Antibody selectivity filter

The limitations of ELISA methods include the specificity of antibodies, the concentrations of primary antibody and antigen, and the type of reaction solution. Nonspecific binding of either of the antibodies to related antigens, unrelated proteins of other bacteria, or even the microtiter plate may lead to false positive reactions.49,52 57 Use of a monoclonal antibody may decrease crossreactivity with other antigens. For detection of low numbers of bacteria, as in drinking water, the sample may be filtered to concentrate the cells or cultured in a selective broth until it reaches the minimum detection limit for ELISA.49,58 Commercial test kits using dipsticks, immunoblots, and sandwich ELISA methods have been developed for the identification of pathogenic bacteria.58,59... 

Biological autofluorescence in mammalian cells due to flavin coenzymes (FAD and FMN absorption, 450 nm emission, 515 nm) and reduced pyridine nucleotides (NADH absorption, 340 nm emission, 460 nm) can be problematic in the detection of fluorescence probes in tissues and cells. Fixation with aldehydes, particularly glutaraldehyde, can result in high levels of autofluorescence. This can be minimized in fixed cells by washing with 0.1% sodium borohydride in phosphate-buffered saline (5) prior to antibody incubation. Problems due to autofluorescence can be minimized by selecting probes and optical filters that maximize the fluorescence signal relative to the autofluorescence. Other factors that limit IF include the performance of the detection instrument (i.e. how well the microscope has been calibrated and set), the specificity of the antibodies, and the specimen preparation. 

Fig. 9.5 Simultaneous identification of binders of two antigens on a single filter, (a) scheme of the screen with two antigens each labeled with a different fluorophore. (b). Similar numbers of colonies from two independent selections (done on each antigen separately) were plated on the Supor master filter (the left half in (b)). The antibodies that diffused down into the cellulose acetate filter were developed with the fluorescently labeled antigens mixed together at 10(lg/ml each. The filters were washed and analyzed using a Typhoon 8600 reader (Molecular Dynamics). The right-hand half in (b) is a mirror image of the left filter with bright spots were green and dimmer spots marked by arrows were red...

Direct detection of antibody against insulin in patient sera using an SPR sensor Biacore 2000 is presented in [50]. Purified human insulin was used as a biorecognition element and immobilized on the sensor surface via amine coupling chemistry. Test sera samples were pretreated to remove insulin and filtered before SPR measurements. Insulin antibodies were detected in eight selected patient sera samples and fell in the range 2.91-16.3 xgmL ... 

---