Anti-IL-6R抗体[B-R6] (ab47215)

I have added the protocol the lab uses to the end of this email for you. Please note that the experiment is done radioactively with Thymidine H3.

Regarding the amount of IL-6 found in the serum, 15pg/ml, it is 60 time less than the amount used in the test to validate the blocking activity of ab47215, so there will not be a problem to block this amount of IL-6 with 1 or 2 µg/ml of antibody ab47215.


1. REAGENTS







Source
Reference

XG1 cell line



hIL-6



RPMI1640
Sigma
R0883

Fetal calf serum



L-Glutamine
Sigma
G7513

Penicillin / Streptomycin
Sigma
P0781

b mercaptoethanol
Sigma
M6250

Tritiated thymidine (Thymidine H3)
Perkin Elmer
NET027A005MC








Performance of ASSAY





XG1 cell line



Culture: the medium used is RPMI1640, 10% FCS, 4mM L-Glutamine, 10U / ml penicillin, 100µg / ml streptomycin, 5.10-5 M b mercaptoethanol, 1ng/ml hIL-6. The cells are cultured at 2.105 cells / ml and split every 3 days.

The cell concentration must not have reach 106 cells/ml the day of the test, if it is the case you have to split the cells to 5.105 cells/ml the day before.



Medium for biological assay :

RPMI1640, 10%FCS, 4mM L-Glutamine, 10U / ml penicillin, 100µg / ml streptomycin, 5.10-5 M b mercaptoethanol, without IL-6.



Notice that the medium contains IL-6. Therefore, before the biological assay performance, the cells are washed two times by centrifugation at 500g in RPMI1640 without IL-6 and deprived 4 hours of IL-6 before to start the assay.
The cells are suspended in the medium at a concentration of 2.105 cells / ml.





hIL-6 standard dilutions: total volume per well: 200 μl

All is done in triplicate straight in the plate assay.
hIL-6 standard dilution is ranging from 10 ng / ml to 10-6 ng / ml (10 fold dilutions)
Prepare a workstock of hIL-6: 8 ng / 400 µl.
Add 100µl of medium to standard wells B4-5-6, C4-5-6, D4-5-6, E4-5-6, F4-5-6, G4-5-6, H4-5-6
Pipet 110µl of the 8 ng / 400 µl workstock into wells A4-5-6. Transfer 10 µl from A4-5-6 to B4-5-6 wells. Mix the content by repeated aspirations and ejections. Repeat this procedure from the well B4-5-6 to wells C4-5-6and from wells C4-5-6 to D4-5-6 and so on up to H4-5-6 creating three parallel columns of hIL-6 standard dilutions. Discard 10 µl from the content of the last microwells used H4-5-6.
Add 50µl / well of medium to adjust the volume to 200 µl / well







Antibodies / hIL-6 coincubation plates: total volume per well: 200 μl

All is done in triplicate straight in the plate assay.



Dilution series of the Abs (see below) (in 100 μl )

Always use a negative control.



MAbs: start with 180 nM = 30µg/ml (final concentration) > 1/10 dilution. Dilute to obtain a 90 nM stock, about 24 µg / 400 µl medium.

Fab: start with 180 nM = 10µg/ml (final concentration) > 1/10 dilution. Dilute to obtain a 9 nM stock, about 8 µg / 400 µl medium.



Add straight in the plate assay, 110 µl of Ab in the first wells of the row (line A) and 100 µl / well of medium in the rest of the rows. Make a 1/10 serial dilution: 10 μl previous dilution + 100 μl medium.



Combine Abs dilution series with a constant amount of hIL-6 solution (in 50µl)

The antibodies are tested against 1 ng / ml of hIL-6 (the concentration at which the plateau is obtained).
prepare a working solution of hIL-6 at 4 ng / ml
Distribute 50µl / well





Add the cells: 104 cells / 50 μl / well in all the plates assay





Template example

Each test is performed in triplicate
Template example:









1
2
3
4
5
6
7
8
9
10
11
12


Ctrl
Standard hIL-6
Ab1 nM
Ab2 nM

A
1 ng/ml IL6
10 ng/ml hIL-6
180
180

B
1
18
18

C
0.1
1,8
1,8

D
0.01
0,18
0,18

E
no hIL-6
0.001
0,018
0,018

F
0.001
0,0018
0,0018

G
0.0001
0,00018
0,00018

H
0.00001
0,000018
0,000018






Development of the assay



hIL-6 Standards wells :



Dispense the hIL-6 standard dilutions in the plate (100µl /well)
Add 50µl / well of medium to adjust the volume to 150 µl / well



Antibodies tested :



Dispense the Abs dilutions in the plate (100µl / well)
Add the 4 ng/ml hIL-6 solution (50µl / well)



The cells : add 104 cells / 50 µl / well in all the plates
The final volume is 200µl / well, complete if necessary
Incubate 96 hours
Harvest of the biological assay :



Add the Thymidine H3 1µCi / 10 µl / well for the last 8 hours of cell culture
Harvest cells and measure the radioactivity with Beta counter