A

SUMMER TRAINING REPORT

ON
LABORATORY TECHNIQUES IN
PREPARATION
OF
TESTOSTERONE-ELISA KIT

SUBMITTED TO
MR. ANIL KUMAR
(For the partial fulfillment of M.Sc. Biotechnology Degree)

SUBMITTED BY
RAVI CHOLIA
ROLL NO. 0709216
M.Sc. Biotechnology

Department of Bio & Nanotechnology

GURU JAMBSHEWARE UNIVERSITY
OF SCIENCE &TECHNOLOGY
HISAR
2007-2009

1
 PREFACE

Practical training is one of the major components for any

professional courses like
Biotechnology. The professional place where a
biotechnologist implements theoretical
Knowledge is a floor of workshop of any industry. With out a
practical knowledge we are worthless.

Being a MSc. Biotechnology student, I took a similar kind of

training at Orbit Biotech. Pvt. Ltd. Mohali. I learnt many a
things over there. I gained a practical knowledge about the
ELISA-KIT development for Testosterone. I was familiar with
the working environment. Overall it was a great experience
for me. This experience will be of immense help to me in my
forthcoming future.

2
 CONTENTS

Sr.no. Particulars Page no.

1 Acknowledgement 4
2 Certificate 5
3 Certificate 6
4 Abbreviations 7
5 Introduction 9
6 ELISA Test & Types 12
7 Preparation of 13
buffers
8 Coating 15
9 Immunization of 17
rabbit
10 Conjugation of 20
hapten with
enzyme (HRP)
11 Characterization of 22
antiserum
12 Ouchterlony 24
double diffusion
test (ODD)
13 Checkerboard 25
assay
14 Fine tuning 28
15 Preparation of 30
testosterone

3
 standards
16 Preparation of 32
standard curve
17 Specificity assay 34
18 Affinity of an 36
antibody with its
antigen
19 Antibody capture 38
assay
20 Protein purification 40
21 conclusion 42

ACKNOWLEDGEMENT

It is my pleasure to take this opportunity to thank all

who helped me directly or indirectly in preparation of this
SUMMER TRAINING REPORT. The blessing of almighty
god, my parents and my elders have enable me to reach
such a standard that I got a chance to do something good
myself.

I am grateful to Dr. J. S. Rana, chairman of the bio &

nanotechnology department for providing a big moral
support. I must pay my gratitude to Mr. Anil Kumar for his
kind support and assurance, I got from him.

I must convey special thanks to Mr. Sudesh Kumar,

instructor of Orbit Biotech Pvt. Lmt. Mohali. Under whose
guidance I completed my summer training successfully. I am
always thankful to Miss Derothy & Jaspreet. I am also
grateful to others for their great guidance and moral support.

4
 The help provided by these personalities have aided a
lot preparation of this summer training report. So, I once
again convey my heartiest thanks to all of them.

RAVI CHOLIA
M.Sc. Biotechnology
Roll no. 0709216
Deptt. Of Bio &
Nanotechnology
GJUS&T
HISAR

GURU JAMBESHWARE
UNIVERSITY
OF SCIENCE & TECHNOLOGY
HISAR

CERTIFICATE

5
 It is certified that Mr. RAVI CHOLIA, a
student of M.Sc. biotechnology, Roll No. 0709216,
session 2007-2009 in my deppt. He is a bonafide
student of my deppt. He has done on the report named
“LABORATORY TECHNIQUES IN PREPARATION OF
TESTOSTERONE-ELISA KIT” from Orbit Biotech Pvt.
Lmt. MOHALI under my supervision. He has worked
well. I wish he get all success in her life.

MR. ANIL KUMAR

(Major Advisor)

Deptt. Of Bio &

Nanotechnology
GJUS&T
HISAR

6
 ABBREVIATIONS

• Ab Antibody

• Ag Antigen

• ARGG Anti Rabbit Gamma Globulin

7
• ANS 8- Amilino Naphthalene sulfonic acid

• BSA Bovine Serum Albumin

• BSB Blocking and Stabilizing Buffer

• 0
C Degree centigrade

• DHT Dihydrotestosterone

• DMF N,N-Dimethylformaimde

• DEAE Diethyl amino ethyl

• EDAC 1-ethyl-3,3-diaminepropylcarbodiamide

• HRP Horse Radish Peroxidase

• Ig Immunoglobin

• NHS N-Hydroxy succinimide

• NRGG Normal Rabbit Gamma Globin

• O.D. Optical density

• PBS Phosphate Buffer Saline

• RT Room temperature

• Rpm Revolution per minute

8
 • SB Serum Buffer

• TMB Tetramethylbenzidine

• TST Testosterone

• v/v volume/volume

• WB Washing Buffer

INTRODUCTION

In clinical endocrinology, measurement of hormonal level in different body especially in

blood and urine has been a general practice to evaluate indirectly the function status of an endocrine
gland. The information thus obtained are extremely helpful in the management of apparent disease
states caused by under production and over production of certain hormones by the respective gland.

9
 In the human male, testosterone is the major biologically active steroid into the blood
stream by the leyding cells of the testis. The ovary in the female also produces certain amount of
testosterone, but the quantitative different observed in the circulating level of the hormone are
responsible for maintaining the sexual characteristics of the male distinct from the female. Any
observations in these levels will result in path physiological conditions in both the sexes.

In order to examine the state of this biologically active compound, testosterone and
its relationship with some of the clinical symptoms both in men and women. It is imperative to know
the intracellular concentrations of the hormone.

