Nouman Shoukat

2014-ag-4040

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Roll no. 807

Sec: E1

MED 404

Submitted to: Dr. Ali Raza

 

 

 

 

 

 

 

Principle and Procedure of different
microbiological tests for virus detection

ELISA

It is enzyme-linked immunosorbent assay.

Principle: If antigen-antibody reaction is
specific, enzymes conjugated with antibodies will utilize the substrate and
produce a color.

Components of ELISA

·    Solid phase: ELISA plates have charged surface which serves as an attachment for antigen or antibody.

·      Antibodies:  these are of 2 types
(1) Against antigen, may or may not be enzyme conjugated (2) anti species
antibodies, always enzyme conjugated.

·      Enzymes: Commonly
used enzymes are

1.  Horse reddish peroxidase

2.  Alkaline phosphatase

3.  Beta D- galactosidase

4.  Urease                                                                       

·      Substrate: It is chosen according to enzyme. They are of 4 types 

1.  ABTS (blue color)

2.  Di amino benzidine (dark brown)

3.  Tetra methyl benzidine(blue)

4.  P- nitrophenyl phosphate (yellow 

·      ELISA reader: It is an advanced form of spectrophotometer.

Procedure

Direct ELISA

·     Coat unknown antigen (before adding substrate after
every step washing is done)

·     Add blocking agent to avoid any nonspecific
agent

·     Add enzyme conjugated antibodies

·     Add substrate

·     Reading of result from ELISA reader

Disadvantage: All antigens attach so anyone of
these can give nonspecific reaction.

Sandwich ELISA

 

·     Coat the surface with known antibodies

·     Add blocking agent

·     Add sample antigen(unknown)

·     Add enzyme conjugated antibodies

·     Add substrate

·     Reading

Advantage: It increases specificity, quality and
precision of overall result.

 

Indirect ELISA (antigen
detection)

·    Add known antibodies

·    Add blocking agent

·     Add unknown antigen sample

·     Add specific antibodies against antigen

·     Add anti species antibodies

·     Add substrate

·     Reading

We are using 2 antibodies against the same antigen.
Antibodies should be from different species.

Indirect ELISA (antibody detection)

·    There is known antigen adsorbed on
well surface

·     Add blocking agent

·     Add unknown antibodies(serum)

·    Add enzyme conjugated anti species
antibodies

·     Add substrate

·     Reading

If reaction is positive, enzyme conjugated anti species
antibodies will attach to antibodies otherwise these will not attach.

 

 

PCR

It is Polymerase chain reaction

Principle: It amplifies a specific region of DNA strand. PCR methods
amplify DNA fragments of between 0.1 and 40 kilo base pairs (kbp). The amount
of product amplified depends on the availability of substrates in the reaction.

Components and reagents:

·       DNA template

·       DNA polymerase (Taq polymerase). It
polymerizes new DNA strands.

·       2 DNA primers complementary to the 3′
ends of the sense and anti-sense strands of the DNA target. Without Primer
there is no initiation site for polymerase binding because DNA polymerase can
only bind to and elongate from a double stranded region of DNA.

·       dNTPs (deoxynucleoside
triphosphates).

·       Buffer solution

·       Mg or Mn ions (bivalent cations)

·       K ions (monovalent cations)

The reaction is carried out in a thermal cycler

Procedure: PCR consists of 20-40 repeated
temperature changes called, cycles. Each
cycle consists of 2 or 3 temperature steps. After cycling there is a single
temperature step at more than 90 Degree Celsius followed by one hold at the end
for final product extension

Steps

·    Initialization: Only required for DNA polymerases that require heat activation by
hot-start PCR. In this reaction chamber is heated at 94-96 Degree Celsius and
then is held for 1-10 minutes

·    Denaturation: In this reaction chamber is breaks
the hydrogen bonds between complementary bases, yielding 2 single stranded DNA
molecules.

·    Annealing: Reaction temperature is lowered to
50-65 Degree Celsius for 20-40 seconds. It anneals the primers to each of the
single stranded DNA templates. 2 different primers are included in the reaction
mixture. The primers are themselves single stranded but are much shorter than
the target region. Determining the temperature is very important in annealing. Temperature must be high enough for hybridization to be
specific and low enough to allow for hybridization

·    Extension: Here the temperature for Taq
polymerase is 75-80 degree Celsius (72 Degree common). In this step DNA
polymerase adds free dNTPs from the reaction mixture that are complementary to
the template in the 5′ to 3′ direction thus it synthesizes a new DNA strand
complementary to the template DNA strand. The time required depends both on the
length of the DNA target region to amplify and on the DNA polymerase used.
Under ideal conditions the number of target DNA sequences is doubled at each
extension step.

