Following is my journal documentation during Master's in Biotechnology completed in 2015. I do understand many changes would've occurred in the curriculum since then, but the basics seldom change. Kindly absorb as per your need.

1. JOURNAL INDEX
PSBTP201:Metabolomics
Ex. ExperimentTitle
No.
Detection ofPhenylalanine for PKU by spot test
and chromatography done with standard
Phenylalanine
Staff Member's
Pg.
No.
Date
Signature
1
-4 6 2014
Isolation of chloroplast by sucrose density gradient
centrifugation 5-10 13 22014
1-12 1o1|2014 e
Uric acid estimation from serum/urine
Determination of acid number and iodine number
13-16 1 |2014
/2/2014
4
of lipids
PSBTP202: Clinical Immunology
Ex. Experiment Title Pg Date Staff Member's
Signature
17-21 2/3|2014 ulye
22-23 17|2014 ye
24-26 23)2014 de
No. No.
Perform Western Blotting
Perform DOT-BLOT for protein
Demonstration practical - Flow cytometry
Demonstration practical - ELISA reader
24-31 P3/2014
PSBTP203:Genomics&MolecularBiology
Ex. ExperimentTitle
No.
Pg.
No.
StaffMember's
Signature
Date
Plasmid Isolation
32-3321/22014 N
Extraction of genomic DNA from bacteria &
blood 3431eblao14
Expression of recombinant protein
38-40 2422o14|
PSBTP204: Advanced AnalyticalTechnique
Ex. ExperimentTitle Date Staff Member's
Pg.
No.
No. Signature
PCR amplification
41-46 5/320l4
Validation of autoclave, micropipette, colorimeter,
spectrophotometer and measuring cylinder 47-59 131220
Calibration of pH meter and weighing balance
60-62 3 2014

2. SEMESTERII
PSBTP202
CCINICALC
1MMUNOLOGY

3. Experiment No. 1 Date: 2/3 2014
WESTERN BLOTTING
AIM
To perform western blotting technique
THEORY
Western blotting is a powerful technique used in the protein detection&
characterization. This technique exploits the inherent specificity of antigen & antibody
interaction to identify specific antigen by polyclonal or monoclonal antibody.
The technique involves following steps:
1. Separation ofproteins using SDS-PAGE.
2. Transfer of proteins from SDS-gel to a nitrocellulose membrane (electric transfer).
3. Binding of primary antibody on the membrane.
4. Use of secondary antibody (this antibody is an antibody-enzyme conjugate, e.g
horseradish peroxidase).
5. Reaction with a substrate.
SDS-PAGE technique is used to separate molecules based on size, shape or
isoelectric point. SDS-PAGE coupled with western blotting is typically used to determine
the presence and/or relative abundance of a given protein.
The gel is a cross linked polymer matrix used to support & separate molecules. The
gel density can be controlled by varying the monomer concentration. Gel can be of
constant density or they can be variable i.e. gradient gels. After cross linking has taken
place, the samples are loaded in wells in the gel & the assembly undergoes
electrophoresis. Electrophoresis involves applying an electric current to the gel and
allowing the proteins to migrate through the matrix. In order for proteins to migrate
through the gel, they are first denatured & negatively charged by exposure to detergent
such as sodium dodecyl sulphate (SDS). The amount of bound SDS is relative to the size
of protein, and proteins have similar charge to mass ratio. Bands in different lanes
separate based on the individual component sizes. A molecular weight marker that
produces bands ofknown size is used to help identify
proteins of interest.
After the protein components have been separated by electrophoresis, they can
be
transferred to a PVDF or nitrocellulose membrane. The transfer process uses the same
principle as SDS-PAGE but this time the electric current is applied at 90 degrees to the
gel & proteins migrate out of the gel onto the membrane. Once the proteins are separated
&bound to membrane support, immunoblottingcan begin.
Western blotting is used to detect a target protein in a sample of mixture of
proteins by using a polyclonal or monoclonal antibody specific to the protein.
1 7 ~

4. Gel
cassete
amilten
Anode
bufer
springe
egative
electrode
Step
1:Lepan
ation
Sample
chamber
Anode
(+)
-Cathode
(3
roteins
by
PA6E
pnasns
Power
source
Separate
pnon
bamds
Tank
Transfer
buffer
Cathode
(
Filter
paper
Gel
Step
2
hansjet
o
Nitroceliulose
membrane
-
frotcins
from
gel
Filter
péper
-
Membrane
Anode
(+)
do
Membh.anewith
ranufeeved
protein
if
proteins
are
hydirophobic.
use
PVDF
membrane
instead.