The body’s immune system provides a smart form of defense against disease. It
identifies antigens. Proteins known as antibodies seek out intruders and destroy them. Antibodies are
clever weapons each binding to a particular antigen. Moreover the specificity of the antibody reaction
can be exploited in wide variety of illness.

With the introduction of radioimmunoassay by Yalow and Berson (1975), the

principle of antigen-antibody reaction has been utilized for developing Radioimmunoassay for number
of hormones.

The coupling of enzymes to the antibodies or antigen les to the development of alternative
labels for detect and measurement of testosterone hormones. Enzymes appear to be more versatile and
promising tracers as the catalytic properties and high turnover no. of enzymes allow quantization of
extremely small quantities of analytes.

Homologous ELISA is using for developing Antigen capture assay. Principle of this
ELISA is utilizing antibody development against testosterone-3-(carboxyl methyl) oxime –BSA and
some derivative labeled with horse radish peroxidase (HRP). Microtitre wells coated anti-rabbit
gamma globulin (20 antibody or ARGG) followed by the primary antibody (anti-testosterone IgG)
provide the solid phase. The enzyme labeled antigen is added in enough quantity to saturate the
binding sites on the antibody. In the presence of serum or std., the enzyme labeled testosterone
competes for binding sites on the antibodies with the testosterone present in the serum or std. this
gives a sose dependent relationship, thus allowing for the construction of a standard. Curve formed
from which testosterone unknown samples can be calculated.

The presents study aims at the indigenous development of Reagents for such a
competitive enzyme immunoassay by----

1. Characterization of polyclonal antibody rose against testosterone in Rabbits.

2. Preparation of enzyme labeled testosterone
3. Development of Enzymes Linked Immunosorbent Assay (ELISA) for testosterone

General information about Immunology

Immunsystem:-

Defense system to protect animals from invading pathogenic microorganisms. It is able to

generate an enormous variety of cells and molecules capable of specific recognizing and eliminating
an apparently limitless variety of foreign invaders.

10
 Functionally, immune response----1.Recognition
2. Response.

Once a foreign organism has been recognized, the immune system recruits a variety of cells
and molecule to mount an appropriate response, called an effectors response. Later exposure to the
same foreign organism induces a memory response.

Immunity – 1. Innate

2. Adaptive.

Antigen:-

Any foreign particle which induces immune response ; substances that can be recognized by
the immunoglobins receptors.

Antibody:-

Produced in response to the antigen and binds with them .

Adjuvant:-

Substances that when mixed with an antigen and injected with it, enhances the
immunogenicity of that antigen .

Hapten:-

These are small organic molecules that are antigenic but not immunogenic. Chemical
coupling of a hapten to a large protein called a carrier yields an immunogenic hapten-carrier
conjugate.

Immunogenicity:-

It is the ability to induce an immune response.

Antigenicity:-

It is the ability to combine specifically with the final product of the response.

Serum:-

Fluid derived from coagulated blood after removal of the clot and components.

11
 Antiserum:-

Serum from an animal containing antibodies reacting with particulate antigens . Sometimes
known as an immune serum .

Plasma:-

Fluid obtained from uncoagulated blood after removal of cellular components. Differs from
serum in containing all the components of the circulation system . Its preparation necessitates the use
of an anticoagulants, e.g. heparin, citrate

Microtitre plate:-

Plastic plate containing many (usually 96) wells in which many types of immunoassay may
be carried out more conveniently than in individual tubes . The wells may be flat bottomed for use in
ELISA, U-shaped for use in RIA or V-shaped for haemagglutination tests. They may flexible or rigid.

ELISA
(Enzyme linked Immunosorbent Assay)

To detect the concentration of analyte (Ag or Abs)

ELISA has three types-

12
 1. Indirect ELISA
2. Sandwich ELISA
3. Competitive ELISA

Indirect ELISA

In indirect Elisa we first coat the antigen on the microtitre well then wash it with tab water to
remove the unbound antigen. Then add the secondary antibodies in it , these antibodies binds with the
antigen already coated on the microtitre well. Then wash it again to remove the unbound antibodies
from it. Then we add enzyme labeled antibodies in it, then again washing with water to remove the
unbound labeled antibodies. Then we add substrate in it .the substrate – enzyme reaction gives colour,
then we take reading on spectrophotometer.

Sandwich ELISA

In sandwich ELISA we first add antibodies in the microtitre well then wash it with water to
remove the unbound antibodies .then we add in it antigen and wash then add enzyme labeled
antibodies in it then wash and then add substrate to it. After coming of colour take readings.

Competitive ELISA

Competitive ELISA has two variant –

In one we first add antigen and in other we add antibody. Next all the procedure is same.

For smaller size analyte we can’t do indirect & sandwich Elisa.

Mainly we use enzyme HRP (Horse Radish Peroxidase). It is cheaper, easily available &
highly stable and the substrate for the HRP is TMB (Tetra methyl Benzedine), also cheaper and easily
available.

There is also another enzyme we use:-

HRP, alkaline phosphatase, β-D- galactosidase, Glucose oxidase, glucoamylase, β- lactamase

.

PREPARATION OF BUFFERS

10mM PO -4 buffer: -
(pH 7.1-7.2)

For 1L

3.3ml of stock A (0.5 M NaH2PO4.2H20) + 6.7 ml of stock B (0.5 M Na2HPO-4 )

13
 Add a pinch of thymersol and xentamycin
Now add distill water to make 1L.

Wash buffer: -

For 500 ml

250 ml of 50mM PO4 buffer + 4.5g NaCl + 25mg thimersal (antibacterial) + 1000ul Tween
20 + 250 ml of distill water

Phosphate buffer saline (PBS)

250 ml of 10 mM PO4 buffer + 0.9 % NaCl

Mix and store

PBS with 0.1 % BSA:-

PBS + 0.1 5 BSA

(Store at 40 C)

Instruction:-

 BSA is temperature sensitive.

