A brief summary of PCR Western Blot ELISA Finger printing Hemagglutination inhibition

1. SPECIAL
INVESTIGATIONS
NAME: FAHIM ASLAM

2. CONTENTS
• PCR
• WESTERN BLOT
• ELISA
• FINGER PRINTING
• HAEMAGGLUTINATION INHIBITION

3. PCR – POLYMERASE CHAIN REACTION
• Polymerase Chain Reaction is used one of the most common
methods to identify the micro-organisms
• Specific Primers are used here for the amplication of the
DNA/RNA material in the specific pathogens
• The procedure involves specific DNA primers binding to the
DNA/RNA strands 30-40 times
• Initially the denaturation of the DNA/RNA strand happens to
break the nucleotides and the bonds between them, 94C for
a minute
• Second step is where the cooling takes place until 54C for a
period of 45 seconds to allow the nucleotides to bind with
the complementary strands formed and primers bounded.
• Final step where the strands are extended with primers, this
happens at 72C for a period of 2 minutes.

4. END-POINT PCR REAL TIME PCR
• This reaction, the results are
obtained on the endpoint of
the PCR.
• This is time consuming and
results are not obtained
immediately for these tests.
• The end point is not fixed for
any sample, it varies from
sample to sample
• The agarose solution used
does not provide a proper
resolution inside the samples
tested and they vary with the
use of the gel.
• The Real time PCR uses
fluorescent primers
compared to normal
primers used in end point
• The PCR monitors the
primer action and plots an
amplification plot
accordingly
• These results can be
obtained quickly as well.
• Can identify several
different types of micro-
organisms involved
• Uses less amount of DNA
compared to end point
PCR to identify the micro
organism.

5. • RESULTS – DIFFERENT BANDS FOUND IN
DIFFERENT SOLUTIONS FOR DIFFERENT
SOLUTIONS.
• DISEASES – MENINGITIS, HEPATITIS, PNEUMONIA
• ERRORS – MORE THAN ONE COPY OF THE GENE
CAN CAUSE RESULTS TO BE ALTERED. WRONG
PRIMER SEQUENCES. FALSE NEGATIVES AND
FALSE POSITIVE RESULTS CAN BE FOUND USING
THE LIKES OF PCR WHICH MAKES IT LESS
RELIABLE.
• APPLICATIONS – GENOTYPING, SEQUENCING,
MUTATION DETECTION.
• CONCLUSION – ISOLATION OF DNA FRAGMENTS
CAN BE DONE THROUGH SELECTIVE
AMPLIFICATION.

6. WESTERN BLOT
• A METHOD MAINLY USED TO DETECT SPECIFIC PROTEINS BINDED TO THE DNA
MATERIAL IN THE ORGANISM.
• THIS METHOD INVOLVES SEPARATION OF THE DIFFERENT PROTEINS ON THE
POLYACRYLAMIDE GEL.
• THE CHARGED PARTICLES MOVE ACROSS TO THE ELECTRODES, BUFFER SYSTEM
WITH DIFFERENT PH VALUES ARE APPLIED ONTO THE GEL ELECTROPHORESIS.
• THE PROTEINS ARE THEN TRANSFERRED ONTO A NITROCELLULOSE PAPER, NON-
FAT DRY MILK IS USED TO WASH OFF THE UNREACTED SITES.
• PRIMARY ANTIBODY INCUBATION IS INTRODUCED, THIS BINDS WITH A SPECIFIC
PROTEIN WHICH SHOWS THE PROTEIN.
• SECONDARY ANTIBODY INTRODUCED WILL HAVE TO WASH OFF THE UNREACTED
PRIMARY ANTIBODIES

7. RADIO IMMUNE
ASSAY
IMMUNOBLOT TEST
• This method uses an immune
reaction, this is used identify
the drugs, enzymes and
hormones inside the body.
• Initially the first antibody is
released, and binds with the
radioactive antibody.
• Unknown antigens are
released the same time,
• A second antibody is released
which binds with the Fc
region of the first antibody
• Using a metal bounded with
the ion the molecule is
• Analyzing of proteins takes
place here, proteins
separated by SDS/PCR.
• Transferring to a synthetic
membrane using wet
blotting methods.
• The electric field generated
allows the charged particles
towards oppositely charged
poles.
• As they move out they are
captured onto nitrocellulose
paper

8. • Results – The nitrocellulose paper taken can
be checked for the specific proteins
according to the molecular weight.
• Diseases - STD’s, Lyme disease
• Errors – Bad protein samples could result in
the Western blot pattern being irregular.
Mixing of the wells, bubbles on blotting gel
• Applications – Protein purification, protein
sequencing.
• Conclusion – This method is useful for
protein detection and it allows the
quantification of the protein expressions.

9. ELISA
• ENZYME LINKED IMMUNO-ABSORBENT ASSAY ALSO KNOWN AS ELISA IS USED TO
LINK UP ENZYMES TO ANTIGENS OR ANTIBODIES.
• DIFFERENT ENZYMES ARE INVOLVED ARE IN ELISA, GALACTOSIDASE AND
PEROXIDASE ARE EXAMPLES.
• THIS METHOD IS USED TO IDENTIFY AND MEASURE THE ANTIBODIES IN THE
BLOOD.