5. PRINCIPLE
Western blotting is a rapid & sensitive assay for detection & characterization of
proteins. This technique exploits the inherent specificity of antigen-antibody interaction
to identify specific antigens by polyclonal or monoclonal antibodies.
The proteins are separated by SDS-PAGE. The gel is placed in contact with
nitrocellulose membrane. The transferred proteins are detected using immunological
reagents. The membrane is then probed with a primary antibody specific to the protein of
interest. The antigen-antibody complex formed on the membrane which is then identified
using an enzyme labelled secondary antibody & a suitable substrate to the enzyme which
results in a coloured band on the nitrocellulose membrane.
APPROACH
Firstly, the proteins are separated by carrying out SDS-PAGE. Blotting is transfer
ofresolved proteins from the gel onto a surface ofa suitable membrane carried out
electrophoretically, referred to as Electroblotting. The gel is placed in contact with
nitrocellulose membrane which is then sandwiched between filter paper, sponge in a
cassette. The entire set is electrophoretically run using the blotting buffer. The proteins
get transferred to the corresponding position on the membrane as resolved on the gel
forming a mirror image of the gel.
The transferred proteins bound to the surface of the nitrocellulose membrane are
detected using immunological reagents. This is known as Immunodetedion. All the
unoccupied sites on the membrane are first blocked with an inert protein, a detergent or
any other suitable blocking agent. The membrane is then probed with a primary antibody
specific to the protein of interest. The antigen-antibody complex formed on the membrane
which is then identified using an enzyme labelled secondary antibody & a suitable
substrate to the enzyme which results in a coloured band on the nitrocellulose membrane.
PROCEDURE
Blocking: The membrane is blocked in order to reduce non-specific protein interactions
between the membrane and the antibody. This is achieved by placing the membrane in a
solution of bovine serum albumin (BSA) or non-fat dry milk (NFDM).
Primary Antibody: The first antibody to be applied (specific for protein of interest) 1s
incubated with the membrane. The antibody is diluted in a buffer solution (PBS)
containing a carrier protein (BSA or NFDM) along with some detergent. The primary
antibody is specific for protein ofinterest&at appropriate concentrations should not bind
to any other protein on the membrane. The membrane is then rinsed.
Secondary Antibody: After rinsingthe membrane to remove unbound primary antibody, a
secondary antibody is incubated with the membrane. It binds to the primary antibody.
This secondary antibody is typically linked to an enzyme that allows for visual
identification by producing fluorescence. An alternative is to use radioactive label.
Developing: The unbound secondary antibody is washed away & the enzyme substrate is
incubated with the membrane so that the positions of membrane bound secondary
18

6. Horseradish
peroxidase
conjugated
secondary
antibodies
Primary
antibody
Slep
3:
hepasation
f
TBST
Blocking
buffer
Wash
buffer
T8ST
membiane
fos
Membrane
Visuali3ation
(with
transferredd
proteins)
Incubate
blots
2
Incubate
blots
hours
at
room
Membrane
overnight
at
4
C
(ready
to
be
visualized)
temperature