 Don’t shake vigorously.
 If frothing occurs, 1st settle down it then use.
 Before use take out the BSA at room temperature for 15 min.

Blocking and Stabilizing Buffer:-

10mM PO-4 buffer + .1 % BSA + 2 % sucrose + 2.5 % lactose + 2.5 % manitol + one pinch of
thimersal and one pinch of n- prophygallate.

Serum buffer (45 ml)

Composition

15 ml of Charcoal stripped filter serum

30 ml of PBS
200 ug of Gentamycein
10 mg of thymersol
45 ug of 8-amino-napthaline Sulphonic acid (ANS)
112 ng of estradiol

14
 45 mg of BSA

First dissolve the ANS into the PBS buffer; it takes about ½ hr to 1 hr to dissolve in the PBS.
In last add BSA.

Calculations:-

Gentamycein stock = 5 mg/2ml

= 5000/ 2000 ul
We need only 200ul so,
For 5000ug-----------------------= 2000ul
For 1 ug-------------------------- = 2000/5000
For 200 ug----------------------- = 2000/5000 * 200

= 80 ul

Estradiol stock = 1ug /2ml

= 1000ng/ 2000ul
We need 112 ng only
For 1000ng ---------------------- = 2000ul
For 1 ng -------------------------- = 2000/1000
For 112 ng ----------------------- = 2000/1000 * 112

= 224 ul

COATING OF ANTIBODIES
(Coated wells ----for characterization of antiserum)

Three types

1. Direct: - In direct we directly add antibodies

2. Indirect:-In indirect we first add NRS then ARGG
3. Covalent: - In covalent first add activator then add NRS and then ARGG

15
 NRS- normal rabbit serum
ARGG-Antirabbit gamma globulin

Direct coating
Strips coated by this method is called Ag-coated strips

Procedure

 take 4 polystyrene strips added 0.5 ug/200ul of test-3-CMO-BSA in eachwell. Stock

solution is 1mg/ml.
 Kept at room temperature for 2 hr. and incubated at 370c for 24hr.
 Add 250ul of blocking and stabilization buffer per well.
 Incubated at 370c for 2 hr.
 Decant off blocking and stabilization buffer and strips placed in air dryer for
overnight in inverted position
 Strips are then incubated at 370c for 2hr.
 Strips packed in zipper bag along with 2 silica bags.

Indirect coating

Called ARGG coated strips; in this we directly add the antibodies and incubate it in incubator
at 370C for 24 hr. then on next day decant off the content.

Procedure

 Take the strip

 Add 200ul of NRS/well. Keep for 2 hrs. at room temperature and then incubate at 370C for 24
hrs. in the incubator and then decant off the content on next day.
 Wash the strip with tap water /distill. Water 4-5 times. After flicking the wells, keep them
inverted on the absorbent paper for the 15 min.
 Then add 200ul of ARGG (15ug/200ul). Keep at room temp. For 2 hrs. And then incubate at
370C for 24 hrs. in the incubator
 Next day decant off the content and wash with tab water for 4-5 times and let the wells dry
for 15 min.
 Then add 250 ul of blocking and stabilizing buffer and incubate the strip for 2 hrs.
 Decant off the content and dry it and place in air drier for 24 hrs.
 Then pack the strips in zipper bag and also add 2-3 bags of silica bags.

COVALENT COATING

Procedure

 Take 1 plate (96 wells).

16
 Add 0.2% glutraldehyde 50 ul / well using 25% stock; it acts as cross linker.
 Add NRS (IgG) 1:75, 50 ul /well using the stock 1:1; for different dilutions of
glutraldehyde & NRS we use 10mM PO-4 buffer.
 Leave for 2hrs. in air
 Incubate overnight at 370C in incubator.
 Wash with tap water & invert for 15 min.
 Then add ARGG (incubate overnight)
 Then washing and tapping.
 Add 100 ul of antibody (incubate for 24 hrs )
 Add 150 ul of blocking and stabilizing buffer and incubate for 2hrs and decant off the
content.
 Place inverted in air dryer for overnight.
 Then pack the strips in the zipper bag and store at 20C to 80C.

IMMUNIZATION OF RABBIT

AIM: - To produce antibodies.

Immunogen: - Which evoke the immune response

17
 Hapten + Carrier = Immunogen

Testosterone + BSA = 2mg dissolve in min. amount of NaOH (it is steroid can’t dissolve
in distill water). Here testosterone act as hapten and BSA is a carrier, both after conjugation form
immunogen.

Adjuvant: - Increase the exposer time by making droplets. And start the immune
response at primary stage.

In order to develop polyclonal antibodies, it is necessary to

a). Choose the type of animal to be used for immunization.

b). Use directly immunogen (direct or after coupling with BSA) / or prepare immunogen.
c). Select the immunization procedure to be adopted.

Choice of animals:

Rabbit are the best choice of animal for laboratory setup, since handling of
animals is easier and the volume of blood that can be collected ranges from 20-40 ml per bleed. It
is also easier to collect blood from ear vein of rabbit with ease and speed. The foreign nature of
the immunogen determines the choice of animal.

