2. 1. What is Disease?
2. What is Diagnostics?
3. What is Diagnosis?
4. Types of Molecular Diagnostics Techniques--
1. Radioimmunoassay
2. ELISA
3. Western Blotting
4. Immunoprecipitation
5. Immunofloresence
6. PCR
5. Examples of Diseases Diagnosed By Molecular Diagnostic Techniques
3. “A disease is a particular abnormal condition that negatively affects the structure or
function of all or part of an organism, and that is not due to any immediate external
injury.”
Diseases are often known to be medical conditions that are associated with specific
symptoms and signs.
“A maladjustment of the human organisms to the environment”– Ecological point of
view.
4. Diagnostics is defined as the identification of diseases by the examination of
symptoms and signs and by other investigations an opinion or conclusion so reached
.
Thorough analysis of facts or problems in order to gain understanding and aid future
planning an opinion or conclusion reached through such analysis .
5. The identification by a medical provider of a condition, disease, or injury made by
evaluating the symptoms and signs presented by a patient.
The act or process of identifying or determining the nature and cause of a disease or
injury through evaluation of patient history, examination, and review of laboratory
data.
6. Molecular Diagnosis -- Is a collection of techniques used to analyze biological markers in the
genome and proteome—the individual's genetic code and how their cells express their
genes as proteins—by applying molecular biology to medical testing.
Clinical Diagnosis -- The estimated identification of the disease underlying a patient's
complaints based merely on signs, symptoms and medical history of the patient rather than
on laboratory examination or medical imaging.
Laboratory Diagnosis -- Diagnosis based on the results of laboratory analyses, including
microscopic, bacteriologic, or biopsy studies.
7. It was developed by S.A. Berson and Rosalyn Yalow in 1959 and win noble prize in 1997.
Radioimmunoassay (RIA) is a very sensitive in vitro assay technique used to measure
concentrations o f antigens (for example, hormone levels in the blood) by use of antibodies.
Sensitivity ranger from 0.0006μg/ml.
Principle:-
It involves three principles which make it most specific.
1. An immune reaction i.e. antigen, antibody binding.
2. A competitive binding or displacement reaction (it gives specificity).
3. Measurement of radio emission (it gives sensitivity)
8.
9. Radiation hazards – use of radio labeled
reagents.
Requires specially trained persons.
Labs require special license to handle
radioactive material.
Requires special arrangements for -
radioactive waste disposal and storage of
radioactive material.
Costly.
Highly specific - immune reactions are
specific, the greater the specificity of the
antiserum, the greater the specificity of the
assay.
High sensitivity - Immune reactions are
sensitive, using antibodies of high affinity
it is possible to detect a few picograms (10-
12g) of antigen in the tube.
Accuracy and precision.
10. 1. The test can be used to determine very small quantities (e.g. nanogram) of
antigens and antibodies in the serum.
2. The test is used for quantization of hormones, drugs, HBsAg, and other viral
antigens.
3. Analyze nanomolar and picomolar concentrations of hormones in biological
fluids.
4. Estimation of vitamins like folic acid, riboflavin, etc.
5. To detect hepatitis and HIV antigens.
6. Determination of Ag concentration in given sample.
11. 1. In 1971, two Swedish scientists, Eva Engvall and Peter Perlman, who died in 2005, invented a
test that revolutionized medicine. Called the ELISA test, the method uses antibodies to seek out
the presence of hormones or viruses.
2. ELISA (enzyme-linked immunosorbent assay) is a plate-based assay technique designed for
detecting and quantifying soluble substances such as peptides, proteins, antibodies, and
hormones.
12. Based on basic immunology response.
Lock and key concept-
Antigen (key) Antibody (lock).
Key fits into lock.
Enzyme conjugate substrates.
Bound to a secondary antibody that binds with the antibody antigen complex.
13. .
1.
• Antigen/sample is added to plate.
2.
• Blocking buffer is added to block remaining protein binding sites.
3.
• Next a suitable primary antibody is added.
4.
• A suitable secondary antibody-HRPO conjugate is then added which recognizes and binds to the primary antibody.
5.
• TMB substrate is added and is converted by HRPO to detectable form.
15. Measurement of enzymes activity can be
more complex than the measurement of
activity of some type of radioisotopes.
Enzymes activity may be affected by
plasma constituents.
Kits are not cheap.
Very specific to particular antigen but
won’t recognize other antigens.
Reagents are relatively cheap and have
long self life.
It is highly specific and sensitive.
No radiation hazards occur during labeling
or disposal of waste.
Easy to perform and quick procedures.
It can be used on most types of biological
samples like plasma, serum, urine, etc.
It is widely available and used for variety
of infections.
16. Screening donated blood for evidence of viral contamination by-
HIV-1 and HIV-2 (presence of anti-HIV antibodies).
Hepatitis B and C (presence of antibody).
Measuring Hormone levels-
HCG (for pregnancy), LH (for ovulation), TSH, T3, T4 (for thyroid function).
Detecting infections-
Sexually transmitted agents like HIV, Syphilis, etc.
Toxoplasma Gondii.
Detecting Illicit Drugs.
17. The term "western blot" was given by W. Neal Burnette in 1981, although the
method itself originated in 1979 in the laboratory of Harry Towbin at the Friedrich
Miescher Institute in Basel, Switzerland.
Western blot is the analytical technique used in molecular biology, immunogenetics
and other molecular biology to detect specific proteins in a sample of tissue
homogenate or extract.
Western blotting is called so as the procedure is similar to Southern blotting. While
Southern blotting is done to detect DNA, Western blotting is done for the detection of
proteins.
Western blotting is also called protein immunoblotting because an antibody is used
to specifically detect its antigen.