7. antibodies will emit light. Bands corresponding to the detected protein of interest appear
as dark regions on the developed film. Band densities in different lanes can be compared
providing information on relative abundance of protein of interest.
REQUIREMENTS
SDS-PAGE Solutions:
1. Stock Acrylamide solution:
Acrylamide 30 gm
Bis-Acrylamide 0.08 gm
Water 100 ml
2. 2X Sample buffer:
Tris-HCl, ph 8.0
Glycerol
130mM
20% (v/v)
4.6% (w/v)
SDS
0.02%
Bromophenol blue
3. 8X Resolving gel buffer: 100 ml
0.8 gm (add last)
36.3 gm
SDS
Tris base (3M)
Adjust pH to 8.9 with conc. HCl.
4X Stacking gel buffer: 100 ml
4. 0.4 gm (add last)
6.05 gm
SDS
Tris base (0.5 M)
Adjust pH to 6.8
10X Running buffer:1 litre
Tris base (0.25 M)
5. 30.3 gm
144 gm
Glycine
SDS (1% add last)
Do not adjust pH.
10 gm
Western blotting Solutions:
10X Blotting buffer: 1 litre
Tris base (0.25 M)
Glycine (1.92 M)
pH should be 8.3, do not adjust.
To make 2 Litre of 1X Blotting
1. 30.3 gm
144 gm
buffer:
400 ml Methanol
200 ml 10X Blotting buffer
1400 ml water
2. Blocking
buffer: 0.5 litre
3% bovine serum
albumin Make up in PBS & Sterile
filter. Then add 0.05% Tween 20.
Keep at 4 degree
Celsius to prevent
bacterial
contamination.
~19~

8. slsiigiitiisll
200
140
30
20
9261
1:1000
9165
1:1000
2579
1:1000
2679
1:1000
2855
1:1000
Weten 8lotinq- AdohadiogAph

9. 3. Phosphate-Buffered Saline, ph 7.4, 1000ml
8gm
0.2 gm
1.44 gm
0.24 gm
Adjust pH to 7.4 with HC1
NaCl
KCI
Na2HP04
KH2P04
4. Phosphate -Free, Azide-Free Blocking Solution, 1000 ml
150 mM NaC1 8.766 gm
6.0657 gm
50 mM Tris-HCl (ph 7.5)
5% (w/v) nonfat dried milk
Adjust pH with 12N HCI
PROTOCOL
SDS-PAGE
A. Preparation of Gel:
1. Assemble the glass plates & spacers (1.5mm thick).
2. Pour an agarose plug (1-2mm)
3. Pour the running gel to about 1 cm below the wells of the comb (approx 20 ml).
4. Seal with 1 ml water saturated 1-butanol.
5. When gel has set, pour off the butanol &rinse with deionized water.
6. Pour the stacking gel (approx 5 ml) and insert the comb immediately.
7. When the stacking gel has set, place in gel rig and immerse in buffer.
8. Prior to running the gel, flush the wells out thoroughly with running buffer.
B. Running the Gel:
1. After flash spinning the samples, load into wells.
2. Be sure to use markers. Run with constant current (30 mA).
C. Preparation of membrane:
1. Cut a piece ofPVDFmembrane/nitrocellulose membrane.
2. Wet for about 10 mins in Ix blotting buffer on a rocker at room temp.
D. Membrane transfer:
1. Assemble "sandwhich" for transblot.
2. Pre-wet the sponges, filter papers (slightly bigger than gel) in lx blotting
buffer. Sponge-filterpaper-gel-membrane-filterpaper-sponge.
3. Transfer for 1 hour at 34 mA.
Bigger proteins might take long for transfer.
4. When finished, immerse membrane in blocking buffer & block overnight.
E. Antibodies & Detection:
1. Incubate with primary antibody diluted in blocking buffer for 60 min at RT
2. Wash 3x lo min with 0.05% Tween in PBS.
3. Incubate with secondary antibody diluted in blocking buffer for 45 min at RT.
20

10. 4. Wash 3x 10 mins with 0.05% Tween 20 in PBS.
5. Incubate with TMB H202 for 10-15 mins.
RESULT
CONCLUSIONS The technigue of Weptenn Blotting was demanstraded
21

