensitivity
Source: https://embryology.med.unsw.edu.au
STEPS
Source- Bio Rad
BINDING TO SOLID SUPPORT
 Plates, tubes, beads etc.
 Must preserve the native structure of the antigen/antibody
 Minimum binding capacity of 400ng/cm2
How antibodies bind?
 Adsorption
 Simple
 passive
 Hydrophobic interaction
 Covalent tether
 Metallic Beads
 Biotin/streptavidin
ADSORPTION OF ANTIBODY
 pH or ionic strength
 Temperature (low temperature increases specificity and reduced
 Time
 Use 2-10ug/ml proteins in buffers such as PBS pH 7.4, or
carbonate bicarbonate pH 9.4)
 Depends on the characteristics of protein
 Prevent nonspecific binding
 Reduce ELISA background signal
 Block nonspecific binding to
adsorbed proteins
 Stabilize proteins adsorbed
to plate for better interactions
Which Blocking reagents can be used?
 2-5% BSA
 5% Skimmed milk
 Casein, calf serum, 5% sucrose containing
1%BSA and 0.01% azide
BLOCKING
Source: https://www.immunochemistry.com/products/elisa-solutions/blocking-buffers.html
METHOD
 Follow the protocol step by step method
 Samples-
 Standards- known concentration of CEA to determine the unknown
concentration of CEA in serum samples
 Serum 1, 2, 3 – PF samples
 Negative and positive control- Ensures experiment is working correctly
 Prepare dilutions
 TASKS
Answer questions in the workbook