Antibody Dilutions

Not only the type (e.g., the cell clone) and the source of availability of an antibody but also its dilution are important in fully utilizing the effectiveness of an antibody as a powerful tool to detect antigens. The optimal antibody concentration for antigen varies, depending on whether the tissue used is aldehyde-fixed or frozen generally higher antibody concentrations are required for sections of aldehyde-fixed tissues (Fig. 4.1). 

different forms of an antigen require different concentrations of the antibody for their maximal detection. This is exemplified by the PC-10 primary antibody, which identifies PCNA antigen at a dilution of 1 1000 in epithelial cells in normal colon tissue, whereas a dilution of 1 400 is required to localize these proliferating cells in adenomatous polyps (Holt et al., 1997). In contrast, some types of antigens (e.g., Ki-67) can be optimally detected in various tissue types at the MIB-1 dilution of 1 50, using the microwave heating antigen retrieval method. However, in a few studies MIB-1 dilutions of 1 20 to 1 100 have been used. 

FIGURE 4.1. Effect of antibody (PC 10) concentration on the immunostaining of PCNA antigen in rectal mucosal proliferating cells. (A) The antibody was used at a concentration of 1 100. (B) The antibody was used at a concentration of 1 400. Note the increased labeling, especially in the upper crypt, when antibody concentration of 1 100 was used. Arrows indicate the upper limit of labeled cells. Reproduced, with permission, from Holt et al. (1997). Copyright 1997 American Association for Cancer Research. 

Some evidence indicates increased labeling efficiency of certain diluted antibodies (e.g., antiamylase antibodies and anti-MHC class II antibodies) when they are exposed to microwave heating prior to their application (Chicoine and Webster, 1998). However, such 

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Optimization of the immunoassay was performed with respect to tracer and antibody concentrations to obtain the required sensitivity. These conditions differed depending on the detection system used photographic detection required higher antibody and tracer concentrations than when the plate luminometer was used. A further complication arose from the very low affinity of the tracer for the antibody when using an antibody dilution of 1 3000 and a tracer dilution of 1 4000 less than 1% of the tracer was bound after a 2-h incubation. This means that the antibody, in the absence of clenbuterol, binds less than 10 pg of the... 

Omission of primary antibody. If positive, it shows unspecific binding of secondary antibodies. Dilute or try another secondary antibody. 

Incubate sections for 30 60 min at room temperature or overnight at +4°C with primary antibody diluted in buffer. If your primary antibody is biotinylated, you may omit the next two steps. 

The number and duration of washing steps can help with the reduction of background. As a rule of thumb, the volume of the wash should be twice the antibody dilution volume, and standard wash time should be 5x 4 min or 3x 10 min. Washing solutions used are PBST or TEST as buffering base, but Tween-20 is sometimes omitted when there is the chance that a low affinity antibody or a weakly bound antigen could be washed away. 

Incubate slides in a moist chamber in primary antibody diluted in TBS plus 1% BSA for 1-2 h at room temperature. Plastic-embedded sections may need to be incubated for 2 h at 37°C. 

If required, incubate 30 min at room temperature in bridging antibody diluted in TBS plus 1% BSA, and rinse five times in TBS plus 1% BSA. 

Primary antibody usually a monoclonal antibody diluted in PBSG with 10% bovine serum albumin see Note 1). 

Primary antibody diluted in PBS without calcium and magnesium, and 1% BSA or TBS with 1% BSA see Note 2). 

Rinse the grids five times in PBS without calcium and magnesium or in TBS. Incubate for 1-2 h at room temperature in primary antibody diluted in PBS without calcium and magnesium containing 1% BS A or in TBS containing 1% BSA. Rinse as in step 10. 

Incubate grids for 60 min with primary antibody diluted with TBS containing 1% BSA or 5% normal goat serum (see Note 4). 

Improper antibody dilutions Use of concentrated antibody solutions can cause high background. This is especially common when changes are made in the incubation times of a procedure. For example, a 2-h incubation at room temperature is lengthened to 18 h at 4°C. It may also be observed when different specimen types (paraffin vs frozen section) are being tested. 

I. Antibody dilution is established empirically. The dilution ratio is usually ranged from 1 500 to 1 10,000. The signal intensity... 