11. Experiment No. 2 Date: 17/12014
DOT-BLOT FOR PROTEIN
AIM
To perform the DOT blot technique for the separation of proteins.
THEORY
DOT-BLOT is a technique for detecting analysing and identifying proteins, similar to
the western blot technique but differing in that proteins samples are not separated by
electrophoresis but are spotted through circular templates directly onto the membrane or
paper substrate. Concentration of proteins in crude preparation (such as culture supernatant)
can be estimated semi quantitatively by using DOT BLOT method if both purified protein
and the specific antibody against it is available.
PRINCIPLE
Dot blot is a simple, convenient method for detection of proteins in crude lysates or
solution without the need for separation by electrophoresis. This method is especially useful
as a simple control because it eliminates problems encountered in Western blotting like
components that interfere with binding or non-specific binding, improper separation of the
proteins and signal interference.
Nitrogen cellulose is type of membrane most commonly used for protein blotting due
to its high binding capacity and low background staining. Nitrocellulose membrane are
however rather brittle and many nylon membranes have been promoted for protein blotting
on the strength of their high flexibility and greater binding capacity. However, problems with
background staining make them less advantageous. Among the various brands of
nitrocellulose available today, Schleicher & Schuell's BA 85, pore size 0.45 um shows
consistently first-rate quality, although it is somewhat more brittle than a similar product
available from Millipore. Millipore has a polyvinyldifluoridene membrane, Immobilon
PVDF, which combines great mechanical strength with high binding capacity and low
background, whereas its wetting properties are less favourable.
APPROACH
The protein sample is spotted on a nitrocellulose membrane and detected by mixing it
With a primary antibody which is further treated with a secondary antibody conjugated with
an enzyme. On addition of the enzyme specific substrate colour development takes place
indicating the presence of the protein, the intensity of the colour depending on the
concentration ofthe protein present.
REQUIREMENTS
a) TBS (20mM Tris-HCI ,150 mM NaCL,pH7.5)
b) TBS-T (0,05% Tween 20 in TBS)
c) BSA/TBS-T (0.1%BSA in TBS-T)
22

12. hotorol
Take Iml o IX ASsay bufel and 5ul eadh oantigeng 1 and 2
in tuo &cponate vialb ix dhohoughly by invenhing
Jns ent the ELISA Bip on in eadh Vial and incubate
at RT tor 20 mins wlh intelmitent haking
Wash dhe sthips 3 times by dipping hem in imt ol
Ix fresh washing bufek for about 3 min(Replace
dhe buffek aftel each Wabh)
Il o Assay bufe in a hesh vial, add
Syl
anthb ody HRP Coojugate and mix thoeoughly
Dip the 6ips sepatately into ials onrtaunung
enzume soluhion and allow the Leation to take
plate for 20 minb with intemettent&haking
LWash 4he Siips n
Lwashing buffe o0 Atep 4
(lhnce)
me+H, 02 60uthioo by dliluching, 100d o 10x substvate
solurbion Lwith 90ul DW Dip the sbps Sepanalcly
nto Vlals Contaunin4 soletion and leaue it for
minb Loith hking
Stop Ahe heaction by washing he ips in D
and obseAue the
heaction for 8ue colowed 3pot5

13. d) Nitrocellulose membrane (BIO-RAD, Trans-Blot, etc)
e) Protein sample
PROCEDURE
1 Have nitrocellulose membrane ready drow grid by pencile to indicate the region to be
blotted.
Used narrow mouth pipette tip, spot 2 micro liter of sample onto the nitrocellulose
membrane at the center of the grid, minimize the area that the solution penetrates
(usually 3-4 diameter) by applying it slowly.
Let the membrane dry.
4
2.
3.
Block nonspecific sites by soaking in 5%BSA in TBS-T(0.5-1 hr,RT). Use 10 cm
petri dish for reaction chamber.
Incubate with primary antibody (0.1-10 microgram/ml for purified antibody, 1:1000
to 1:100000 dilution for antis era, 1:100 to 1:10000 for hybridoma supernatant)
5.
dissolved in BSA/TBS-T for 30 mines at RT.
6. Wash three times with TBS-T(3x 5 min)
Incubate with secondary antibody conjugated with HRP (for optimum dilution) for 30
7
min at RT.
8 Wash three times with TBS-T (15 minx 1,5min x2), then once with TBS (5 min)
9 Incubate with ECL reagent for 1 min, cover with saran wrap (remove excessive
solution from the surface), and expose X-ray film in the dark room. Try several
different lengths of exposure.
10. Compare the signal from unknown sample to that of the standard and estimate the
concentration.
RESULTS: esence opot jn the positive zone and no spot in
heqative Z0ne Js an indiuation o prope petoemance olest
Absence Spot in tebt z0ne jpditates that antigen ointexest
is absent in qiveh 6ample
CONCLUSTONS omnle 1 howed no (oloued Spot Which n dilales
that the sample does not haue the antiqen bound to
irmrnobilised anibgdy
whetal dhcltest ofSample 2 showed the ptebeneea 4pot
in
clikepting dhe pesence oantiqen bound to imo abilised
23

