2 antibody controls nonspecific binding

For the negative control, primary antibody is replaced with an irrelevant, isotype-matched antibody. This step controls nonspecific binding of the secondary antibody. The... 

Blocking may need to take place for longer than 2 h if nonspecific binding of the antibody takes place. This can be determined by examining the negative control wells (5). 

First and foremost, the antibody being used should recognize denatured antigen. Nonspecific binding of antibodies can occur, so control antigens and antibodies should always be run in parallel. Time of transfer and dilutions of primary antibody and conjugate should always be optimized. 

NOTE A special situation to be aware of is when two or more antibodies are applied to serial sections. In this case, negative stain areas of one slide may serve as the negative/nonspecific binding background control for other antibodies. 

In the control experiment to test for nonspecific binding, the cells are treated with nonspecific rabbit serum prior to Incubating in goat anti-rabbit Ig antibody-latex conjugate. 

The specificity of the immunolatex markers can be demonstrated in control experiments designed to measure the amount of nonspecific binding to cell surfaces. RBC, which have not been sensitized with rabbit antihuman RBC antibodies, are treated with goat anti-rabbit Ig antibody-latex conjugates (20 mg/ml) and washed in the usual manner. As shown in Figure 7b, only a few spheres adhere to the surface of the red blood cells. 

Validation of the result is essential and is achieved through the use of positive and negative controls to assess the method (e.g., labeling with an alternative antibody, using a known positive system as positive controls and determining nonspecific binding as a negative control). 

Besides choosing the appropriate Ab, the assay method can be designed or manipulated to improve assay specificity using (a) protein precipitation, (b) liquid/liquid or solid-phase extraction, (c) HPLC separation of the analyte from the interfering compounds, (d) sample dilution with buffer or control matrix, or (e) an affinity solid phase (e.g., antibody-coated microtiter plate or polystyrene beads) to capture the analyte followed by wash steps. Affinity-purified antibodies and protein blockers are used in EIA to decrease nonspecific binding in plate assays. Increasing incubation time to reach equilibrium also improves binding specificity. 

The optimal number of cells, as well as the primary antibody concentration should be determined in preliminary experiments by serial-dilution assays. A range of 0.1-1 x 105 cells/ well and primary antibody at 1-10 pg/mL for purified antibody. When the antibody is hybridoma supernatant, a 1 2 dilution may be used. Both negative and positive controls should be included to account for any nonspecific binding. 

Cons Require species-specific reagents, which can present problems in terms of suitable assay controls, particularly when using an animal semm as a positive antibody control for the assay. May suffer from nonspecific binding. 

Coronal cryostat sections (10 pm thick) are cut after cryoprotec-tion and processed for indirect IMF. Sections are first incubated for 60 min with 10 % NGS or 10 % normal donkey serum in PBS to block nonspecific binding. They are then incubated with antibodies diluted according to manufacturers recommendations 1 h with primary antibody and then 1 h with secondary antibody diluted 1 100. Double IMF is performed according to standard protocols [48]. Mounting media contain DAPI to stain nuclei. Negative controls are performed each time immunostaining was done and consisted of preincubation with PBS, substitution of nonimmune isotype-matched control antibodies for the primary antibody, and/or omission of the primary antibody. Further details can be found in [48]. 

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