18. The technique consists of three major processes:
1. Separation of proteins by size (Electrophoresis).
2. Transfer to a solid support (Blotting)
3. Marking target protein using a proper primary and
secondary antibody to visualize (Detection).
Electrophoresis used to separate proteins according to their electrophoretic mobility which
depends on charge, size of protein molecule and structure of the proteins. Proteins are
moved from within the gel onto a membrane made of Nitrocellulose (NC) or
Polyvinylidene difluoride (PVDF). Without pre-activation, proteins combine with
nitrocellulose membrane based on hydrophobic interaction (Blotting). For Detection of the
proteins primary antibody and enzyme conjugated secondary antibody are used. On addition
of substrate, a substrate reacts with the enzyme that is bound to the secondary antibody to
generate colored substance, namely, visible protein bands.
20. Prone to False or Subjective Results- In
spite of its sensitivity and specificity, a
western blot can still produce erroneous
results.
High Cost and Technical Demand- The cost
of a western blot is a composite of the large
individual expenditures for tagged
antibodies, skilled analysts and laboratory
equipment .
Sensitivity- One of the biggest arguments
in favor of western blot is its sensitivity.
Because of its ability to detect as little as
0.1 nanograms of protein in a sample.
Specificity- The western blot technique
owes its specificity to two big contributing
factors. First, is gel electrophoresis and
second is antigen-antibody interactions.
21. 1. Identification of a specific protein in a complex mixture of proteins. In this method,
known antigens of well-defined molecular weight are separated by SDS-PAGE and
blotted onto nitrocellulose. The separated bands of known antigens are then probed with
the sample suspected of containing antibody specific for one or more of these antigens.
Reaction of an antibody with a band is detected by using either radiolabeled or enzyme-
linked secondary antibody that is specific for the species of the antibodies in the test
sample.
2. Estimation of the size of the protein as well as the amount of protein present in the
mixture.
3. It is most widely used as a confirmatory test for diagnosis of HIV, where this procedure
is used to determine whether the patient has antibodies that react with one or more viral
proteins or not.
4. Demonstration of specific antibodies in the serum for diagnosis of neurocysticercosis
and tubercular meningitis.
22. Immunoprecipitation (IP) is the technique of precipitating a protein antigen out
of solution using an antibody that specifically binds to that particular protein.
This process can be used to isolate and concentrate a particular protein from a
sample containing many thousands of different proteins.
Immunoprecipitation requires that the antibody be coupled to a solid substrate
at some point in the procedure.
Antigens isolated by IP are analyzed by SDS-PAGE or Western blotting.
What is the mechanism of immunoprecipitation?
23. .
Immunoprecipitation
Direct capture method-
Antibodies that are specific for a particular
protein are immobilized on a solid-phase
substrate such as superparamagnetic
microbeads or on microscopic agarose
beads.
Indirect capture method-
The antibodies have not been attached to a
solid-phase support yet. The antibodies are
free to float around the protein mixture and
bind their targets.
24.
25. 1. Isolate/ detect proteins of interests.
2. Enrrichment of low abundant proteins.
3. Study proteins-protein interaction and protein complexes.
4. Identify unknown proteins in a protein complex .
5. Verify protein expression in a specific tissue.
26. Immunofluorescence is a technique allowing the visualization of a
specific antigen by binding a specific antibody chemically conjugated
with afluorescent dye such as fluorescein isothiocyanate (FITC).
The specific antibodies are labeled with a compound (FITC) that makes
them glow an apple-green color when observed microscopically under
ultraviolet light.
Fluorescence is the property of certain molecules to absorb light at one
wave length and emit light at longer wave length when it is illuminated
by light of a different wavelength.
The fluorescence can be visualized using fluorescence microscopy.
27. .
1. Immunofluorescence is an assay which
is used primarily on biological samples
and is classically defined as a
procedure to detect antigens in cellular
contexts using antibodies. The
specificity of antibodies to their
antigen is the base for
immunofluorescence.
28. A. Direct immunofluorescence: Staining in which the primary antibody
is labeled with fluorescence dye.
B. Indirect immunofluorescence: Staining in which a secondary
antibody labeled with fluorochrome is used to recognize a primary
antibody.
29. A. Immunofluorescence can be used on tissue sections, cultured cell lines, or
individual cells, and may be used to analyse the distribution of proteins and small
biological and nonbiological molecules.
B. Immunofluorescence can be used in combination with other, non-antibody
methods of fluorescent staining, for example, use of DAPI, (4',6-diamidino-2-
phenylindole) is a fluorescent stain that binds strongly to A-T rich regions in
DNA, to label DNA.
C. The technique has a number of different biological applications including
evaluation of cells in suspension, cultured cells, tissue, beads.
D. It also play a key role in the diagnosis of autoimmune disorder.
30. 1. PCR was invented in 1984 by
the American biochemist kary Mullis at Cetus
Corporation.
2. Polymerase chain reaction (PCR) is a method
widely used to rapidly make millions to billions of
copies of a specific DNA sample, allowing
scientists to take a very small sample of DNA and
amplify it to a large enough amount to study in
detail.
3. It is fundamental to much of genetic testing
including analysis of ancient samples of DNA and
identification of infectious agents.
4. The majority of PCR methods rely on thermal
cycling.
.
32. 1. Identification and characterization of infectious agents--
Direct detection of microorganisms in patient specimens
Identification of microorganisms grown in culture
Detection of antimicrobial resistance
Investigation of strain relatedness of a pathogen of interest
2. Genetic fingerprinting (forensic application/paternity testing).
3. Detection of mutation ( investigation of genetic diseases).
4. Cloning genes.
5. PCR sequencing.