14. Kesut
Barmple 1
Larople 2
one
Negatiive
Test
fosithive
Neqative - Negaive
Jut pot Jat (Spot)
Posit've PositVe,
Sauiple Soupe
1 2

15. Experiment No. 3 Date: 2/3/2014
FLOW CYTOMETRY
AIM
To perform Flow Cytometry
THEORY
Flow cytometry is a technique for counting, examining and sorting microscopic
particles suspended in a stream of fluid. It allows simultaneous multiparametric analysis of
the physical and/ or chemical characteristic of the single cell flowing through an optical and
electronic detection apparatus.
PRINCIPLE
A beam of light( usually laser light) is directed towards onto hydrodynamically
focused stream of fluid a number of detectors are aimed at the point where the stream passes
though the light beam, one in line with the light beam forward scatter( FSC) and several
perpendicular to it [Side scatter (SSC)] and one or more florescent detector each suspended
particle passing through the beam scatters the lightin some way, and fluorescent chemicals
found in the particle or attached to the particle may be excited into emitting light of higher
wavelength than the light source. This combination of scattered and fluorescent light picked
up by the detectors and by analyzing fluctuation in brightness at each detector ( one each
fluorescent peak) it is then possible to derive various type of information abqut the physical
and chemical structure of each individual particle FSC correlates with the cell volume and
SSC depends on the inner complexity of the particle ( i.e. shape of the nucleus ,amount and
type of cytoplasmic granules or the membrane roughness)some flow cytometers in the market
have eliminated the need for fluorescence and use of only light scatter for measurement.
Other flow cytometers form images of the cell's fluorescence scattered light and transmitted
light.
CONSTRUCTION
Modern flow cytometers are able to analyze several thousand particles every second
in "real time" and can actively separate and isolate particles having specified properties. a
flow cytometry is similar to a microscope except that instead of producing an image of the
cell, flow cytometer provides a "high throughput" (for a large number of cells) automation
quantification of set parameters. To analyze solid tissues single cell suspension must first be
prepared.
A flow cytometerhas five main components
1. A flow cell- liquid stream (sheath fluid) carries and align the cell so that they pass single
through the light beam for sensing
2. A light source- commonly used are lamps( mercury, xenon); high power
water cooled
laser(argon krypton, dye laser); low powered air cooled lasers [argon (488 nm), red HeNe
(633 nm), green
HeNe ,
HeCd (U V); diode laser ( blue, green, red, violet).
3. A detector and a analogue to digital
conversion system- generating FSC and SSC as a
fluorescence signal.
2 24

16. Flow Cytometer Scheme
Laser
Forward scatter detector
Mirrors
Side scatter detector
Source of liquids
to keep the flow
Waste container
Direction offluid flow
Tube with cells or other analysed particles
(a) Phinciple of Flow aytomcthy
Fluidics system
Filters Detectors
635 nm
4 8 8 n m
Lens
PMT
FL-1)
Lasers
PMT
FL-2
Filters
PMT
FL-3
FL-
Screen
Detector
6)Dedection ystem in a low aytontheh
suojp010p
J0uO

