PRODUCTIONAL ASPECTS OF F(ab) 2

FROM HUMAN IgG THROUGH PEPSIN

DIGESTION

By
Alekhya robbi
V.N.U.Sowmya
P.Naga Chaitanya
P. shushma
 INTRODUCTION
• Antibodies (also known as
immunoglobulins,abbreviated Ig) are gamma globulin
proteins that are found in blood or other bodily fluids of
vertebrates, and are used by the
immune system to identify
and neutralize foreign objects,
such as bacteria and viruses.
• Each antibody binds to a specific
antigen; an interaction similar to a
lock and key.
 Structure of IgG and subclasses
IgG antibodies are large
molecules of about 150 kDa
composed of 4 peptide chains. It
contains 2 identical heavy
chains of about 50 kDa and 2
identical light chains of about 25
kDa, thus tetrameric quaternary
structure.
There are four IgG subclasses
(IgG1, 2, 3 and 4) in humans,
named in order of their
abundance in serum
 PROPERTIES OF IgG
• Most versatile immunoglobulin because it is capable of carrying
out all of the functions of immunoglobulin molecules.
• a) IgG is the major Ig in serum - 75% of serum Ig is IgG
• b) IgG is the major Ig in extra vascular spaces
• c) Placental transfer - IgG is the only class of Ig that crosses
the placenta. Transfer is mediated by receptor on placental
cells for the Fc region of IgG. Not all subclasses cross equally;
IgG2 does not cross well.
• d) Fixes complement - Not all subclasses fix equally well; IgG4
does not fix complement
• e) Binding to cells - Macrophages, monocytes, PMN's and
some lymphocytes have Fc receptors for the Fc region of IgG.
Not all subclasses bind equally well; IgG2 and IgG4 do not bind
to Fc receptors.
DIGESTION OF IgG WITH
PEPSIN AND PAPAIN
 PEPSIN DIGESTION OF IgG
METHODOLOGY

• Blood collection and plasma separation

• Animal Blood is carefully collected in the
presence of an anticoagulant [Tri sodium citrate]
and it is kept undisturbed in refrigerator for a
whole night. The next day a light yellowish
brown colored supernatant is observed over the
RBC mass in collection vial.
 DIGESTION
• 0.5 ml of plasma was taken and 0.5 ml of phosphate buffer was added
• A light centrifugation was done at 3000 rpm
• The pellet was discarded, supernatant was transferred to other vials
• Precipitation was done with 20%W/V ammonium sulphate.
• Then the precipitate was centrifuged at 3800-4800 rpm for 6 minutes
• Then the pellet was collected and constituted with 0.5ml of phosphate
buffer
• Then 1ml of pepsin was added to the constituted mixture .
• Then the mixture was incubated overnight at 37 degrees C
• Then negative precipitation was observed to have occurred
• The pellet was discarded and super natent was taken for further
analysis
 SDS PAGE
• CASTING THE GEL

• The glass plate sandwich of the electrophoresis apparatus is assembled using

two clean glass plates and there 1mm 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. Acryl amide
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 and also to cut off
the 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
• Now the stacking gel solution is prepared by mixing the reagents in correct
proportions. On pouring into the plates Teflon comb is slowly inserted into
the plates so that no air bubbles get trapped between the two gels. This
arrangement is also left for 15 minutes. After the gel solidifies the comb is
removed and the wells are washed thoroughly with the tank buffer in order
to remove any unpolymerized acrylamide.
• The sandwich is fitted onto the vertical slab unit after removing the bottom
spacer. Care should be taken to see that there are air bubbles between the
bottom of the sandwich and the tank buffer, which is filled in the tank of the
vertical slab unit. The protein samples are 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 microlitre syringe. The entire unit is connected
to a powerpack 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 that is
produced during the electrophoresis, which might cause the gel to melt.
When the proteins enter the separating gel the current is raised to 13mA.
When the electroporesis is completed the gel is stained
 COOMASSIE BLUE STAINING
PROCEDURE
• Place the gel ready to be stained in d/w for 2 hours. This will lead to diffusion of
unreacted proteins and salts into the d/w and will also help in separating the gel from
the glass plate
• Take a gelatin paper and cut it to size that is little excess to one size of the plate
• Wet the gelatin paper and cover the glass plate. Avoid trapping of air bubbles
• After 2 hours the gel will slip from the slide and will float in water. Remove the glass
plate and slip carefully the glass plate covered with gelatin paper under the gel
• 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 Whatmann filter paper and wet it with water
• Slowly place the Whatmann 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 Whatmann filter paper and the gel.
• Place the slide on filter papers and dry it in the incubator overnight at 370C
• All the remaining salts and proteins will diffuse by capillary action
• Next day place the dried gel plate directly in Phosphate Buffer Saline with
azide for 6 hrs
• This will further remove the salts and proteins from the gel
• After 6 hrs remove the gel plate and place it in Coomassie Blue stain
• Stain for 10 minutes
• Remove the gel plate and transfer after decanting into a tray background
gets cleared
• Change the destaining fixative regularly
• Finally place the gel slide in d/w to wash off the fixative for 15 mins
• Decant the d/w from the slide and place it over filter papers for drying at
room temperature, overnight
• Now the gel slide is ready and the bands can be seen
RESULTS
RESULT OBTAINED BY SDS
PAGE
 CONCLUSION
• Precipitation agents like ammonium
sulphate or octanoic acid or PEG are used
for precipitating IgG from Human plasma.
As per the norms of FDA of Indian
government these precipitation agents
have faesibility to produce effective
precipitation results with minimum side
effects.