Immunogen:

Small molecular weight compound (less than 1000) need to be coupled to bovine serum
albumin before being used as an immunogen. Protein above 10,000 molecular weight can be used
directly for immunogen; the immunogen is dissolved fresh in isotonic saline (0.9 % sodium
chloride). Concentration of immunogen will vary, this being 2mg/ml for BSA coupled compounds
whereas proteins are used 100-200 ug/injection. Prior to injection, the immunogen in saline is
mixed with Freund’s complete adjuvant (1:1). The emulsion is best prepared in the injection
syringe itself. By taking the mixture into the syringe and rapidly expelling it 20-30times, a stable
emulsion is formed.

Immunization Procedure:

It appears that each laboratory and each investigator has their own injection schedule. the
production of high titre, high affinity antibodies are as much an art, with some luck involved, as it
is a science.
IMMUNIZATION WITH A HAPTEN- BSA CONJUGATE

Rabbit 2 to 3 months old are generally used for immunization with hapten-BSA conjugate.
Preferably, these are immunized in the morning hours after overnight fasting.

Material Required

18
 1. Standard rabbit box
2. Electric animal clippers
3. Xylene or toluene
4. Vacutainer needle
5. 10cc syringe with 22g needle
6. Rectified sprit
7. Cotton
8. Ice and bucket
9. 50 ml beaker

Collection of control serum

Prior to immunization of the rabbit, control serum is collected by inserting a vacutainer

needle into the ear vein of the rabbit. Swabbing the ear with cotton soaked in the xylene/toluene and
tapping the vein gently dilates the ear vein, making needle insertation easy. The blood is collected in a
small beaker and kept at room temperature for 1 hr. The sides and top of the blood are mildly
disturbed with the spatula or a rod, so that the serum gets separated at the top. After the serum
separates into a layer, it is aspired with a Pasteur pipette into a centrifuge tube and centrifuged at 1500
xg for 15 minutes. The serum , thus, separated is aliquot into vials, stoppered and stored at -200c . This
serum is used as control.

Primary injection

Hapten-BSA conjugate 2 mg/ml, prepared and emulsified (fresh) with complete Freund’s
adjuvant is drawn into a 2 ml syringe with 19g’’ needle and injected into the thigh muscles of hind
legs and front legs of the rabbit in four equal portions.

The following schedule is followed for the primary injections:

Day

1- 2mg immunogen with complete Freund’s adjuvant intramuscular

14- Same as above
21- Same as above

Booster injections

Booster injections are given intravenously in isotonic saline or intramuscularly in incomplete

Freund’s adjuvant plus saline in 1:1 proportion.

19
Week

6 – Intravenous administration of 0.5mg/0.2ml saline

7 – Collect 2-3ml blood from ear vein. Separate serum and test the serum antibody
development.
12 – Intravenous administration of hapten-BSA 0.5 mg/0.2ml saline.
13 – Bleeding and testing for antibody development

NOTE:

 For monoclonal antibodies production we use mice.

 For polyclonal antibodies production we use rabbit.

 Antibodies produced is stored at -200 C or 40C

CONJUGATION OF HAPTEN WITH HRP

Hapten: - Low molecular weight, less immunogenic compounds.

20
 Hapten is Testosterone having molecular weight – 288 i.e. < 5000.

Methods for the conjugation:-

1. Carbodiamide method
2. Periodate method
3. Gutraldehyde method.

All these chemicals in these methods requires cross linking agents.

Carbodaimide method:-

In this method cross linking agent is 1-Ethyl-3,3-Diamine Propyl Carbadimide (EDAC).

It is called zero length cross linking agent as there is no formation of bridge. All other
methods are with 1, 2, 3……..length; depends on the no of Carbon atom presents.
It is used to convert hapten to some active group. There is formation of imide’s bond.

Material required

1. Sucrose
2. Ammonium sulphate
3. 1-Ethyl-3(3-DimethylaminePropyl) Carbadimide (EDAC)
4. Hapten (testosterone)
5. Horseradish peroxidase
6. Dioxane
7. Dimethyl formamide
8. N-hydroxy/ succinimide
9. 10mM Phosphate buffer pH 7.0
10. Ethyl glycol
11. Bovine serum albumin
12. Sephadex G25 or G-75

Procedure:-

 Take testosterone 5 mg in vial 1st and dissolve in 100 ul of dioxane and 100ul of
Dimethyl Formamide (DMF). Take EDAC 200mg and N- hydroxysucunamide
(NHS) 10 mg in vial no 2. And dissolve in minimum amount of distilled water (app.
200ul).
 Then add the content of vial no 2 into the vial no 1 and keep for 2hrs. at room temp.
With occasionally shaking.
 Then take HRP-3mg in vial no 3 and dissolve in 15 mM PO-4 in minimum amount
and add the content of vial no 1 into the vial no 3. Store the vial in the freezer over
night.
 After this we get in vial unbound testosterone and unbound HRP and bound T-HRP.

The molecular weights of these are:

21
 Testosterone = 299 Daltons

HRP = ~44000 Daltons

T-HRP = ~44288 Daltons

To separate the T- HRP from unbound testosterone and unbound HRP . We perform Gel
filtration chromatography and separate the T-HRP and HRP from the testosterone .and HRP is
separated from the T-HRP by washing solution as HRP alone can’t binds to wells.

GEL FILTRATION CHROMATOGRAPHY

We can separate T-HRP from unbound testosterone by using the technique gel filtration
chromatography.
This technique based on the molecular size and shape exploits the molecular sieve property of
porous materials.
1st of all a column of glass is filled 2/3 with sephadex G 25 gel and wash the gel with PBS
buffer. Then this porous gel is equilibrated with the mobile phase for the analytes to be separated.
Large analyte that are completely excluded from the pores will pass through the interstitial spaces
between the gel particles and will appear in the elute first. Gel should not dry during the whole
procedure.

