An Introduction to

molecular diagnostics
Dr Gloria Amegatcher
 ELISA
Enzyme Linked Immunosorbent Assay (ELISA)
Different Types
Direct
Indirect
Sandwich
Competitive
Can Be Used To Detect Both Antibody and Antigen
Very Sensitive, pg/mL
Relies on Monoclonal Abs
 Sandwich ELISA
2 Antibodies Required
Must Recognize Different Epitopes
1st Antibody Is Referred To As Capture Ab
2nd Antibody Detection Ab
2nd Antibody Is Biotinylated
Enzymes Commonly Used: HRP (Horse Radish
Peroxidase) And AKP (Alkaline Phosphatase)
Substrate is TMB (Chromogen)
 ELISA Plate
96 well plate
Made of plastic on which protein can be adsorbed
(bind) easily
Usually done overnight @ 4C
Special buffer used that will not denature Ab and
maximize binding
Blocking step ensures no empty spaces are left
Blocking reagent is often 10% FBS
Assume You Have A Stock Tube @ 2000pg and transferred
some into tube 1 to serve as neat.
Usually Remaining Standard Is Thrown Away
Thawing-Unthawing Affects standard
Addition of detection Ab
Avidin/streptavidin has very high affinity for biotin
(B vitamin)

B vitamin is conjugated on the detection Ab

Add Working Detector @ 100 L/well

Incubate for 60 mins @ R.T

Wash 6x
Addition of substrate
Prepare Substrate by Mixing 1:1 volume

Add 100 L/well

Incubate for 10 mins, formation of excessively

bright color (Spec will not be able to read)

Terminate Reaction by Adding 0.5 M H2SO4 (color

changes from blue to yellow)
Enzyme-Linked Immunosorbent Assay (ELISA):
immunological detection

A. Bind sample to the support (commonly plastic or a membrane)

B. Add primary antibody; wash
C. Add secondary antibody-enzyme conjugate; wash
D. Add substrate
enzyme linked
secondary antibody
bound primary E
Y E
Y
antibody Y Y antigenic site
colorless substrate
Y
Y

Y
Target molecule Y
E
E

iiiiiiiiiiiiiii colored product

Support
Target antigens and polyclonal versus monoclonal
antibodies
2 3 4

Target antigen 5
with various antigenic
1 determinants (epitopes)

7 6

Polyclonal antibodies are made against and react with

multiple antigenic sites (epitopes) on a target antigen.
Monoclonal antibodies are directed against a particular
antigenic site.
Procedure for producing a
monoclonal antibody to
protein X

Note: B lymphocytes or B cells produce

antibodies but do not reproduce in culture.
Some B cells can become cancerous and
are known as myelomas which can
reproduce in culture.

See animations
http://bcs.whfreeman.com/lodish7e/#800
911__811944__

https://www.youtube.com/watch?v=O-SrP
qJuEVg

https://www.youtube.com/watch?v=0A99p
Targets for diagnostic monoclonal antibodies
• Polypeptide hormones (human chorionic
gonadotropin (hCG), growth hormone)
• Tumor markers (Prostate-specific antigen)
• Cytokines (interleukins 1-8)
• Drug monitoring (cyclosporin)
• Miscellaneous targets (Vitamin B12)
• Infectious diseases (Chlamydia, Herpes, Rubella,
Hepatitis B, HIV)
See examples of hCG pregnancy test:
https://www.youtube.com/watch?v=aOfWTscU8YM
http://www.sumanasinc.com/webcontent/animation
s/content/pregtest.html
 DNA diagnostic systems
1. Bind ssDNA (target) to membrane
2. Hybridize to labeled ssDNA or RNA (probe)
3. Wash membrane to remove unbound probe
4. Detect hybrid sequences formed between the
probe and target DNA

membrane
Nonradioactive Hybridization Procedures