Interfering HA (e.g. HAMA) Mouse IgG
Mouse IgG
Why use blockers such as Mouse IgG?
IgM RF
Blockers are used in ELISA and LF assays to reduce interference from proteins in patient samples that could produce false results and an incorrect diagnosis. Specifically in double mouse monoclonal assays, blockers are required to remove particular types of HA interference called human anti-mouse antibodies (HAMA) and Rheumatoid Factor (RF). Passive blocking reagents such as Mouse IgG work by preventing interfering antibodies from binding to the capture or detection antibodies by providing alternate binding sites. Mouse IgG must be added in excess concentration and the effectiveness depends on the affinity of interfering antibody for the animal IgG.
Detection Antibody (mouse origin) Capture Antibody (mouse origin) Antigen
Blocking Buffer
Figure 2. Blocking antibodies such as mouse IgG neutralize the interfering antibodies present in serum samples resulting in accurate binding of the analyte of interest by the capture and detection antibodies.
Mouse-FREE IgG Performance Data
A) HAMA Interference
100
100
100
80
80
69
66
60
60
42
40
40
20
20
0
0
Mouse-FREE IgG
Native Mouse IgG
Competitor Blocker #1
Competitor Blocker #2
Blocker concentration ( µ g/ml)
B) RF Interference
100
100
100
80
80
60
60
40
40
20
20
14
14
11
0
0
Mouse-FREE IgG
Native Mouse IgG
Competitor Blocker #1
Competitor Blocker #2
0.01
0.1
1
10
100
Blocker concentration ( µ g/ml)
Native mouse IgG | Mouse-FREE IgG | Competitor Blocker #1 | Competitor Blocker #2
Figure 3. A double mouse monoclonal sandwich assay was used to compare the performance of Meridian’s Mouse-FREE IgG (100% animal-free, recombinant Mouse IgG, Cat No. BN1300) against serum-derived purified native mouse IgG and two competitor HAMA blockers. (A) Human HAMA-positive or (B) RF-positive serum was preincubated with the individual blockers for 20 minutes at room temperature. Blocker concentrations ranged from 0.0003-1.25 μ g/mL for the HAMA assay and 0.0195-80 μ g/mL for the RF assay. The effectiveness of each blocker was determined by comparing the relative suppression of the HAMA or RF signal – lower EC50 values indicate greater interference suppression. The results demonstrate that Mouse-Free IgG is significantly more effective than native mouse IgG and the two competitor blockers at blocking HAMA and RF interference.
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