As the T-HRP is large in size than the testosterone, so are easily separated.

CHERACTERISATION OF ANTISERUM

An antiserum for being used in an immunoassay should satisfy the following

requirements:

22
 1. Titre
2. Specificity
3. Affinity for binding the antigen

ANTIBODY TITER ASSAY

It is a quantitative test. The concentration of antibody is often expressed as titre. This is the
dilution of antiserum used in the assay tube or microwell and should be able to bind 30-70% of
enzyme labeled antigen. Titre of the antibody is estimated by incubating progressive dilutions of the
antiserum with a fixed amount of the labeled antigen. At increasing dilution of the antibody, the
binding decreases and antibody is saturated at a particular point, part of antigen will appear as
unbound. Usually the antiserum dilution that binds 50% of the added antigen is taken as the titre for
the assay.

Material required

1. Antiserum (Anti-testosterone antibody)

2. Secondary antibody coated on microwell plate
3. Testosterone-HRP
4. 10 mM PBS assay buffer having BSA and thimersol
5. Dispensers 100ul, 1000ul
6. Dispenser tips
7. Distilled water
8. TMB/H2O2 (20x)
9. 0.5 M H2SO4
10. Crude testosterone antiserum is used for making serial dilution with the assay buffer

Stocks:

Antibody stock: - 1:50

T-HRP -: 1:1000
Dilute using PBS with 0.1% BSA buffer

Table:

23
 ARGG coated Dilutions T-HRP O.D Mean
wells (add 100 ul/well) (add at 450 nm
100 ul/well)
A 1:1,000 1:20,000 0.325 0.283
B -do- -do- 0.241
C 1:2,000 -do- 0.230 0.258
D -do- -do- 0.286
E 1:4,000 -do- 0.231 0.224
F -do- -do- 0.218
G 1:8,000 -do- 0.191 0.165
H -do- -do- 0.139

S
R

Procedure

 Add analyte according to the table.

 Incubate for 1 hr. in incubator and wash with washing buffer.

 Then add substrate 1x and then again incubate for 15 min. in incubator at 370C.

 Then add stop solution in it to stop the reaction.

 Then finally take the reading at 450 nm.

Draw a graph between the O.D. & concentration of antibodies in the dilutions. Taking O.D on
the x-axis & dilution on the y-axis.

RESULT

The titre concentration of antibody in the given crude serum comes out to be
1:40,000.

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OUCHTERLONY DOUBLE DIFFUSION TEST (ODD)

This is a Qualitative test only. This can be used for testing titre and specificity of antibody.
This test is named after the inventor Ouchterlony.

Used for testing titer and specificity of antibody.

Materials used

Beaker-50 ml, glass rod , Petri dishes, well borer, antiserum (20 antibody or ARGG),
Normal rabbit gamma globulin(NRGG), 10mM PO-4 buffer saline , sodium azide 0.3%

Procedure

 Add agrose to 50 ml of 10mM PO-4 buffer containing 0.15M saline. Sodium azide is
added to the buffer to block the bacterial growth.
 Heat the solution with stirring to boiling point for 4-5 min. or more to get a clear solution.
 Pour the hot solution in Petri dishes up to half mark avoiding formation of air bubbles
and having uniform thickness (4-5mm).
 Let the agrose cool and get as a thick gel. Using a glass borer or plastic tip- bore wells at
the centre and sides, clean the well properly.
 Prepare normal rabbit gamma globulin (NRGG). Make antiserum in 1:10, 1:20, 1:50, and
1:100 dilutions with PO-4 buffer.
 Place 100ul of NRGG in the central well and 100 ul each of four dilutions into side wells.
 Keep the Petri dish over wet cotton or filter paper in a big dish cover with aluminum foil
at 40C in the refrigerator for 48 hrs.
 Precipitin lines are formed between antigen and antibodies, the intensity of which will
vary with the titre of the antibody.

RESULTS

Precipitin lines is formed between antigen and antibody.

25
` CHECKER BOARD ASSAY

Used to determine the concentration of antibody .

In this we use 4 different concentration of anti testosterone (0.25ug/100ul, 0.5 ug/100ul, 0.75
ug/100ul, and 1.0ug/100ul)

Procedure:

Make different dilutions and add according to the table.

Table:

Sr. no. Antibody concentration Dilutions T-HRP OD

of ARGG (in ug/100ul) (100ul /well) (100ul/well)
coated wells

1 0.25 1:20,000 1:20000 0.329

2 -do- 1:40,000 -do- 0.323
3 -do- 1:60,000 -do- 0.311
4 -do- 1:80,000 -do- 0.280
5 0.50 1:20,000 -do- 0.324
6 -do- 1:40,000 -do- 0.382
7 -do- 1:60,000 -do- 0.262
8 -do- 1:80,000 -do- 0.245
9 0.75 1:20,000 -do- 0.317
10 -do- 1:40,000 -do- 0.230
11 -do- 1:60,000 -do- 0.235
12 -do- 1:80,000 -do- 0.199
13 1.0 1:20,000 -do- 0.289
14 -do- 1:40,000 -do- 0.224
15 -do- 1:60,000 -do- 0.202
16 -do- 1:80,000 -do- 0.217

 Then incubate for 1 hr. at 370C.

26
  Then wash with 1x wash buffer.

 Then add 1x substrate 100ul/well.

 Then incubate at 370C for 15min. in incubator for colour development.

