Laboratory Techniques In

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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 BYRAVI CHOLIA

ROLL NO. 0709216

M.Sc. Biotechnology

Department of Bio & Nanotechnology

GURU JAMBSHEWARE UNIVERSITY

OF SCIENCE &TECHNOLOGY

HISAR

2007-2009

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PREFACE

Practical training is one of the major components for anyprofessional courses likeBiotechnology. The professional place where abiotechnologist implements theoreticalKnowledge is a floor of workshop of any industry. With out apractical knowledge we are worthless.

Being a MSc. Biotechnology student, I took a similar kind oftraining at Orbit Biotech. Pvt. Ltd. Mohali. I learnt many athings over there. I gained a practical knowledge about theELISA-KIT development for Testosterone. I was familiar withthe working environment. Overall it was a great experiencefor me. This experience will be of immense help to me in myforthcoming future.

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CONTENTS

Sr.no. Particulars Page no.

1 Acknowledgement 42 Certificate 53 Certificate 64 Abbreviations 75 Introduction 96 ELISA Test & Types 127 Preparation of

buffers13

8Coating

159 Immunization ofrabbit

17

10 Conjugation ofhapten withenzyme (HRP)

20

11 Characterization ofantiserum

22

12 Ouchterlonydouble diffusiontest (ODD)

24

13 Checkerboardassay

25

14 Fine tuning 2815 Preparation of

testosterone30

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standards16 Preparation of

standard curve32

17 Specificity assay 34

18 Affinity of anantibody with itsantigen

36

19 Antibody captureassay

38

20 Protein purification 4021 conclusion 42

ACKNOWLEDGEMENT

It is my pleasure to take this opportunity to thank all

who helped me directly or indirectly in preparation of thisSUMMER TRAINING REPORT. The blessing of almightygod, my parents and my elders have enable me to reachsuch a standard that I got a chance to do something goodmyself.

I am grateful to Dr. J. S. Rana, chairman of the bio &nanotechnology department for providing a big moralsupport. I must pay my gratitude to Mr. AnilKumar for hiskind support and assurance, I got from him.

I must convey special thanks to Mr. Sudesh Kumar,instructor of Orbit Biotech Pvt. Lmt. Mohali. Under whoseguidance I completed my summer training successfully. I amalways thankful to Miss Derothy & Jaspreet. I am alsograteful to others for their great guidance and moral support.

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The help provided by these personalities have aided alot preparation of this summer training report. So, I onceagain convey my heartiest thanks to all of them.

RAVI CHOLIAM.Sc. BiotechnologyRoll no. 0709216Deptt. Of Bio &

Nanotechnology

GJUS&THISAR

GURU JAMBESHWAREUNIVERSITY

OF SCIENCE & TECHNOLOGYHISAR

CERTIFICATE

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It is certified that Mr. RAVI CHOLIA, astudent 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 namedLABORATORY TECHNIQUES IN PREPARATION OFTESTOSTERONE-ELISA KIT from Orbit Biotech Pvt.Lmt. MOHALI under my supervision. He has workedwell. I wish he get all success in her life.

MR. ANIL KUMAR(Major Advisor)

Deptt. Of Bio &Nanotechnology

GJUS&THISAR

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ABBREVIATIONS

Ab Antibody

Ag Antigen

ARGG Anti Rabbit Gamma Globulin

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ANS 8- Amilino Naphthalene sulfonic acid

BSA Bovine Serum Albumin

BSB Blocking and Stabilizing Buffer

0C 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

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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.

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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 andits relationship with some of the clinical symptoms both in men and women. It is imperative to know

the intracellular concentrations of the hormone.

The bodys 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 alternativelabels 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 formedfrom 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.

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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.

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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 labeledantibodies 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 cant 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, -D- galactosidase, Glucose oxidase, glucoamylase, - 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 )

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45 mg of BSA

First dissolve the ANS into the PBS buffer; it takes about 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 ---------------------- = 2000ulFor 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

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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).