The processes of
extension, annealing and denaturation constitute a single cycle. Multiple
cycles are required. The formula for calculating number of DNA copies after a
given number of cycles is 2n, where n is the number of cycles.

·   

Final hold: Cooling of reaction chamber to 4-15 Degree Celsius for an
indefinite time.

 

 

 

CFT

It is complement
fixation test

Principle:

                        
It is an immunological test to detect the presence of specific antigen
or antibody in the serum of patient.

Materials:

·       Sheep erythrocytes suspension

·       Hemolysin (rabbit anti sheep red cell
antibody)

·       Guinea pig complement (without
antibodies)

·       Barbital buffer diluents

·       Plastic microtiter plate

·       Water bath

·       Color standards for hemolysis judging

 

Procedure:

Steps

First step (complement
fixation stage):

A known antigen and patient’s serum (inactivated) are
incubated with limited amount of complement (Serum of patient is heated for 30
minutes at 56 Degree Celsius to inactivate endogenous complement)

Ø The complement will be activated by
the antigen-antibody complex if the serum contains specific complement
activating antibody.

Ø If no antibody in serum, no formation
of antigen-antibody complex and complement not fixed and will remain free.

Second step (Indicator
stage):

It detects whether the complement was utilized in the first
step or not (done by adding the indicator system)

Ø If the complement was fixed in the
first step now there will be no complement left to fix to the indicator system.
No lysis of RBCs

Ø If the complement was not fixed in
the first step, now the complement will be present free or unfixed. This free
complement will react with the antibody coated sheep RBCs to bring about lysis.

Results:

 
Positive CFT            
Presence of antibody in serum                    No lysis of sheep RBCs

   Negative CFT            Absence of antibody in serum           Lysis of sheep RBCs

 

 

 

 

 

 

 

 

HAI

It is hemagglutination
inhibition test.

Principle: Nucleic acids of certain viruses have
surface proteins that agglutinate the RBC of variety of species.

For example: Influenza virus has an envelope protein called
the hemagglutinin (HA) which binds to RBCs and thus forms a lattice. This
property is called hemagglutination. Reaction of hemagglutinins of virus with
RBCs results in a lattice of agglutinated cells which settle in a microtiter well.
Un-agglutinated cells settle in a compact button.

HAI Titer: Highest dilution of serum
(antibodies) that prevents hemagglutination.

The sera should be treated before use or false positive or
negative results may arise because HAI test may be complicated by the presence
of non-specific inhibitors of viral hemagglutination and naturally occurring
agglutinins of RBCs.

 

 

 

 

 

 

 

 

 

Materials and reagents:

·      
RBCs
from an appropriate species (guinea pig, chicken, goose) collected in heparin.

·      
Diluent
(Bovine albumin veronal buffer) at an appropriate pH.

·      
Solutions
for removing non-specific hemagglutinins.

·      
Infected
cultural fluid or standard antigen.

Procedure:

Get a virus preparation (e.g. Influenza virus with known HA
titer or find its HA titer.

·      
Prepare
2-fold dilution of test serum to be tested e.g. from 1:4 to 1:1024.

·      
Add
a determined amount of virus to each well of a 96-well plate equal to 4 HA
units (depends on virus), except for the serum control wells.

·      
The
plate is put at room temperature for 60 minutes (varies according to specific
requirements)

·      
Add
RBCs and incubate at 40 Degree Celsius for 30 minutes.

·      
Read
wells.

Results:

           A smooth shield of cells or an irregular button
indicates agglutination. Observe the movement of the button of RBCs when the
plate is tilted. It helps to clarify the end point.

Virus sample has an HAI titer of 1280, which means the
greatest dilution of antibody that still blocked hemagglutination was at 1280
dilution. At this dilution antibodies were still capable of binding to antigens
on the virus and recognizing them.

 

Latex agglutination test

Principle:

                 It is observed when a sample having
the specific antigen or antibody is mixed with an antigen or antibody which is
coated on the latex particles surface.

Latex particles:

                           The procedure which is applied for
the preparation of latex particles is Emulsion Polymerization. First the
styrene is mixed with the surfactant solution which is sodium dodecyl sulfate, forms
billions of uniform emulsified micelles. Then a small amount of potassium
persulfate is added which is water soluble polymerization initiator. When
polymerization is finished, polystyrene chains are arranged into the micelles.
Hydrocarbon part of chain attaches to the center and terminal sulfate ion to
the surface of spheres which is exposed to the water phase.