 Then add stop solution (0.5 M H2SO4) 100u/well to stop the reaction and then take the
readings at 450 nm.

Calculations

For antibody stock = 1mg/1ml

= 1000ug/1000ul
= 1ug/1ul

For 0.25 ug/100ul:-

1/1 * V1 = 0.25/100 * 5000

= 12.50ul of antibody stock solution

And add 4987.50 ul of 10mM PBS buffer with 1% BSA to make the total volume 5000ul.

For 0.50ug/100ul:-

1/1 * V1 = 0.50/100 * 3000

= 15.0 ul of stock antibody solution

And add 2985.0 ul of 10mM PBS buffer with 1% BSA to make the total volume 3000ul.

For 0.75ug/100ul:-

1/1 * V1 = 0.75/100 * 3000

= 22.50ul of stock antibody solution

And add 2977.50ul of 10mM PBS buffer with 1% BSA to make the total volume 3000ul.

For 1ug/100ml:-

1/1 * V1 = 1/100 * 3000

= 30ul of stock antibody solution
And add 2970 ul of 10mM PBS buffer with 1% BSA to make the total volume 3000ul.

27
For T-HRP stock = 1:1000

Make dilution = 1: 20,000

1/1000 * V1 = 1/20000 * 1000

= 50 ul of stock antibody solution

And add 950 ul of 10mM PBS buffer with 1% BSA to make the total volume 1000ul.

The result of this assay will confirm the concentration of antibody and enzyme conjugate
required for optimizing the assay.

RESULT

Antibody dilution 1.0ug/100ul is found to be useful antibody coated strips.

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 FINE TUNNING

AIM: - This assay is used to find out the exact concentration of T-HRP.

Procedure:

Make different dilutions and add according to the table.

Table:

Anti testosterone T- HRP Readings at Mean

coated wells (stock 1:1000) 450nm
Add 100 ul/well
A 1:5,000 >3.00
B 1:5,000 >3.00
C 1:10,000 >3.00
D 1:10,000 >3.00
E 1:20,000 >3.00
F 1:20,000 2.824
G 1:40,000 2.676 2.450
( i.e.
1:40,000)
H 1:40,000 2.224

 Then 1 hr incubation in the incubator at 370C

 Then wash with the wash buffer (1x)

 Then add substrate 100ul / well

 Then give 15 min. incubation

 Then add stop solution to stop the reaction

29
  Then take the readings at 450 nm.

Calculations:-

For 1:5000

1/1000 * V1 = 1/5000 * 800

V1 = 160 ul of the stock T-HRP and add 10mM PBS with 1% BSA

Now serial dilutions for the next dilutions:-

1:10000 = 400ul of the 1:5000 + 400ul of PBS buffer with 1% BSA

1:20000 = 400ul of the 1:10000 + 400ul of PBS buffer with 1% BSA

1:40000 = 400ul of the 1:20000 + 400ul of PBS buffer with 1% BSA

RESULT: - We get the concentration 2.450 which is in the range of standard value 2.3 to 2.8

30
 Testosterone standards
Standards are made for estimating testosterone concentration in the blood sample.

Normal concentration in male is 2 to 20 ng and in female is 0.2 to 2 ng .

Stock is 2 ug/ ml, we dilute to form 2000ug/ml

Different standards of testosterone:

1. 40ng/ml
2. 20ng/ml
3. 6ng/ml
4. 2ng/ml
5. 0.2ng/ml
6. 0.6ng/ml
7. ‘o’ dose

Taking the 40ng/ml sample we can prepare the 20ng/ml, using 20ng/ml make 6ng/ml and 2ng/ml,from
6ng/ml make 0.6ng/ml, from 2ng make 0.2 ng/ml

Calculations and Procedure :-

For 40 ng/ml

2000 * V1 = 40 * 2200
V1 = 44 ul of the stock testosterone solution and add in this 2156 serum buffer.

For 20 ng/ml

40 * V1 = 20 * 2000

31
 = 1000 ul of the stock testosterone solution and add in it 1000ul of serum buffer.

For 6 ng ml

20 * V1 = 6 * 2000
= 600 ul of the stock testosterone solution and add in it 1400ul of serum buffer.

For 2 ng/ml

20 * V1 = 2 * 2000
= 200 ul of the stock testosterone solution and add in it 1800 ul of serum buffer.

For 0.2 ng/ml

2 * V1 = 0.2 * 1200
= 120 ul of the stock testosterone solution and add in it 1080 ul of
serum buffer.

For 0.6 ng/ml

6 * V1 = 0.6 * 1200
= 120 ul of the stock testosterone solution and add in it 1080 ul of serum buffer.

For ‘0’ dose

We take only 1000ul of serum buffer only.

Vortex all the samples and packed in pastry box and store in refrigerator.

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 PRPARATION OF STANDARD CURVE

T-HRP
Strips Sr. No. Standards Readings
(100ul/well)
A ‘0’ dose 100 2.608
B 40ng/ml 100 0.553
C 20ng/ml 100 0.787
D 6 ng/ml 100 1.512
E 2 ng/ml 100 2.029
F 0.6ng/ml 100 2.342
G 0.2ng/ml 100 2.734
H ‘0’ dose 100 2.868

Procedure

1ST we take the antitestosterone coated strips. Then add the 50ul of the standard in each well.
In 1 well and the last well we add 50 ul of the ‘0’ dose. In the 2 nd well we add 40 ng/ml and then in 3rd
st

20 ng/ml, in 4th 6ng/ml, 5th 2ng/ml 6th 0.6ul/ml, 0.2 ng/ml.