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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

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Hapten + Carrier = Immunogen

Testosterone + BSA = 2mg dissolve in min. amount of NaOH (it is steroid cant 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 ofthe 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 % sodiumchloride). 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 Freunds 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. theproduction 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

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1. Standard rabbit box

2. Electric animal clippers

3. Xylene or toluene

4. Vacutainer needle

5. 10cc syringe with 22g needle

6. Rectified sprit7. 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 1500xg 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 Freunds

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 Freunds adjuvant intramuscular

14- Same as above

21- Same as above

Booster injections

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

Freunds adjuvant plus saline in 1:1 proportion.

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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.

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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 imides 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 albumin12. 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:

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Testosterone = 299 Daltons

HRP = ~44000 Daltons

T-HRP = ~44288 Daltons

To separate the T- HRP from unbound testosterone and unbound HRP . We perform Gelfiltration 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 cant 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 spacesbetween 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:

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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 H2SO410. 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:

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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 be1:40,000.

24

ARGG coated

wells

Dilutions

(add 100 ul/well)

T-HRP

(add

100 ul/well)

O.D

at 450 nm

Mean

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


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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.

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` 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, 0.75ug/100ul, and 1.0ug/100ul)

Procedure:

Make different dilutions and add according to the table.

Table:

Then incubate for 1 hr. at 370C.

26

Sr. no.

of ARGG

coated wells

Antibody concentration

(in ug/100ul)

Dilutions

(100ul /well)

T-HRP

(100ul/well)

OD

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


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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.

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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:

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

Anti testosterone

coated wells

T- HRP

(stock 1:1000)

Add 100 ul/well

Readings at

450nm

Mean

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


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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



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

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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

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= 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

Procedure

1ST we take the antitestosterone coated strips. Then add the 50ul of the standard in each well.

In 1st 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

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

Table:

33

Strips Sr. No. StandardsT-HRP

(100ul/well)Readings

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


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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

glycoproteins 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 :

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= 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

in 1

st

strip50ul/well

DHT in

2

nd

strip50ul/well

T-

HRPul

Wash

bufferin ul

Substrate

in ul

O.D

ForTestosterone

at 450 nm

O.D

ForDHT

at 450

nm

%

bindingfor

testo

sterone

%

bindingfor

DHT

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.

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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 labeledhormone 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]

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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 forimmunoassay. 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.

no.

X

ng/ml

Y %

binding

100-Y Log

X

Logit

YIn(Y/

100-

Y)

X nmol/l

1 / M.W*1000*X

Y nmol/l

X*Y/100

Y=

mX+C

100-Y Y/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

38

Sr. no. X ng/ml O.D

Strip

1

O.D

Strip

2

Mean

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 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


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RESULT

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

ANTIBODY CAPTURE ASSAY

These are mainly designed for quantifying specific antibodies. Antigenare 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.

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Table:

Abwells

Stds. I ARGG -HRP

I Substrate StopSoln.

OD MeanOD

1 1

ug/100ml

N 100ul N 100ul 100ul 1.793 1.754

2 1

ug/100ml

C 100ul C 100ul 100ul 1.714

3 0.8

ug/100ml

U 100ul U 100ul 100ul 1.651 1.591

4 0.8ug/100ml B 100ul B 100ul 100ul 1.531

5 0.4

ug/100ml

A 100ul A 100ul 100ul 1.380 1.332

6 0.4

ug/100ml

T 100ul T 100ul 100ul 1.283

7 0.1

ug/100ml

I 100ul I 100ul 100ul 0.655 0.707

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8 0.1

ug/100ml

O 100ul O 100ul 100ul 0.760

9 0.05

ug/100ml

N 100ul N 100ul 100ul 0.344 0.333

10 0.05

ug/100ml

100ul 100ul 100ul 0.321

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 1ug/100ml

100ul 100ul 100ul 1.772 1.882

16 1ug/100ml

100ul 100ul 100ul 1.992

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 varietyof 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.

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

The pH is adjusted between 7 and 8 with ammonium hydroxide which isgradually 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-sephdexequilibrated 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.

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CONCLUSION

Estimation of testosterone by immunoassay using HRP have beendeveloped . 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.

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