I am working in developing ELISA for anti-HCV detection. I am using biotinylated peptides as a capture on streptavidin coated plates. My detection system is protein-A conjugated to HRPO.
Now I have encountered one sample which is giving false positive in the above system. Further investing, I find that the sample is having anti-streptavidin antibodies and hence giving false reaction.
Does anyone have any suggestions for blocking anti-streptavidin antibody?
The possible interferences: 1) cross-reactivity with endogenous and exogenous non antibody-structured substances; 2) cross-reactivity with endogenous and exogenous antibody-structure substances; 3) the hook effect; and 4) the matrix effect.
Special attention must be paid to assays with bedside measurements where is also reported about interference of heterophilic antibodies.
Protein-A sepharose pre treatment corrected the nonlinear dilution and revealed an interference trend of falsely decreased results, as measured by sandwich assay, and falsely elevated results, as measured by competitive assay.
To date, there has been no report in the literature of interference from endogenous anti-streptavidin antibodies; however, such antibodies would potentially affect multiple diagnostic platforms.
Protein-A sepharose pre treatment corrected the nonlinear dilution and revealed an interference trend of falsely decreased results, as measured by sandwich assay, and falsely elevated results, as measured by competitive assay.
Removal of interfering antibody
1) Extraction of analyte from sample
2) Immuno extraction by addition of murine or other animal species serum
3) immobilized onto Sepharose beads or immobilized Protein A suspension Polyethyleen glycol precipitation (PEG 6000)
4) Heating to 70-90°C for heat-stable analytes
Addition of blocking agent from the same species as the antibody reagents The inclusion of one or more blocking agents in manufacturers’ immunoassay
1) Reagent may be insufficient to overcome the interference Non-immune serum, species-specific polyclonal IgG, anti-human IgG
2) Polymerized mouse IgG Non-immune mouse monoclonals
3) Species-specific fragments of IgG (Fc, Fab)
4) Heterophilic blocking reagents (HBR), immunoglobulin inhibiting reagent (IIR) and antibody blocking tubes
It is important to recognize the potential for interference in immunoassay and to put procedures in place to identify them wherever possible. Most important is consideration of the clinical picture.
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