Table:

33
  Then add T-HRP 100 ul /well. Incubate for the 1 hr in the incubator.

 Then wash with the wash buffer (250ul/well).

 Then add 100 ul of substrate 1x in each well.

 Then 15 min incubation at 370C in the incubator.

 Then add stop solution to stop the reaction.

 Then take readings at 450 nm. And plot a graph.

Now finding the % binding:-

Take ‘0’ as 100 % binding

Formula used: -

A1/A0 * 100

Whereas:
A1 = reading of 40, 20, 6, 2, 0.6, 0.2
A0 = reading of ‘0’

For 40ng/ml

0.553/2.868 * 100
= 19.28 %

For 20ng/ml

0.787/2.868 * 100
= 27.44 %

For 6ng/ml

34
 1.512/2.868 * 100
= 52.71 %

For 2ng/ml

2.029/2.868 * 100
= 70.74 %

For 0.6ng/ml

2.342/2.868 * 100
= 81.66 %

For 0.2ng/ml

2.734/2.868 * 100
= 95.33 %

RESULT :- we get a sigmoid curve while plotting % binding against testosterone standards.

SPECIFICITY ASSAY
(Also called cross reactivity)

Aim: - To determine the specificity of antibody.

Antibody raised against a given ligand (antigen or hapten) will usually react with varying
affinity with structural analogues, which are biochemically similar to the substance being assayed.
With polyclonal antibodies, pituitary hormones pose a problem. This is specially so with
glycoprotein’s such as LH, FSH, TSH and also hCG (produced by placenta). Since they are
structurally similar in their alpha subunits composition, they cross-react with antibodies against any of
these hormones. This is overcome by using beta-subunit for immunization or by developing
monoclonal antibodies against specific antigenic determinates.

Many of the steroid hormones are closely related in structure. By taking advantages of
functional groups or introducing groups at new position on the molecule, which are different in some
of closely related compounds. The haptens are conjugated with the carrier protein to raise antibodies.
The steroids are usually bound to proteins in blood stream and need to be stripped prior to the assay.
The relative activities of various steroids with the particular serum under investigation can be
determined from the inhibition curves plotted for them under similar assay conditions used for the
antigen in question.

Taking arbitrary cross reaction of antigen (testosterone) as 100% binding at ‘0’ dose, the
percent reaction of closely related analogue (DHT) is calculated at 50% displacement of T-HRP as :

35
 = Mass of antigen (testosterone) required to displace 50% T-HRP / Mass of analogue (DHT)
required displacing 50%T-HRP * 100

Procedure:-

Take two testosterone coated strips. In 1st strip add 50 ul of testosterone standards and in 2nd
strip add 50 ul of DHT standards. And add according to the table.

Table :

Testosterone DHT in T- Wash Substrate O.D O.D % %

in 1st strip 2nd strip HRP buffer in ul For For binding binding
50ul/well 50ul/well ul in ul Testosterone DHT for for
at 450 nm at 450 testo DHT
nm sterone

‘0’ ‘0’ 100 250 100 1.896 1.657 100 100

0.2 0.2 100 250 100 2.075 1.584 91.37 95.59
0.6 0.6 100 250 100 1.784 1.580 85.97 85.56
2 2 100 250 100 1.177 1.183 56.72 55.80
6 6 100 250 100 0.860 0.907 41.44 40.39
20 20 100 250 100 0.557 0.839 26.84 25.99
40 40 100 250 100 0.379 0.547 18.26 18.19
‘0’ ‘0’ 100 250 100 1.689 1.722 100 100

Than draw graph and determine the specificity.

36
 RESULT :-

Specificity = amount of testosterone required to displace 50% T-HRP / amount of DHT

required to displace 50% T-HRP.

= 2.5 / 7 * 100

= 35.7 %

AFFINITY OF ANTIBODY WITH ITS ANTIGEN

This is an important characteristics of the antibodies and determines the avidity with which
antiserum binds the antigen and is related in turn to the sensitivity of immunoassay, while some
variations in the dilutions of the binding protein or the specific activity and mass of the labeled
hormone may affect sensitivity with the affinity constant of the binding protein. Thus, in a general
way, the sensitivity of the assay gives some indication of this association constant.

The reaction between antigen and antibody obeys law of mass action:

Km = K1/K2 = [Ab.Ag] / [Ab] [Ag]

Km is equal to the free antibody concentration at 50% saturation or for antigens to the free
antigen concentration at 50 % concentration at 50% saturation

 Affinity:-when a monovalent antibody binds with monovalent antigen.

 Avidity: - when a polyvalent antibody binds with the monovalent antigen.

Affinity: -

Ka = [Ka / Kd]

37
 Whereas: -

Ka is association constant
Kd is the dissociation constant

Generally Ka values of 10-9 Moles/L or above indicates antiserum with acceptable affinity for
immunoassay. This can be tested by adding increasing concentration of unlabeled antigen to the assay
of binding system containing antibody and enzyme labeled antigen. The inhibition of binding of
enzyme labeled antigen to the antibodies is recorded and plotted.

By plotting Y against Y’/100-Y’ bound antigen in molar concentrations, slop of the curve is
plotted and Ka can be calculated.

The normal range of the Ka = 104 to 1011]

If Ka = 104 to 107 i. e. weak binding strength

If Ka = 107 to 1011 i.e. strong binding strength

Higher the Ka values higher the sensitivity. In testosterone Ka ranges from 107 to 1010.