17. 4. An amplification system- linear or logarithmic.
5. A computer for analysis of the signals.
SPECIMEN ANALYSIS
Specimen Preparation
Specimens are often whole blood but may be other tissues. For analysis, the specimen must
contain greater than 95%% single cells for accurate interpretation of the data. Whole blood is a
single cell suspension, but tissues must be disaggregated into single cells before analysis.
Often antibodies are used to stain cells and select subpopulations or evaluate protein
exDression. In most cases, these antibodies are commercial products with robust staining
Droperties. However, the staining pattens should be confirmed in the laboratory on each new
lot of antibody using appropriate control materials. Unknown antibodies must be carefully
evaluated for specificity and efficacy of staining. This is best done under a microscope
initially because nonspecific staining or unusual patterns can be better distinguished with a
microscope than with a flow cytometer. Once approximate staining characteristics are
determined, the procedure can be fine-tuned for the flow cytometer.
Data Processing
The initial light signal is first converted by the detectors into an electric current, which is
amplified and measured as an analog quantity, such as 5 V of a maximum of 10 V. Because
this signal is analog, it must be converted into a digital signal for computer processing. The
signal then becomes an event with a specific digital channel value such 512 on a 1,024-
channel scale. The digital data can be displayed as 1-parameter histograms or as 2-parameter
The information on a l-parameter histogram is described either as the percentage of
plots
cells within a set of markers or as the mean fluorescence intensity of a population. For
logarithmically amplified signals, the geometric mean fluorescence is used, since it is less
influenced by the extreme ends of the distribution. parameter plot is usually divided into 4
quadrants, each containing
distinguish between fluorescent and non fluorescent cells. It also defines expression and non
expression of a cell molecule marked by a fluorescent antibody or other fluorochrome.
Because the specimen that is analyzed may be complex with several populations plus debris,
Such as lysed whole blood, analysis of the total specimen would give variable and confusing
results. Therefore, it is often necessary to analyze a single population, such as lymphocytes.
Electronic gating allows separation of the total population into parts, which can be
individually analyzed.
percentage ofthe total population. This division is used to

18. 8.1%
FL1-H3)s FL2-H(4)
45 5%
1 FL1-H(3) vs FL2-H(4
10
21 1%
0
8% 25.6%
25.6%
0.8%
44 5
FL1-H3)vsFL2-H(4)
IUngatedtotal particl
Ga
10 10
FL1-H
U
0.f
28.1%%
FSC-H(1)vsSSC-H(2)
FL1-H (3) vs FL2-H (4)
24 %
0 %
Granulocytes
:Monocytes
11
Debris
65 %
Lymphocytes
023
0
10
RESULT
CONCLUsION The tecbrdque oHouw utomethuy Lwab &tudied
dheonchjcalli
26

19. Experiment No. 4
Date: 2/3 2014
PREPARATION OF ELISA PLATE
AIM
To perfornm Enzyme Linked Immunosorbent Assay (ELISA)
THEORY
Enzyme Linked Immuno Sorbent Assay also called ELISA, is a biochemical
technique used mainly in immunology to detect the presence of an antibody or an antigen in a
sample. It has been used as a diagnostic tool in medicine and plant pathology, as well as a
quality control check in various industries. In simple terms, in ELISA an unknown amount of
antigen is affixed to a surface, and then a specific antibody is washed over the surface so that
it can bind to the antigen. This antibody is linked is linked to an enzyme, and in the final step
a substance is added that the enzyme can convert to some detectable signal. Thus in the case
of fluorescence ELISA, when light of the appropriate wavelength is shone upon the sample,
any antigen/antibody complexes will fluoresce so that the amount of antigen in the sample
can be inferred through the magnitude of the fluorescence.
Types of ELISA
1. "Indirect"ELISA: In this the antigen to be tested is coated on a microtiter plate. A
primary antibody specific for this antigen is then added which could be serum of the
patient to be tested. A secondary antibody labeled with an enzyme is added which
binds to the primary antibody. Substrate which gives a coloured product on enzyme
interaction is added.
2. Sandwich ELISA: This technique is used to detect sample antigen. A surface is
prepared to which a known quantity of capture antibody is bound. The antigen-
containing sample is applied to the plate. A specific antibody is added that binds to
antigen. Enzyme-linked secondary antibodies are applied as detection antibodies. A
substrate is added to be converted by the enzyme into a color or fluorescent signal
which can be measured.
3. CompetitiveELISA: Unlabeled antibody is incubated in the presence of its antigen.
These bound antibody/antigen complexes are then added to an antigen-coated well.
The more antigen in the sample, the less antibody will be able to bind to the antigen
in the well, hence "competition". A secondary antibody, specific to the primary
antibody, is added. This second antibody is coupled to the enzyme. A substrate is
added, and remaining enzymes elicit a chromogenic or fluorescent signal.
ELISA can be performed to evaluate either the presence of antigen or the presence of
antibody in a sample and hence is a useful tool for determining serum antibody
concentrations eg. HIV test. It can also be used to detect potential food allergens. ELISA can
also be used in toxicology as a rapid presumptive screen for certain classes of drugs. ELISA
test can also be used to detect various diseases, such as malaria, Chagas disease, and Johne's
disease. ELISA tests also are used as in in vitro diagnostics in medical laboratories.
27

