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Pertemuan ke-3 part 2

The quality of Ab produced

Depend on purity and dose of ligand


Route of administration
Kind Adjuvant
Timing of blood collection
Animal species
Also depend of MW (Berat Molekul/
Molecul Weight) of ligand

Media
1. PZ = 9 g NaCl + 1000 Aqd (Aquades)
2. Prog-11-BSA-Sol = 100 ug P4-11-BSA +
1 ml PZ
3. P4-11-BSA Emulsion = 0.5 sol P4-11BSA + 0.5 ml FCA
Mix well by using 2 syringe that
connected each other with small canule,
pumping several times emulsion?
Tester (??)
Why should apply FCA?
There are containing with oil, the mechanism?
There are also with weaked mycobacterium,
what the function and mechanism?
How to collect blood serum in rabbit
Clean up one ear
A small incision at V marginale
The ear is entered into graded
Erlenmeyer glass connected with canule
and vacuum machine
The blood will come up smoothly and
stops it after 15 20 ml
The blood let clotting for 2 3 hours
One hour after collected, release the
cloth by puncturing at glass wall
Another 1 2 hours later the serum will
come out proportionally, max 60% of
blood can be harvested as NRS.

Immunitation

Fixation the rabbit at dorsal thorax and


dorsal pubic
Clean up by cutting using a scissor all
hair at dorsal last thorax until dorsal
lumbal
Antiseptic should be washed up at
location
Injectio should be done intradermal

Intradermal Injection

By using insulin syringe, the 1 ml P4-11BSA emul (mentioned above) injected


intradermally at those locations without
hair, 25 30 ul eac, to be divided to 3040 site of injection.
The next 12-14 D the booster injection
should be done with 1 ml P4-11-BSA
solution IP
The next 14-21 D following booster the
blood serum must be collected to
analyze the anti hormone
The blood collected as the same
technique as well as mentioned when
withdrawal NSR above, but here found
A/S.

Clinical Response

1st response
Laten period there take 5 7 D
No any Ab increase
Body temp arise
2. Primary Ab response 10 30 D, just
immature response of increasing Ab,
mostly due to of B-cell response
3. 2nd response, a optimum to mas
response of increasing Ab mostly by Tcell
2nd response, (following booster)

1. Unsignificant Body Temp arise


2. Latent period much shorter
3. Produce Ig M and Ig G much
higher
4. Second booster be able done
following 30 D of first booster

Result
X cpm A/S x cpm NRS
% B = -------------------------------- x 100%

Titration of A/S

X cpm TC x cpm NRS

Media PBS:
A. 1.42 g Na2HPO4 1000 cc aquades
B. 1.38 g Na2HPO4.H2O 1000 cc
aquades
Take 600 ml A + 400 ml B mix
Put in 9 g NaCl and 1 g sod azide
A part (500 ml) of this sol add with 500
mg gelatin
NRS serial dilution (1 10)
1:50, 1:100 1:25600 (2 x each Step
Lower)
A/S serial dilution (110)
1:50, 1:100 1:25600 (2 x each Step
Lower)
Protein A-sol
P4-11-succinyl125I diluted 20.000
CPM/ minute (gamma counter)

Protocol P4 A/S Titration


Tube
TC
TC
NRS
1:50
NRS
1:50
A/S
1:50
A/S
1:50

PBSG Diluted Tracer Prot


(ul)
SRA
(ul)
A (ul)
(ul)
0
0
100
0
0
0
100
0
500
200
100
100

Cold
PBS
0
0
3

500

200

100

100

500

200

100

100

500

200

100

100

Find the highest solution with percent binding >


27%, that the real result be able to use as good
anti hormone.
Titration Continued
Tube
NRS
1:100
NRS
1:100
A/S
1:100
A/S
1:100

PBSG Diluted
(ul)
SRA
(ul)
500
200

Tracer Prot Cold


(ul)
A (ul) PBS
100

100

500

200

100

100

500

200

100

100

500

200

100

100

And soon
Tube

PBSG Diluted Tracer


(ul)
SRA
(ul)
(ul)
500
200
100

NRS
1:25600
NRS
500
1:25600
A/S
500
1:25600

Prot Cold
A
PBS
(ul)
100 3

200

100

100

200

100

100