Isotype control

Hummasti, S., and Tontonoz, P. The peroxisome proliferator-activated receptor N-terminal domain controls isotype-selective gene expression and adipogenesis. Mol Endocrinol 20... 

Add diluted mouse anti-CCL21 and rabbit anti-CCR7 antibodies (in the Antibody Diluent provided with the PLA Probe kit) to the tissue. As negative controls, isotype-matched control IgG antibodies were added at same dilution. 

Compared to people in a noncontaminated area, plasma IgG levels were also significantly decreased in proportion to increasing plasma levels of TCDD in a cohort exposed in an industrial accident in Seveso, Italy.118 There was no effect on IgM or IgA levels, or on complement levels IgE was not measured. In separate studies, in vitro exposure to TCDD enhanced the spontaneous production of IgE by B cells isolated from atopic but not non-atopic individuals, but did not affect the levels of other isotypes.119 Other recent studies have reported small changes in immune cells from individuals exposed occupationally to PHAH.120121 For example, compared to unexposed controls, a cohort of men exposed occupationally to TCDD had diminished IFNy production in a recall response to tetanus toxin, while IFNy production following polyclonal activation was unaffected.120 This observation is consistent with mouse studies, in which antigen-specific responses are highly suppressed by TCDD, but mitogen-driven T cell responses are less susceptible to impairment.83 88122123... 

Figure 7.9. FcyRI expression in blood and synovial-fluid neutrophils. Neutrophils were isolated from the blood (trace ii) and synovial fluid (trace iii) of a patient with rheumatoid arthritis. Expression of CD64 (FcyRI) was then measured by FACS analysis. Trace (i) indicates the fluorescence distribution of a non-immune isotype control monoclonal antibody. Similar results were obtained in 6 out of 11 patients with rheumatoid arthritis.

Plasma clearance (Cl), blood-brain barrier permeability surface area product (PS) and accumulation as % injected dose detected in brain tissue (%ID tissue) at 1 h after administration. Results show free [ H]-daunomycin (Daunomycin), [ H]-daunomycin encapsulated in conventional liposomes (Liposomes), sterically stabilized liposomes (PEG-liposomes), immunoliposomes (29 0X26, where 29 designates the number of 0X26 mAb conjugated per liposome) and control immunoliposomes where the 0X26 mAb was replaced by a non-specific isotype control antibody (IgG2a). Values are means SEM of n = 3 experiments. 

Positive selection is not recommended as these procedures may induce signaling pathways in CLL cells due to antibody/antigen interactions. Purity of CLL cells is assessed by flow cytometry using anti-CD19, anti-CD5, and matching isotype control... 

Wash three times with PBST, and determine the amount of radioactivity bound Include MAbs of known isotype as controls (see Note 13)... 

Isotype-matched unconjugated or fluorochrome-labeled antibodies unreactive with mammalian cells are available as control reagents from many suppliers. 

Dispense 100 pL of fluorochrome-labeled antibody solution (10 pg/mL) into tubes (see Note 3) Control fluorochrome-labeled antibody of the same isotype should be used m control tubes at the same concentration... 

MAb to PCNA and Ki-67 with the appropriate isotypic antibody controls—secondary antibodies if a two step staining is required... 

Resuspend pellet in 90 pL of antibody staining buffer containing BSA (Section 2), and add 10 pL (see Note 7) of MAb The recommended antibodies are PC 10-FITC (DAKO F863) and Ki-67-FITC (DAKO F788). The appropriate directly conjugated isotypic control antisera are also available from DAKO The suspensions are incubated for 1—2 h at room temperature and protected from light... 

Examples of typical staining patterns are shown in Fig 2 These profiles are analyzed for positivity by setting a lower limit of detection based on the isotypic control sample In our laboratory, we would normally set a region on the control that includes < 1 % of positive events and transpose this region to the M Ab-stained samples. 

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

Fig. 6.4. The fluorescence histogram of an isotype control sample is used to decide on the fluorescence intensity that indicates positive staining.

Such a control antibody is known as an isotype control because it is of the same immunoglobulin isotype (subclass) as the staining antibody used in the experiment. It will allow you to determine how much background stain is due to irrelevant stickiness (dead cells, Fc receptors, and so forth). The only trouble with this scenario is that exactly correct isotype controls are not usually available. Various manufacturers of monoclonal antibodies will sell so-called isotype controls and will certainly recommend that they be used. These are, however, general purpose isotype controls that will be of an average... 

Fig. 6.5. The use of a phycoerythrin (PE) isotype control to help in deciding where, in a dual-color plot, to draw the horizontal line between fluorescein-stained cells to be considered positive and those to be considered negative for the PE stain. Misplacing of the horizontal line will affect the number of CD19 cells determined to express the CD5 antigen in the stained sample. Data courtesy of Jane Calvert.

For monoclonal primary antibodies, nonspecific negative reagent controls may be developed by different methods. The optimal method is an antibody of the same isotype, present in the same immunoglobulin concentration, using the same diluent and exhibiting no specific reactivity with the given human tissues tested. A less optimal alternative is to use mixtures of antibodies representing all or most relevant IgG subtypes. Finally, the diluent itself may also be used as an alternative which, however, is neither efficient nor desirable. 

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