to raise rabbit anti-human t-cell receptor polyclonal antibodies

8/8/2019 To Raise Rabbit Anti-human T-Cell Receptor Polyclonal Antibodies

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TO RAISE RABBIT ANTI-HUMAN T-CELL

RECEPTOR POLYCLONAL ANTIBODIES

FOR IN-VIVO NEUTRALIZATION OF T-CELLS IN

GRAFT REJECTION PATIENTS


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INTRODUCTION

Polyclonal anti-thymocyte globulins preparations are

heterologous preparations.

Animals (rabbits (rATG) and horses (eATG) ) are

immunized with human T cells and thymocytes.

Antisera is then collected and antibodies are purified.

These polyclonal preparations are directed at

multiple different epitopes on the T cell (CD2, CD3,

CD4, CD8, CD28, and the T-cell receptor) as well as

CD16 found on natural killer cells and macrophages. These antibodies cause depletion of T cells by

apoptosis, antibody mediated cytolysis and

internalization of the cell surface receptors.


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These antibodies are highly useful in

transplantation where it is used as

immunosuppressive agent. ATGs are used either alone or along with traditional

immunosuppressive agents in different

combinations.

Organs used in clinical transplantation are eitherisografts or allografts. As in most cases the

transplanted organ is allograft, graft rejection is

obvious phenomenon.

These antibodies (rATG) are currently in clinical

trails, and most clinical trails have shown ATGshave more benefits and fewer side effects than

traditional immunosuppressive agents.


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PROCEDURE

1. THYMUS CELL SUSPENSION Thymus is cut in two to three

pieces in center of either lobe.

These pieces are pressed inbetween glass slides in circularmotion until it is dissociated.

The dissociated cells were passed

through a fine mesh nylon filter. Dissociated cells was mixed

thoroughly with Freund'sComplete Adjuvant [40% v/v].Both were mixed thoroughly for 15minutes in sterile injection vial.

This thymus cell suspension and Adjuvant mixture was filteredthrough bacterial filter.

The filtered mixture can now betermed as Antigen which can betaken for immunization procedure.


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2. IMMUNIZATION

The prepared antigen was injected into the rabbit[Done by NIN Hyderabad].

Dose 1 - 2.6 ml [1ml per Kg wt of animal body]

Dose 2 - After 15 days from Dose 1. [Volume

same as Dose 1] Dose 3 - After 15 days from Dose 2. [Volume

same as Dose 1]

Bleed 1 - After 10 days from Dose.3. [75ml per

rabbit] Every successive bleed can be performed with 10

days gap between each bleed


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3. BLOOD COLLECTION

In case of rabbits the Cardiac (anesthetized only)and marginal ear vein sites are used for blood

collection.

Regardless of the method of collection used, an

animal may not be returned to its cage untilcomplete hemostasis has been achieved (there is no

more blood coming from the collection site).


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4. SERUM SEPARATION

Animal Blood was carefully collected carefully in the

presence of an anticoagulant [Tri sodium citrate]

and it was kept undisturbed in refrigerator for a

whole night.

The next day a light yellowish brown coloredsupernatant was observed over the RBC mass in

collection vial.

This clear supernatant was serum which was used

for the further procedures.


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5.PEG BASED PRECIPITATION OF SERUMPROTEINS

Equal volumes of serum and phosphate buffer weretaken into container.

They were kept in refrigerator for 15-20 min. Acalculated percentage of pre-chilled polyethyleneglycol was added with continuous mixing on

magnetic stirrer. Mixing was done until PEG gotdissolved and a visual precipitation was observed.

After a continuous mixing this mixture was kept inrefrigerator for 20 min for a good precipitation.

Mixture was transferred to centrifuge tubes and

they were centrifuged at 5000 rpm for 6 minutes.The pellet was separated and supernatant wasdiscarded.

Pellet was taken for dialysis in dialysis bag. It wasdialyzed against phosphate buffer.


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6. SDS-PAGE

SDS-PAGE of separated proteins was performed toanalyze the presence of rATG by comparing with

standard rATG preparations.

2.6 ml of acrylamide, 8 l of 10% SDS and 10l of

TEMED was used in separating gel and stackinggel had acrylamide (0.532ml), SDS 10% (40l) and

TEMED of 10l.

Before running the samples on SDS-PAGE they

were treated with treatment buffer containing B-

mercaptoethanol and bromophenol blue.