10. • DIRECT ELISA – INVOLVES THE ATTACHMENT OF ANTIGEN ONTO THE SOLID PHASE
FOLLOWED BY THE ENZYME LABELLED ANTIBODY. (PRIMARY ANTIBODY)
• INDIRECT ELISA – INVOLVES THE ATTACHMENT OF ANTIGEN ONTO THE SOLID PHASE
FOLLOWED BY THE ENZYME LABELLED ANTIBODY WHICH IS THE SECONDARY
ANTIBODY.
• COMPETITIVE ELISA – INVOLVES MULTIPLE ANTIBODIES OR PROTEINS THAT ARE
ADDED SIMULTANEOUSLY.
• SANDWICH ELISA – ANTIBODIES ATTACH TO THE SOLID PHASE, SAMPLES
CONTAINING KNOWN OR UNKNOWN ARE ADDED TO THE BUFFER, ENZYME LABELLED
ANTIBODY INTRODUCED FOR DETECTION.

12. • Results – a graph of absorbence against protein
concentration is drawn, this way the duplicates
obtained are calculated.
• Diseases – HIV, Pernicious Anemia, Rocky mounted
spotted fever, Syphillis
• Errors – The errors arised from ELISA techniques
vary significantly, the buffer solution used needs to
be the proper standard. Callibration needs to be
done before the test carried out.
• Applications – Food allergen tests, identification of
an antibody/antigen.

13. PLASMA FINGERPRINTING
• A METHOD USED TO IDENTIFY MICROBIAL SPECIES.
• USING STRAINS OF THE SPECIES, THE MOLECULAR WEIGHTS ARE COMPARED.
• THE PROCESS INVOLVES THE STRAINS OBTAINED, THE CELLS ARE LYSED INTO
THE AGAROSE GEL AND SEPARATED BY ELECTROPHORESIS.
• THE PLASMIDS ARE SEPARATED AND USING ETHIDIUM BROMIDE THE PLASMIDS
ARE COMPARED, ETHIDIUM BROMIDE PROVIDES THE ILLUMINE FOR THE
SAMPLES.

14. • RESULTS – THE BANDS IN THE STRAINS ARE ANALYZED AND WITH THE PRESENCE
OF ETHIDIUM BROMIDE THE STRANDS ARE VISIBLE.
• DISEASES – CORONARY SYNDROME
• ERRORS – THE STRAINS OBTAINED NEED TO BE SET WELL INTO THE AGAROSE
SOLUTION IF NOT THE RESULTS WILL NOT BE OBTAINED EASILY.
• APPLICATION – IDENTIFICATION OF DNA AND ITS TYPE.

16. HAEMAGGLUTINATION INHIBITION
• HAEMAGGLUTINATION IS THE PROPERTY WHERE THE RBC’S ARE AGGLUTINATED
BY THE SURFACE PROTEIN OF VIRUSES.
• THE PRINCIPLE OF HAI IS TO USE ANTIBODIES OF THAT PARTICULAR VIRUS TO
PREVENT THE ATTACHING OF VIRUS TO THE RBC.
• THERE ARE TWO POSSIBILITIES WHICH CAN TAKE PLACE, IF SERUM CONTAINS NO
ANTIBODIES THEN HAEMAGGLUTINATION WILL TAKE PLACE.
• IF THERE’S AN ANTIBODY, HAEMAGGLUTINATION WILL NOT BE OBSERVED.

18. • RESULTS – USING THE AGGLUTINATION OR THE CLUMPING THE RESULTS CAN BE
DETERMINED.
• DISEASES – VIRAL DISEASES, SUCH AS INFLUENZA
• ERRORS – THE ANTIBODIES TAKEN NEEDS TO BE SUITABLE FOR THE RBC’S
TESTED, CAN LEAD TO FALSE RESULTS.
• APPLICATIONS – IDENTIFICATION OF VIRUSES.

19. CONCLUSION
• PCR IS USED TO INCREASE DNA FRAGMENT AMOUNTS MAINLY.
• WESTERN BLOT IS USED TO IDENTIFY THE DIFFERENT TYPES OF PROTEINS IN THE
DNA STRAND
• ELISA INVOLES AB-AG REACTIONS MAINLY
• FINGERPRINTING IS USED TO RECOGNIZE THE DNA BANDS WITHIN SPECIES FOR
IDENTIFICATION OF SAMPLES.
• HAEMAGGLUTINATION INHIBITION INVOLVES THE RBC AND VIRAL BINDING.

20. REFERENCES
• GIBALDI, M. AND PERRIER, D. (1982). PHARMACOKINETICS. NEW YORK: M.
DEKKER.
• HIV. (2002). JAMA, [ONLINE] 288(2), P.253. AVAILABLE AT:
HTTP://DX.DOI.ORG/10.1001/JAMA.288.2.253-JBK0710-5-1 [ACCESSED 28
OCT. 2016].
• TATRO, D. (2012). DRUG INTERACTION FACTS 2013. ST. LOUIS, MO.: WOLTERS
KLUWER HEALTH/FACTS & COMPARISONS.