Table:

Sr. X Y% 100-Y Log Logit X nmol/l Y nmol/l Y’= 100-Y Y’/100-Y

n ng/ml binding X Y 1 / M.W X*Y/100 mX+C
o. In(Y/ *1000*X
100-
Y)
A 0
B 0.2 72.03 27.97 0.694 0.500 62.101 37.90 1.64
C 0.6 72.77 27.23 2.083 1.516 61.564 38.44 1.60
D 2 56.15 43.85 6.944 3.899 59.68 40.32 1.48
E 6 40.22 59.78 20.83 8.378 54.311 45.69 1.19
F 20 24.41 75.60 69.44 16.95 35.508 64.49 0.55
G 40 16.276 83.73 138.888 22.605 8.646 91.34 0.0946
H 0

Sr. no. X ng/ml O.D O.D Mean

Strip Strip
1 2
A 0 2.903 2.098 2.5005
B 0.2 2.342 1.840 2.091
C 0.6 2.234 1991 2.1125
D 2 1.700 1.560
38 1.63
E 6 1.043 1.292 1.1675
F 20 0.816 0.601 0.7085
G 40 0.561 0.384 0.4725
H 0 2.363 2.449 2.406
RESULT

The affinity comes out from graph is 1.38 * 1010 moles.

ANTIBODY CAPTURE ASSAY

These are mainly designed for quantifying specific antibodies. Antigen

are allowed to be coated in excess to the solid phase. Usually, diluted test serum is added
to excess antigen immobilized on the solid phase. If the antigen is a protein, it can be
coupled to a carrier protein like bovine serum albumin.

Procedure

Stock of testo-3-carboxy methyl oxime (CMO) – BSA = 1mg/ml

Working is 0.5ug/200ul

Add different dilution into the micro well strips according to the table.

Washing should be done after each incubation.

39
Table:

Ab Stds. I ARGG - I Substrate Stop OD Mean

wells HRP Soln. OD
1 1 N 100ul N 100ul 100ul 1.793 1.754
ug/100ml
2 1 C 100ul C 100ul 100ul 1.714
ug/100ml
3 0.8 U 100ul U 100ul 100ul 1.651 1.591
ug/100ml
4 0.8 B 100ul B 100ul 100ul 1.531
ug/100ml
5 0.4 A 100ul A 100ul 100ul 1.380 1.332
ug/100ml
6 0.4 T 100ul T 100ul 100ul 1.283
ug/100ml
7 0.1 I 100ul I 100ul 100ul 0.655 0.707
ug/100ml

40
8 0.1 O 100ul O 100ul 100ul 0.760
ug/100ml
9 0.05 N 100ul N 100ul 100ul 0.344 0.333
ug/100ml
10 0.05 100ul 100ul 100ul 0.321
ug/100ml
11 ‘0’ 370C 100ul 370C 100ul 100ul 0.198 0.176
12 ‘0’ 100ul 100ul 100ul 0.155
13 Control 40 100ul 30 100ul 100ul 1.427 1.424
14 Control Min 100ul Min 100ul 100ul 1.420
15 1 100ul 100ul 100ul 1.772 1.882
ug/100ml
16 1 100ul 100ul 100ul 1.992
ug/100ml

Result:

The reading of control comes out is 1.424, so the testosterone concentration from graph is
0.5ug/100ml

PROTEIN PURIFICATION
(IgG)

A. salt fractionation

Precipitation by salting out to remove non-specific protein is a highly effective

method of purification of proteins. The procedure to be followed depends on a variety
of experimental conditions, e.g., degree of saturation, pH and temperature.

Procedure

Solvent used is ammonium sulphate use for the precipitation.

 Take 5ml of human serum in glass cup.

 Add 5ml of normal saline in it. To dilute the crude serum in 1:1 proportion.

 Then add 20 ml of ammonium sulphate drop wise.

41
 The temperature is lowered to 200C or 40C overnight.

 The pH is adjusted between 7 and 8 with ammonium hydroxide which is

gradually added with stirring.

 Then keep the content on the magnetic stirrer for 2 hrs.

 Then centrifuge the content at 6000 rpm at 40C (cold centrifuge) for 20 minutes.

 The supernatant is discarded and the precipitate is resuspended in the original

volume of antiserum with 15 mM phosphate buffer.

 The solution is then dialyzed against the same 15mM phosphate buffer for 24 hr
giving 2-3 changes of buffer to remove ammonium sulphate. For dialysis, 10-12
kd dialysis membrane is the one most commonly used.

B. Ion exchange chromatography

DEAE-sephadex gel , an anion exchanger, is mainly used for its high flow rate. It is
employed for removing contaminating proteins, while IgG passes unabsorbed. The
dialysate obtained from salt fraction, is allowed to be adsorbed on DEAE-sephdex
equilibrated with 15mM phosphate buffer in a closed column. The unabsorbed
fraction is then eluted and dialyzed for 24 hr against ammonium carbonate buffer
(0.09%) and then lyophilized.

42
 CONCLUSION

Estimation of testosterone by immunoassay using HRP have been

developed . It is comparable in terms of recoveries, precision and for
estimating normal sample in performances with available imported
kits. In this method HRP has been used due to its cost effectiveness &
comparatively low molecular weight as compared to other enzymes.
While developing assay, is essential to find out the optimal
concentration of Ab & available enzyme that are required for
determining sensitive assay.

Testosterone assays of today are more sensitive & require smaller

quantities of serum & are performed more rapidly. The enhanced
efficiency & reduced cost , improved sensitivity , ease of performance
have made them more available to clinicians & researchers , thereby
facilitating both clinical care & research.

43