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STEPS-

The glass plate sandwich of the electrophoresisapparatus was assembled using two clean glass plates

and there 1mm acrylic spacers. The sandwich was locked in the positioned and then

sealed along the three sides using molten 1% agar.

The separating gel solution was prepared by mixingthe reagents in correct proportions.

Care was taken that the freshly prepared APS andTEMED are added last, as they are responsible for thepolymerization.

The solution was mixed thoroughly and filled into thesandwich to the required height. A small amount of

carbon tetrachloride was poured on top in order to levelthe top layer to cut off the oxygen supply.

This arrangement was left for 15 minutes so that thegel solidifies. The top layer of carbon tetrachloride wasthen poured off and the surface was washed withdistilled water.


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Now the stacking gel solution was prepared by mixingthe reagents in correct proportions. On pouring into theplates Teflon comb was slowly inserted into the plates

so that no air bubbles get trapped between the two gels. This arrangement was also left for 15 minutes. After

the gel solidifies the comb was removed and the wellsare washed thoroughly with the tank buffer in order toremove any unpolymerized acrylamide.

The sandwich was fitted onto the vertical slab unitafter removing the bottom spacer.

The protein samples were mixed with treatment bufferin the ratio 2:1 and boiled for 5 minutes.

Appropriate amount of sample was then loaded into the

wells using a microlitre syringe. The entire unit wasconnected to a power pack and a current of 12mA madeto pass through the gel by applying potential differencebetween the two electrodes.

When the proteins enter the separating gel the currentis raised to 13mA. When the electrophoresis was

completed the gel is stained.


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7. COOMASSIE BLUE STAINING

The gel to be stained was placed in a glass tray in

D/W for 2 hours. This will lead to diffusion of un-reacted proteins and salts into the D/W and will

also help in separating the gel from the glass plate.

After few hours the gel slipped from the plate and

was floating in water.

The plate was removed and the glass plate covered

with gelatin paper was slipped under the gel. The

gel plate was removed from the water and was

placed on an inverted 50ml beaker.

Whatmann filter papers was wetted with waterand placed on top of the gel.

Care was taken that air should not get trapped in

between.


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A slide on filter papers was placed and was dried

in the incubator overnight at 37C. All the

remaining salts and proteins diffuse by capillary

action.

Next day the dried gel plate was placed directly in

Phosphate Buffer Saline with azide for 6 hrs. This

further removes the salts and proteins from the

gel. After 6 hrs the gel plate was removed and placed

in Coomassie Blue stain. It was stained for 10

minutes.

The gel plate was removed and transferred afterdecanting into a tray background gets cleared.

The destaining fixative was regularly changed.


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The gel slide was then placed in D/W to wash off

the fixative for 15 mins. The D/W was decanted

from the slide and filter papers were placed onslide for drying at room temperature, overnight.

The gel slide is ready and the bands can be seen.


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8. DOUBLE IMMUNODIFFUSION

1% agarose was prepared and it was boiled. A

clean dry glass plate was taken and 12 ml of themolten agarose was poured onto the slide.

This forms a gel of 1.5mm thickness. Gel was

placed in a chamber covered with moist filter

paper.

The gel was kept at 4 degree Celsius till further

use.

Six punches were made in a circular pattern with a

central punch on the gel.

The central well has an antibody. The surroundingwells have decreasing concentration of the

corresponding antigen, labeled Neat (1:1), 1:2, 1:4,

1:8, 1:6 and 1:32, clockwise from top.


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Using this method it can be determined whether

the antibody developed in the animal is for a

particular antigen or not and also if the animal is

responding to the specific antigen which was

injected into it.

After the antigen and the antibody had been

loaded the plate was kept in a moist chamber butnot in the fridge. The precipitin bands appeared

with in 6hrs after loading the plate.

If the precipitin rings do not appear after 12hrs,

then the plate is kept in 1%Tannic acid solution(i.e.1gm Tannic acid in 100ml D/W) until the

precipitin rings develop.


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RESULTS

1. SDS-PAGE

Several distinct bluecoloured bands were

observed in the gel.

A match between the

52 KD bands of 2, 3, 4,and 5 was seen.


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In double immuno-diffusion continuous precipitation

line was formed between concentrations 1:1 to 1:8.


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THANK YOU.

to raise rabbit anti-human t-cell receptor polyclonal antibodies

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