20. ) Indirect ELSA
wash wash
wash
Add enzyme
conjugated
secondary
antibody
Add substrate (S)
Antigen
coated well
Add speciic
antibody to be
measured
and measure
color
b) Sandwich ELSA
wash
wash
wash
Add substrate
Add enzyme
conjugated
secondary antibody
Addantigen
Antibody-
coated well and measure
to be measured
color
(O Competitve ELISA
Y wash
wash
Incubate
antibody with
antigen to be
Add substrate
Add Ag-Ab Add enzyme
and measure
Conjugated
secondary
measured
mixture to
color
antigen-coated well
antibodv
() ypes of ELISA dechniques
()
(6) ELISA mionotitre plate

21. PRINCIPLE
As a heterogenous assay, ELISA separates some component of the analytical
reaction mixture by adsorbing certain components onto a solid phase which is physically
immobilized. In ELISA, a liquid sample is added onto a stationary solid phase with special
binding properties and is followed by multiple liquid reagents that are sequentially added,
incubated and washed followed by some optical change (e.g. color development by the
product of an enzymatic reaction) in the final liquid in the well from which the quantity of
the analyte is measured. The qualitative "reading" usually based on detection of intensity of
transmitted light by spectrophotometry, which involves quantitation of transmission of
some specific wavelength of light through the liquid. The sensitivity of detection depends
on amplification of the signal during the analytic reactions. Since enzyme reactions are very
well known amplification processes, the signal is generated by enzymes which are linked to
the detection reagents in fixed proportions to allow accurate quantification -
thus the name
"enzyme linked".
The analyte is also called the ligand because it will specifically bind or ligate to a
detection reagent, thus ELISA falls under the bigger category of ligand binding assays. The
ligand-specific binding reagent is "immobilized", i.e., usually coated and dried onto the
transparent bottom and sometimes also side wall of a well, which is usually constructed as a
multiple-well plate known as the "ELISA plate". Conventionally, like other forms of
immunoassays, the specificity of antigen-antibody type reaction is used because it is easy to
raise an antibody specifically against an antigen in bulk as a reagent. Alternatively, if the
analyte itself is an antibody, its target antigen can be used as the binding reagent.
Dilution scheme for various solutions
1IX PBST (phosphate buffer saline-tween)
Take Iml of 10X PBST provided in kit and distilled water upto 10 ml
a.
2. Dilution buffer
To 1ml of blocking buffer, add 29ml of 1X PBST to make final volume
30ml. Use the solution for dilution ofantigen and antigen HRP conjugates.
a.
Dilution of standard antigen
3
The concentration ofantigen provided in kit is 1000 ul/ml
a.