A series of antibody dilution tests prior to the combined ISH/ IHC can give a good hint of optimal antibody dilution. Best fluorescence images are obtained when the intensity of the individual fluorophores are balanced. 

When blocking is finished, blots can be dried on air, are stored frozen, or are incubated with the proper antibody dilution in Soln. A. If antisera are used, the dilution should be 1 100 at least to prevent non-specific adsorptions. Purified polyclonal or monoclonal antibodies are mostly diluted much higher. 

Incubation of blotted proteins with antibody dilutions should not last less than 30 min and not more than 2 h at RT or may be kept in the refrigerator overnight. 

Dilute the affinity-purified capture antibody to lO-lOOpg/ml in Soln. A. Incubate a new piece of blotting membrane with the antibody dilution (0.2-0.5 ml/cm ) at RT for 30 min. 

Wipe off remaining antibody dilution after incubation with filter paper and incubate the wet membrane in Soln. B for 30 min. Rinse with Soln. C and stop the fixation by incubation with Soln. D for 2 min. Block with Soln. E for 30 min, wash three times with Soln. C, and dry in vacuo. 

Incubate the wet strip, or parts of it, in antibody dilution in Soln. A at RT for 30 min. The incubation volume should be 0.5-1 ml per cm. This volume maybe reduced if the incubation is done in a lockable tube on a roller desk. Wash the strip in a sufficient volume of PBS, at least three times for 5 min each. 

Pipet 100 pi of the antibody or antiserum dilution (for titer determination a serial dilution in PBS), blank (buffer without antiserum) and, if available, controls into the respective wells and incubate on a shaker at RT for 30 min. Remove the antibody dilution and wash three times with 250 pi of Soln. B each. 

Incubate with the biotinylated secondary antibody (30 min, antibody dilutions of 1 200 are often suitable)... 

To saturate serum transferrin with iron, 10 pi of serum and 10 pi 200 pM Fe(III)-citrate are added to 30 pi of double-distilled water. After incubation for 10 min at room temperature the mixture is diluted 1 50 with double-distilled water. A 1 -pi aliquot is loaded onto the PhastGel sample applicator 8/1 and IEF is performed as described above. After separation, gels are incubated with rabbit anti-human transferrin antibody (dilution 1 3 in 150 mM NaCl) for 40 min followed by washing in... 

Empty the wells, and add 100 pL of antibody diluted in TMT/well. Suitable starting dilutions are 1 in 50 for antisera, 1 in 2 for hybndoma culture supernatants, and 1 in 500 for hybndoma ascites fluids Serially dilute antibody in doubling dilutions down one row of the microtiter plate (l e., eight dilutions)... 

Dilute the enzyme-conjugated secondary antibody in blocking buffer. Follow any recommendations that may be provided by the supplier regarding antibody dilution (see Note 13). 

The concentration of cells or nuclei is important. The procedure outlined in this chapter is designed for a starting density of 2—3 million cells. The volume of HC1 should be increased and the antibody dilution decreased if more cells are used. A cell or nuclei count is usually performed prior to step 6 in Section 3.3. 

Dry around the section with a soft tissue and incubate in the freshly prepared primary antibody (diluted 1/25 with PBS) at room temperature in a moist chamber. Allow 100 pL of antibody for each section (see Notes 6 and 7)... 

Wipe around the sections as before and apply 100 pL of liquid containing both secondary antibodies diluted 1 100 in PBS-BSA and incubate in the humid chamber for 1 h. 

The dilution of the antibodies has to be determined empirically Polyclonal antibodies can usually be diluted between 1.50 and 1.250 Monoclonal antibodies in tissue culture supernatant may need to be concentrated by centrifuging a frozen sample m a microfuge for 10 min and discarding the top third of the solution. This solution (100 pL) can then have the other primary antibody diluted into it in place of PBS-BSA Monoclonal antibodies produced as ascites can usually be diluted between 1-200 and 1 1000. 

Incubate the sections with an appropriate dilution of specific antibody diluted with BSA-PBS for 1 h The concentration of antibody is found by immunolabeling at different concentrations until a satisfactory signal noise ratio is achieved Suitable starting dilutions for monoclonal antibodies are 1 5, 1 10, and 1.20 For polyclonal antibodies, 1.10,1 50, and I T00 are recommended. 

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