Examples of hydrocarbons used for latex particles production
are: polyvinyl toluene, styrene vinyl toluene, styrene-dlvinyl benzene.

Materials:

·       Microcentrifuge

·       Glycine saline buffer

·       Blocking buffer

·       Vials (1.5 ml)

·       Microtips

·       Pipette

·       Refrigerator

·       Latex beads

·       Glass slides

·       Beaker

·       Antigen for coating

·       Antiserum

·       Tooth pick

Procedure:

Latex coating:

·      
Add
40 µl of glycine saline buffer to 20 µl of latex beads taken in 1.5 ml of vial.

·      
Add
60 µl of antigen to latex.

·      
Incubate
at 37 Degree Celsius for 2 hours.

·      
Spin
at 5000 rpm for 10 minutes.

·      
Aspirate
the supernatant.

·      
Resuspend
the pellet in 1 ml of blocking buffer.

·      
Spin
at 5000 rpm for 10 minutes.

·      
Repeat
washing.

·      
Add
90 µl of blocking buffer to the pellet and mix.

·      
Incubate
at 40 Degree Celsius for night.

Agglutination Test:

·      
Add
4 µl of test antisera (diluted 50 times) to 200 µl pf glycine saline buffer
taken in a vial.

·      
Add
50 µl of antigen to 50 µl of diluted antiserum in 1.5 ml vial.

·      
Mix
well.

·      
Incubate
at 37 Degree Celsius for 10 minutes.

·      
Pipette
10 µl of latex (coated) onto glass slides.

·      
Add
10 µl of diluted test antiserum to A slide.

·      
Add
10 µl of antiserum (mixed with antigen) to B.

·      
Add
10 µl of glycine saline buffer to C.

·      
Mix
contents in each slide with tooth pick (use separate tooth picks for each
slide).

·      
Wait
for 2 minutes and observe result.

Result:

·      
Presence
of white clumps       Positive       Suspended particle present.

·      
Absence of
white clumps       Negative      Suspended particles absent.

Glycine saline buffer
(0.5 M):

·      
Dissolve
14 g glycine, 0.7 g sodium hydroxide, 17 g sodium chloride, 1 g sodium azide in
500 ml distilled water and adjust 8.6 ph. Make volume up to 1000 ml with
distilled water.

 

1.          
RAI

It is radio immune
assay.

Principle:

                It measures the presence of an
antigen of very high sensitivity. It is an in vitro method. Any biological
substance can be measured for which a specific antibody exists, even in small
concentrations.

It analyzes the picomolar and Nano-molar concentrations of
hormones in biological fluids.

Substances and
equipment:

·      
Specific
antiserum to the antigen which is to be measured

·      
Radioactive
labeled form of the antigen

·      
A
method to separate unbound tracer from antibody bound tracer

·      
Radioactivity
counting instrument

Procedure:

                  Target antigen is radioactively
labeled and bound to its particular antibodies (a known limited specific
antibody should be added). Blood serum taken as sample is then added to
initiate a competitive reaction of the antigens (labeled) from the preparation,
and the antigens (unlabeled) from serum samples, with particular antibodies. Competition
for the antibodies will emit a certain amount of antigen (labeled). This amount
is proportional to the labeled unlabeled ratio of antigen. Then a binding curve
can be generated which allows the antigen in the serum of patient to be
derived.

As the unlabeled antigen concentration is increased, more of
it binds to the antibody, displacing the labeled variant. The unbound antigens
are then separated from the bound antigens and the radioactivity of the free
antigens left in the supernatant is measured.

Radioactivity:

 Usually applied are 125-I labels although H3 and C14 are also used.

Separation techniques:

Bound
and free labeled antigen are separated by following techniques:

Ø Double antibody technique (most
common combined with polyethylene)

Ø Chromatography

Ø Cellulose technique

Ø Charcoal technique

Ø Solid phase techniques

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

IFA

It is Immunofluorescence
antibody technique.

Principle:

                         
It is a technique that uses fluorescent dyes to recognize the presence
of antibodies bound to specific antigens.

An
individual who is exposed to infectious agents, in that individual IFA is very
beneficial to find the serological response.

Types

Primary (Direct):

               It uses a primary antibody (single)
which is linked to a fluorophore. The primary antibody identifies the antigen
and binds to it’s epitope. The attached fluorophore can be identified by
fluorescent microscopy. Depending upon the messenger used, a light of specific
wavelength will be emitted.

It is less
common.

Secondary (Indirect): 

                 Two antibodies are used in this. The
primary antibody is unlabeled and it binds to the target molecule and secondary
antibody (carrying the fluorophore) identifies the primary antibody and bind
it. It is possible that multiple secondary binds a single primary antibody.

