Special investigations used in the biomedical field

1. S.Srilakshman
BS/15/11/29

2. • WESTERN BLOT
• ELISA
• PCR
• Heamaglutination inhibition
test
• Plasmid Finger printing

3.  Western blot is an analytical technique used to detect specific
proteins in a given sample of tissue homogenate or extract
 The transferred protein is detected using specific primary and
secondary enzyme labeled antibody.
 Antibodies bind to specific sequences of amino acids, known as the
epitope.
 Because amino acid sequences are different from protein to
protein, antibodies can recognize specific proteins among a group
of many.

4.  Denature proteins by boiling in detergent
 Seperation of the proteins on polyacralamide gel by electrophoresis
 Transfer (blotting) of proteins from the gel to the membrane (nitrocellulose
or nylon) and identification of the protein with a specific Ab.

6.  Positive result- bands found in 2 location
 Negative result- no bands are present
Related diseases – Confirmation Of HIV
Lyme disease

7. APPLICATION
 Used for evaluating levels of protein
expression in cells
 For monitoring fractions during
protein purification.
 For comparing expression of a target
protein from various tissues, or
seeing
 how a particular protein responds to
disease or drug treatment.
ERRORS
 All proteins may not be transferred
to membrane
 Transferred proteins may not have
adhered correctly to membrane
 Dyes added interfere with antibody
binding and detection

8.  ELISA test means Enzyme Linked IMMUNO SORBANT Assay test
 This technique is done to measure the concentration of specific serum
antibodies. And it is used to detect various antigenic properties such as
proteins, polysaccharides and nucleic acids

10.  Antigen is added
 Excess is washed off
 Antibody is added and excess is washed
 Next enzyme-linked secondary antibody is added and again
excess is washed
 Finally enzyme substrate is added to it and the excess is rinsed
 Finally the antibodies binds to the antigen, the substrate react with
enzyme and produce a color

11. False positive and false negative can occur.
Related Diseases – Lyme disease, Rocky
Mountain Spotted Fever, Rotavirus,
Syphilis, Toxoplasmosis and HIV

12. Application
 Virus test
 Home pregnancy test
 Food industry
 Toxicology
 Immunology
 Biological pharmacy
 Diagnostic industry
Error
 No signal
 Large coefficient of variation
 Low sensitivity

13.  It is an efficient and cost-effective way to copy or amplify small segments of DNA
or RNA.
 Sequence specific primers are used in PCR
 It allows for the detection even if only a few cells are present and can also be
used on viable nonculturables.

14. (Paxson, 2008)

15.  Related Diseases – Detection of Chlamydia pneumoniae in CSF.
Lyme Disease
Adenovirus myocarditis
Anaplastic lymphoma Kinase
Avian Influenza A
Breast and ovarian cancer
Dengue etc.
 Errors – Sequence Errors
No product
Multiple or Non specific products

16.  The agglutination test can be modified to be used for the measurement of soluble
antigens. This test is called hemagglutination inhibition.
 It is called hemagglutination inhibition because one measures the ability of soluble
antigen to inhibit the agglutination of antigen-coated red blood cells by antibodies.
PROCEDURE
 Prepare two-fold dilutions of test serum to be tested
 Add a fixed amount of virus to every well except for the serum control wells.
 The plate is then allowed to stand at room temperature for 60 minutes.
 Add red blood cells and incubate at 40 ⁰C for 30 minutes.
 Read the wells
 If the sample contains the antigen, the soluble antigen will compete with the antigen
coated on the red blood cells for binding to the antibodies, thereby inhibiting the
agglutination of the red blood cells.

17. Related Diseases - Influenza, measles, mumps, mononucleosis, and other viral infections
Results Interpretation – No hemolysis considered as positive test. Hemolysis of RBC
indicative of a negative test.

18.  Plasmid fingerprinting identifies microbial species or similar strains as related strains
often contain the same number of plasmids with the same molecular weight
Procedure
 The bacterial strains are grown, the cells lysed and harvested.
 The plasmids are separated by agarose gel electrophoresis
 The gels are stained with EtBr and the plasmids located and compared
Application
 Plasmid of many strains and species of E. coli, Salmonella, Camylobacter and
Psseudomonas has demonstrated that this methods is more accurate than phenotypic
methods such as biotyping, antibiotic resistance patterns , phage typing and serotyping.

19.  Chinnabathini, A., 2012. Western blotting. Available at:
http://www.slideshare.net/doctorrao/westernblotting [Accessed October 30, 2016].
 Acharya, T. (2015) API 20E Test System: Introduction, Procedure Results and Interpretations -
microbeonline, [online] Available from: http://microbeonline.com/api-20e-test-system-
introduction-procedure-results-interpretations/ (Accessed October 30,2016).
 Butler, J. M. (2005) Forensic DNA Typing: Biology, Technology, and Genetics of STR Markers,
Academic Press, [online] Available from:
https://books.google.com/books?id=gwDyBq2xLjIC&pgis=1 (Accessed 01 November 2016).
 Crowther, J. R. (1995) ELISA: Theory and Practice, Springer Science & Business Media, [online]
Available from: https://books.google.com/books?id=AodkPkz_7NEC&pgis=1 (Accessed 01
November 2016).
 Symons, R. H. (1989) Nucleic Acid Probes, CRC Press, [online] Available from:
https://books.google.com/books?id=v90E2P6_UmQC&pgis=1 (Accessed 2 November 2016).

20. Any questions?