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ELISA develop problem, Please help me.

ELISA capture antibody

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#1 ylu

ylu

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Posted 28 January 2015 - 01:04 PM

We are developing an ELISA assay by using a purified mouse mAb as coating Ab(capture Ab). First we ran a chaseboard which we diluted our Ab from 20ug/ml to 0.02ug/ml and a sample (which is mouse serum contains our Ag) from 1:150 to 1:9600. The result are as folows.

sample(1:x) Ab: ug/ml     20     10          5       2.5       1.25    0.625    0.312   0.1562   0.078    0.039     0.02      0

150                            0.313   0.363   0.416   0.427   0.473     0.501   0.477   0.451     0.446     0.437   0.454   0.445

300                            0.268   0.292   0.299   0.302   0.334    0.322    0.293   0.283     0.264     0.261    0.268  0.265

600                            0.224   0.218   0.2       0.199   0.192    0.176   0.149    0.135     0.131    0.125     0.123   0.126

1200                          0.188   0.172   0.158   0.155   0.158    0.139   0.115    0.106     0.091    0.092      0.088   0.09

2400                          0.175   0.162   0.15     0.144   0.135    0.118   0.093    0.079     0.072    0.069      0.069   0.069

4800                          0.171   0.156   0.139   0.134   0.13      0.107   0.082    0.068     0.063    0.058     0.057    0.057

9600                          0.17     0.154   0.137   0.131  0.128     0.101   0.074    0.062     0.055    0.053     0.051    0.051

0                                0.181   0.163   0.136   0.127   0.12      0.09      0.064   0.053     0.049     0.047     0.046   0.044

 

binding ratio:

0.703   0.816   0.935   0.960   1.063   1.126   1.072   1.013   1.002   0.982   1.020   1.000

1.011   1.102   1.128   1.140   1.260   1.215   1.106   1.068   0.996   0.985   1.011   1.000

1.778   1.730   1.587   1.579   1.524   1.397   1.183   1.071   1.040   0.992   0.976   1.000

2.089   1.911   1.756   1.722   1.756   1.544   1.278   1.178   1.011   1.022   0.978   1.000

2.536   2.348   2.174   2.087   1.957   1.710   1.348   1.145   1.043   1.000   1.000   1.000

3.000   2.737   2.439   2.351   2.281   1.877   1.439   1.193   1.105   1.018   1.000   1.000

3.333   3.020   2.686   2.569   2.510   1.980   1.451   1.216   1.078   1.039   1.000   1.000

4.114   3.705   3.091   2.886   2.727   2.045   1.455   1.205   1.114   1.068   1.045   1.000

 

There is no plateau on the Ab curve. Then we increased the concentration of our coating Ab to 80ug/ml in the first wells. We still did'nt see the plateau.

I think 80ug/ml is a lot and we couldn't increase the Ab concentration anymore. I dont' know what's the problem and what we should do next step. Please help.

Thank you.


Edited by ylu, 28 January 2015 - 01:16 PM.





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