Anti-ID antibody case study

Case study 1: Anti-idiotype monoclonal antibody

A1. Affinity ranking of antibodies by BIAcore.
A2. Affinity determination of clone 2 antibody in different concentration (nM)

A. ProBio can not only provide mouse anti-ID antibodies, but also provide rabbit anti-ID antibodies that usually have a high affinity at the picomolar.

B. Blocking assay

B. Competitive ELISA with different concentration of drug target could set as an optional step to distinguish the type of anti-ID antibodies. Ab1 and Ab2, which could bind to the antibody drug when competed with drug target, are non-blocking anti-ID antibodies, while Ab3 and Ab4 are antigen-blocking antibodies.

Capture mAb, 2μg/ml	Concentration of Antibody Drug (ng/ml)				Detection mAb,1μg/ml
Capture mAb, 2μg/ml	1000	100	10	0	Detection mAb,1μg/ml
mAb1	2.011	0.468	0.329	0.367	mAb1-biotin
mAb2	2.167	0.950	0.214	0.104
mAb3	2.108	0.335	0.154	0.135
mAb4	2.066	0.294	0.187	0.163
mAb5	2.044	0.342	0.119	0.102
mAb1	2.703	2.767	0.948	0.261	mAb2-biotin
mAb2	0.382	0.117	0.093	0.085
mAb3	2.904	2.662	0.799	0.305
mAb4	2.776	2.944	2.190	0.793
mAb5	2.977	2.711	1.347	0.565
mAb1	1.156	0.210	0.133	0.127	mAb3-biotin
mAb2	2.270	0.618	0.138	0.081
mAb3	1.332	0.210	0.102	0.087
mAb4	1.210	0.158	0.115	0.112
mAb5	1.290	0.218	0.097	0.089
mAb1	1.606	0.258	0.143	0.137	mAb4-biotin
mAb2	1.886	0.612	0.176	0.110
mAb3	1.802	0.297	0.126	0.117
mAb4	1.691	0.217	0.150	0.141
mAb5	1.711	0.320	0.142	0.133
mAb1	1.464	0.654	0.583	0.624	mAb5-biotin
mAb2	2.406	0.944	0.222	0.151
mAb3	1.343	0.265	0.169	0.156
mAb4	1.039	0.236	0.198	0.201
mAb5	1.278	0.251	0.118	0.104

C. Sandwich ELISA could be performed to select anti-ID pair which could largely increase the success rate of immunoassay development. As it was shown in the table, mAb2 is able to pair with other four clones (mAb1, mAb3, mAb4, mAb5).

Sensitivity (Limit of detection, LOD)
Coating Antibody	anti-ID-1
Detection Antibody	anti-ID-2-Biotin
Sample	Zero standard
OD 450 nm	0.053	0.054	0.052
	0.052	0.053	0.051
	0.052	0.053	0.05
	0.053	0.054	0.05
	0.053	0.055	0.053
	0.053	0.053	0.052
	0.052	0.053	0.051
Mean	0.053
SD
Mean+2SD	0.055
Standard Curve	y=0.16534x+0.09116 r^2 =0.99576
LOD	<0.2 ng/mL (~0.042 ng/mL)

D. After the antibody sensitivity analysis, the limit of detection (LOD) of the anti-idiotype Ab is typically less than 0.2ng/ml.

Case study 2: Anti-idiotype polyclonal antibody

A. Affinity-purified anti-idiotype polyclonal antibodies showed specific binding to antibody drug. The cross rate with isotype control is less than 2%.

B. After the antibody sensitivity analysis, the limit of detection (LOD) of the anti-idiotype pAb is typically less than 10ng/ml.

Sensitivity (Limit of detection, LoD)
Coating Antibody	Drug
Detection Antibody	Drug-Biotin
Sample	Zero standard
OD 450 nm	0.048	0.048	0.049
	0.046	0.047	0.049
	0.047	0.047	0.048
	0.049	0.047	0.047
	0.047	0.05	0.048
	0.048	0.047	0.049
	0.047	0.051	0.048
	0.048	0.048	0.046
Mean	0.048
SD	0.001
Mean+2SD	0.05
Standard Curve	y = 0.01719x + 0.04010, R² = 0.99987
Sensitivity (LoD)	0.576 ng/mL

Antibody stability

The antibody is generally stable for more than 5 years by accelerating the stability experiment, covering the time of the customer's clinical declaration process.