ROLE OF ATG POLYCLONAL ANTIBODIES(13)

ANTIBODIES(13)

Antithymocyte globulins (ATGs), the immunoglobulin G (IgG) division of sera starting rabbits or horses immunized with human thymocytes or T-cell lines, are used in conditioning regimens for bone marrow transplantation, in the treatment of acute graft-versus-host virus. In non human primates, ATGs induce rapid, dose-dependent, T-cell depletion in marginal lymphoid tissues, where apoptotic cells can be demonstrated in T-cell zones.

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ROLE OF ATG POLYCLONAL ANTIBODIES(12)

Antigen Coating

Ag diluted in the ELISA coat buffer is added into the polystyrene wells. The dilution of the Ag should be in the such a pattern that the amount of Ag absorbed to the polysterine plate must be max at such dilution. The concentration taken should be 10^g/ml. Incubation for the 2 hours overnight at 370c. Store the Ag solution until the immunoassay is performed. Before performing the immunoassay the wells should be washed in the following way PBS-TWEEN wash. Add 300 ц1 of the PBS-TWEEN buffer mix it well and leave it for 2 min after that aspirate the buffer from the wells.

BSA-ELISA Wash

Add 300 ц1 of the BSA-ELISA coat buffer to the wells and incubate at RT for about two hours. Repeat the PBS-TWEEN wash and also repeat the BSA_ELISA wash

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ROLE OF ATG POLYCLONAL ANTIBODIES(11)

ANTIBODIES(11)

Avoid shaking the gel or the water to prevent the gel from breaking. Slowly remove the gel plate from the water and place it on an inverted 50ml beaker. Put a few drops of water on top of the gel. Take a whatman filter paper and wet it with water. Slowly place the whatman filter paper on top of the gel. Avoid any air bubbles in between the gelatin paper and the glass plate, gelatin paper and the gel and between the whatman filter paper and the gel.

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ROLE OF ATG POLYCLONAL ANTIBODIES(10)

The protein samples are the mixed with treatment buffer in the ratio 2:1 and boiled for 5 minutes. Appropriate amount of sample is then loaded into the wells using a microliter syringe. The entire unit is connected to a power pack and a current of 12mA is made to pass through the gel by applying potential difference between the two electrodes. The electrophoresis is preferably run at 40C. This is to counter the heat is produced during the electrophoresis, which might cause the gel to melt. When the proteins enter the separating gel the current is raised to 13 mA. When the electrophoresis completed the gel is stained.

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ROLE OF ATG POLYCLONAL ANTIBODIES(9)

ANTIBODIES(9)

Procedure for Casting the Gel

The glass plate sandwich of the electrophoresis apparatus is assembled using two clean glass plates and there 1 mm acrylic spacers. The sandwich is locked in the positioned and then sealed along the three sides using molten 1% agar. The separating gel solution is prepared by mixing the reagents in correct proportions. Care should be taken that the freshly prepared APS and TEMED are added last, as they are responsible for the polymerization. Acrylamide being neurotoxic should be handled with care. The solution is mixed thoroughly and filled into the sandwich to the required height. A small amount of carbon tetrachloride is poured on top in order to level the top layer also to cut off oxygen supply. This arrangement is left for 15 minutes, so that the gel solidifies. The top layer of carbon tetrachloride is then poured off and the surface is washed with distilled water.

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ROLE OF ATG POLYCLONAL ANTIBODIES(8)

PEG based Preparation of Plasma Proteins

Equal volumes of plasma and phosphate buffer are taken into a container and their pH is adjusted between 7.2 – 7.4. Keep it in refrigerator for 15-20 minutes. A calculated percentage of pre-chilled poly ethylene glycol is added with continuous mixing on magnetic stirrer. Mixing is done until PEG got dissolved and a visual precipitation is observed. After a continuous mixing this mixture is now kept in refrigerator for a good precipitation. Mixture is now transferred to centrifuge tubes and they are centrifuged at 5000 rpm for 6 months. Now the pellet is separated and supernatant is stored for the comparative study in the future proceedings. Pellet is now taken to the dialysis beaker where they dialyzed against phosphate buffer.

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ROLE OF ATG POLYCLONAL ANTIBODIES(7)

ANTIBODIES(7)

Immunoregulatory role has been qualified to the discrete subset of major histocompatibility complex class I-restricted NK1+ mature heat-stable antigen- (HSA-) thymocytes expressing an unusual Vp8-biased T cell receptor repertoire. The cytokine interferon-y-inducing factor (IGIF) supplements natural killer (NK) cell activity in cultures of human outlying blood mononuclear cells (PBMC), likewise to the structurally unrelated cytokine interleukin (IL). IGIF is been found to develop the production of interferon-y (IFN-y) and granulocyte/macrophage colony-stimulating factor (GM-CSF) while preventing the production of IL-10 in concanavalin A (Con A)-stimulated PBMC. The antibody bound to CCR3 is used to isolate T cells from peripheral blood that give rise to TH2-polarized cell lines and to identify TH2 cells derived from naiDve T cells In vitro. Eotaxin stimulated increases in intracellular calcium and chemotaxis of CCR3+ T cells.

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