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FEATURE Laboratory diagnostics


destroy it. In other instances an antigen may contain unknown or conformational epitopes, which can only be preserved by purifying the antigen in its native state using complicated and time-consuming procedures. The use of in vivo expressed antigens circumvents these hurdles and enables the diagnostic application of assay parameters that might otherwise languish in research laboratories. A further advantage of cell-based


assays is that they are relatively fast and easy to develop. So when a new target antigen is identified by research groups, a corresponding cell-based IIFT system can be developed and brought to market much faster than immunoassays based on purified antigens. Thus, serological diagnostics and ultimately patients benefit much sooner from advances in antigen characterisation.


“The use of in vivo expressed antigens circumvents these hurdles and enables the diagnostic application of assay parameters that might otherwise languish in research laboratories”


In some cases antibody binding can be


boosted by modifying the target antigen. Where beneficial, the antigens used in cell- based assays are specially designed and/or adapted by molecular biological techniques to enhance their performance. For example, by selecting only relevant antibody-binding epitopes and deleting protein regions that cause unspecific reactions, the sensitivity and specificity of a test can, in some cases, be significantly increased.


COMPREHENSIVE, STANDARDISED ANALYSIS Often it is advantageous to produce a detailed patient antibody profile to aid disease diagnosis and exclude other causes. To facilitate this, new cell-based substrates can be analysed side by side with classic tissue sections using proven BIOCHIP Mosaic technology. BIOCHIP Mosaics consist of miniature sections


of different substrates positioned next to each other in the test fields of a microscope slide (figure 2). The substrates are incubated in parallel, which reduces workload and enhances reproducibility. Hence, an extensive antibody analysis can be obtained quickly and reliably. Further standardisation is achieved by choosing a microscope that is tailored to the requirements of immunofluorescence. State-of-the-art IIFT microscopes incorporate a controlled LED, which ensures highly reproducible results while providing cost-effectiveness through a long life span and low power consumption. The efficiency and convenience of IIFT can be further boosted by automation. Virtually the entire IIFT procedure, from sample dilution and incubation to evaluation and archiving of results, can be automated using specialised devices and software.


AUTOIMMUNE DIAGNOSTICS The detection of characteristic autoantibodies is a central element in the diagnosis of many autoimmune diseases. Autoantibody analysis is also frequently employed to monitor the activity of an autoimmune disease or a patient’s response to treatment. Below are some examples of how cell-based IIFT systems are now being used to support the diagnosis of autoimmune diseases of the skin, nervous system, gastrointestinal tract and kidneys.


AUTOIMMUNE BULLOUS DERMATOSES Autoimmune bullous dermatoses are a group of severe blistering diseases, which are characterised by circulating autoantibodies directed against various structural proteins of the skin. Autoantibodies against the desmosomal proteins desmoglein 1 and desmoglein 3 occur in patients with pemphigus diseases, notably pemphigus foliaceus and pemphigus vulgaris. Two new IIFT systems based on transfected cells expressing tailored recombinant antigens allow these autoantibodies to be determined with ease (figure 3). In clinical studies the assays yielded sensitivities of 100% for pemphigus vulgaris and 90% for pemphigus foliaceus at specificities of over 99%. Moreover, this analysis allowed a prima vista differentiation between these two disease forms.


FIG 1


PRINCIPLE OF IIFT USING CELLS TRANSFECTED WITH PLASMIDS EXPRESSING NOVEL ANTIGENS


FIG 2 BIOCHIP MOSAIC


Slide


Combination of BIOCHIPS


Autoantibodies against the glycoprotein BP230 occur in bullous pemphigoid and pemphigoid gestationis. Their determination enriches the diagnosis of these diseases by an additional, independent parameter, supplementing existing markers such as anti-BP180. A new cell-based immunoassay for determination of these autoantibodies yielded a sensitivity of 44-55% and a specificity of 100% for bullous pemphigoid in clinical studies. BIOCHIP Mosaics combining the new


cell-based substrates with classic tissue sections provide the most wide ranging analysis of autoantibodies occurring in autoimmune epidermal blistering diseases in a single test, and thus represent a powerful diagnostic tool for dermatologists. 


MedLab Issue 2 2011 27


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