 

 

Western blotting

Principle:

                          It is a technique
used for the analysis of individual proteins present in a mixture. The protein
mixture is applied in a gel electrophoresis in a matrix. That will sort the
proteins by charge, size or other differences. The protein bands which are
separated are then transferred to a carrier membrane (PVDF, nylon or
nitrocellulose). This process is referred to as blotting. The proteins get
adhered to the membrane similarly as they are separated due to interaction of
charges. The proteins are then detectable for antibody binding.

Procedure:

Types of possible gel
electrophoresis for proteins

·       SDS-PAGE

·       Native PAGE

·       Isoelectric focusing

 

Blotting:

                      
The polypeptide bands are transferred to the membrane carrier after the
proteins are separated. The membrane is attached to a gel and this sandwich
type is transferred to an electrophoresis chamber. It is a possibility that
protein re-natures again. The electric charge causes the proteins to travel out
from the gel to the direction they traveled in on the gel, onto the membrane.
In this protein bands get bound to the membrane.

Immunodetection:

The specific antibodies are identified only after the
proteins are separated and blotted. Specific antibodies bind to their band of proteins.
We can remove un specifically binding antibodies by washing with buffers
(detergent containing). First primary antibodies are applied and then these are
detected by a secondary antibody. The secondary antibody is color or enzyme
conjugated for detection. Polyclonal antibodies have an advantage that these
can recognize several epitopes.

 

 

Virus isolation

For
isolation of virus we can use:

·       Cultured cells

·       Eggs

·       Laboratory animals

In most
laboratories Cultured cells is the only option for isolation of virus.

For the
preparation of cell cultures collagenase or trypsin first dissociates the
tissue fragments. Then the suspension of cells is placed in a glass container
(test tube, bottle, petri dish) with a liquid medium. After a variable lag
cells will give rise to a primary culture. Solid support attachment is
essential for normal cells.

Primary and secondary cultures:

If we change
the fluid 2 or 3 times a week, the primary cultures will be maintained. EDTA or
trypsin detaches the cells from the vessels when the cultures become over
crowded, and the portions are used to start the secondary cultures. The cells
retain some of the characteristics of the derived tissue.

Cell strains and cell lines:

From primary
cultures, cells can be transferred a number of times. The cells then continue
to divide at a constant rate for many successive transfers. A time reaches when
the cells cannot be transfer anymore.

Cell cultures:

There are 3
types of cell cultures:

1.  
Primary cells: Prepared from human or animal tissues
directly and can be cultured once or twice.

2.  
Semi-continuous diploid cells: Derived from human fetal tissue.

3.  
Continuous cells: Derived from animal or human tissue.

Cell cultures have different susceptibility for different viruses. When
you take the sample for cell culture, transport it to the laboratory as soon as
possible. Tissues and body fluids must be put in a sterile container.

When received, depending upon the clinical presentation and specimen
nature, the sample is inoculated into many different types of cell cultures.
After 1 hour, change the maintenance media. Incubate the tubes at 35 to 37
Degree Celsius in a rotating drum.

The inoculated tubes should be read every next day for CPE. Urine and
feces might be toxic to some cell cultures. We need to passage the inoculated
cells if toxic effects are extensive. Pass the cell cultures through bacterial
filter if contaminated with bacteria. Keep the cell cultures for 1 or 2 weeks.
Regularly re feed the maintenance medium in cell cultures.

 

 

Virus neutralization

Principle:

                         When the virus is reacted with a
specific antibody, there is a loss of infectivity.

 

Reversible neutralization:

If we dilute the antigen-antibody mixture within 30 minutes of formation
of antigen-antibody complexes, the neutralization process can be reversed. It
is due to the interference with the attachment of virions to the cellular
receptors.

Stable neutralization:

As the time goes on (several hours), Ag-Ab complexes become stable and
then the process cannot be reversed. Antibodies and virions do not change
completely in stable neutralization so, the unchanged components can be
recovered. Reactivation of neutralized virus can be possible by proteolytic
cleavage. For stable neutralization, smaller number of antibody molecules are
required. A virion can even be neutralized by a single antibody molecule.
Neutralization of polio viruses is the example of stable neutralization.

We must standardize the known
components which are to be used before carrying out neutralization test. A
known (pretitred) antiserum should be used. If we are to titrate a known virus
tenfold serial dilution of the isolate is prepared and then it is inoculated
into s host system such as animal or cell culture. The reciprocal of the
highest dilution that infects 50% of the system of host will be the endpoint titer
of virus.