22. b. Dilution table
Final
Designation of Antigen Dilution buffer Totalvolume concentration
dilution (ul) (ul) (ul)
(uI)/ml)
From stock 20 480 500 40
From A -200 800 1000 8
From B-500 500 1000
From C-500 S00 1000
E From D-500 500 1000
From E-500 500 1000 0.5
From F-500 500 1000 0.25
From G-500 500 1000 0.125
I Dilution of test sample
Dilute the 5 micro lit of sample in Iml of dilution buffer
IXTMB/H202
To 0.6 ml of 10XTMB in 5.4ml of distilled water
I.
1X stop solution
To 12 ml 5X stop solution add 48 ml of distilled water
III.
PROCEDURE
A. Coating of antibody on wall of microtitre plate
1. Concentration of reconstituted antibody of coating is 0.Img/ml, dilute to
Imicrogram/ml with coating buffer i.e. 50 ul of sample in 4.95 ml of coating
buffer.
2. Pipette 200 ul of diluted antibody into each well of micro titre plate allow it
spread eventually.
3. Incubate the 96 well micro titre plate at 4 Covernight.
B. Blocking residual binding site
1. Discard the content. Rinse with distilled water three times
2 Fill each well with 200 ul of blocking buffer and incubate at room temperature for
3. Rinse with distilled water three times drain out the water completely by aping on
blotting paper
1 hour.
C. Addition of antigen to the well
1. Add 100 ul ofdiluted sample as shown in table along with PBST
Dilute 1000X HRP-conjugate to 1X using diluent i.e. 3 ul ofstock in 3 ml diluents
1
D. Addition of HRP-conjugate antigen
solution.
Add 100 ul of 1XHRP-conjugate
to each well
29

23. Discard the content. rinse with distilled water three times
E. Addition of substrate and measurement of absorbance
1. Add 200 ul of 1X TMB/H202 to each well
2. Incubate at room temp for 10 minute
3. Add 100 ul of 1X stop solution to each well
4. Transfer content of each well into individual tube containing 2ml of 1X stop
solution
5. Prepare substrate blank by adding 200ul of 1X TMB/HO2 in 2.1 ml of stop
solution. Take O.D. at 450 nm.
F. Preparation of blank
Add 100 ul of 1X substrate and 2.1ml of 1X stop solution to a test
tube Measure O.D at 450nm
RESULTs
cONCLUSIONS Reparation o ELI6A plate was shudied theoNeically
30

24. Experiment No. 5 Date: 2132014
ELISA READER
AIM
Demonstration of ELISA
THEORY
Enzyme Linked Immuno Sorbent Assay also called ELISA. Enzyme Immuno Assay
or ElA is a biochemical technique used mainly in immunology to detect the presence of an
antibody or an antigen in a sample. The ELISA has been used as a diagnostic tool in
medicine and plant pathology, as well as a quality control check in various industries. In
simple tems, in ELSA an unknown amount of antigen is affixed to a surface, and then a
specific antibody is washed over the surface so that it can bind to the antigen. This antibody
is linked is linked to an enzyme, and in the final step a substance is added that the enzyme
can convert to some detectable signal. Thus in the case offluorescence ELISA, when light of
the appropriate wavelength is shone upon the sample, any antigen/antibody complexes will
fluoresce so that the amount of antigen in the sample can be inferred through the magnitude
ofthe fluorescence.
PRINCIPLE
Performing an ELISA involves atleast one antibody with specificity for a particular
antigen. The sample with an unknown amount of antigen is immobilized on a solid support
i.e on microtiter plate either non-specifically or specifically. After the antigen is immobilized
the detection antibody is added, forming a complex with the antigen. The detection antibody
can be convalently linked to an enzyme, or can itself be detected by a secondary antibody
which is linked to an enzyme through biconjugation Between each step the plate is typically
washed with a mild detergent solution to remove any proteins or antibodies that are not
specifically bound. After the final wash step the plate is developed by adding an enzymatic
substrate to produce a visible signal, which indicates the quantity ofantigen in the sample.
ELISA READER
Main features
-Flexibility and precision in microplate strip washing, Top loading: provides easy
handling and operation, Low noise pump, Aspiration Time: continuous aspiration
reduces risk ofwell to well overflow and cross, Compact size
RESULTs
CONCLUSIONS he technigpue of ElISA Lwas demonsirated