Identifying Autoimmune Disease Biomarker Panels Using Human Protein based Microarrays - 0 views

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  #1 Stef Barnes on 21 Jun 12

   

  INTRODUCTION
  
  [Need an autoimmune certain introduction].
  
  Nevertheless the use of serological methods remains attractive because they are relatively non-invasive and can be performed quickly. For several many years, our group has been creating a high-throughput proteomic microarray platform which allows thousands of protein gene products or antigens being printed on a glass slide and used to interrogate sera from humans or animals (2). The arrays can be produced rapidly and to date we now have used with considerable success to recognize diagnostic and vaccine targets candidates in a number of pathogen systems including, tuberculosis (3) brucellosis [4] Chlamydia (5) together with Lyme disease (6). Lately we have used the strategy to express human antigensidentify brand-new targets of pemphigus auto-antibodies (7). The purpose of this study was to generate a preliminary assessment of that utility of proteomic microarrays printed with human autoantigens inside management of lupus.
  
  MATERIALS AND METHODS
  
  Serum Samples.
  Sera were studied with patients attending the autoimmune rheumatic disease clinic at the University College Hospital within the last 25 years. All patients with lupus met the revised criteria in the American College of Rheumatology (8). Those considered to have kidney involvement had to have had a confirmatory biopsy. People with Sjögren’s syndrome found the American European Complete Criteria (9); those with myositis had three out of the four of the requirements proposed by Bohan and Peter (10) and the ones with rheumatoid arthritis all had four or more of the revised criteria of the American Rheumatism Association (11).
  
  Antigen Selection.
  Human protein antigens in the following categories were selected for printing over the microarrays (i) proven autoantigens from autoimmune rheumatic diseases (ii) established autoantigens from organ-specific autoimmune diseases (iii) autoimmune condition associated molecules as referred to in recent literature (e. g. MHC molecules, complement components, signalling molecules) (4) immunological targets using disease modifying potential (i. g. cytokines, chemokines, linked receptors, co-stimulatory molecules, etc.) and (v) proteins without the need of known immune reactivity (since controls). In entire 797 proteins were picked for these initial trials.
  
  Human Protein Microarray Manufacture. Human gene clones were from the National Institutes associated with Health’s (NIH) Mammalian Gene Collection (MGC) since cDNA clones. Amplicons with the human genes were secured by PCR amplification associated with human genes from cDNA imitations. The primers (Sigma-Aldrich; Saint. Louis, MO) are made up of 20 base sets (bp) with gene-specific sequence and 20 bp with adapter sequences. The adapter sequences were that will be homologous to the cloning site with the linearized T7 expression vector pXT7 and allowed the PCR products to become cloned by homologous recombination within Escherichia coli DH5a cells. At the 5' end of the fusion protein, a polyhistidine (poly-His) fragment had been incorporated. The amplicons while using the flanking adapter sequences were raised for in vivo recombination cloning to a T7 promoter based plasmid expression vector. Once the expressible library was verified to support the correct inserts, the plasmids with human open reading frames (ORFs) were expressed using an in vitro transcription-translation system following the manufacturer's instructions (RTS 100 product; Roche; Indianapolis, IN). Microarrays have been printed onto nitrocellulose coated glass slides FAST slideshow (Whatman Inc., Piscataway, NJ) utilizing an OmniGrid Accent microarray lazer printer (DigiLab Inc., Holliston, MA). Protein expression levels were monitored in the microarrays by using anti-poly-His (clone His-1; Sigma-Aldrich; St. Proteome Microarray

   

  <div class="cArrow"> </div><div class="cContentInner">INTRODUCTION<br /><br />[Need an autoimmune certain introduction].<br /><br />Nevertheless the use of serological methods remains attractive because they are relatively non-invasive and can be performed quickly. For several many years, our group has been creating a high-throughput proteomic microarray platform which allows thousands of protein gene products or antigens being printed on a glass slide and used to interrogate sera from humans or animals (2). The arrays can be produced rapidly and to date we now have used with considerable success to recognize diagnostic and vaccine targets candidates in a number of pathogen systems including, tuberculosis (3) brucellosis [4] Chlamydia (5) together with Lyme disease (6). Lately we have used the strategy to express human antigensidentify brand-new targets of pemphigus auto-antibodies (7). The purpose of this study was to generate a preliminary assessment of that utility of proteomic microarrays printed with human autoantigens inside management of lupus.<br /><br />MATERIALS AND METHODS<br /><br />Serum Samples.<br />Sera were studied with patients attending the autoimmune rheumatic disease clinic at the University College Hospital within the last 25 years. All patients with lupus met the revised criteria in the American College of Rheumatology (8). Those considered to have kidney involvement had to have had a confirmatory biopsy. People with Sjögren’s syndrome found the American European Complete Criteria (9); those with myositis had three out of the four of the requirements proposed by Bohan and Peter (10) and the ones with rheumatoid arthritis all had four or more of the revised criteria of the American Rheumatism Association (11).<br /><br />Antigen Selection.<br />Human protein antigens in the following categories were selected for printing over the microarrays (i) proven autoantigens from autoimmune rheumatic diseases (ii) established autoantigens from organ-specific autoimmune diseases (iii) autoimmune condition associated molecules as referred to in recent literature (e. g. MHC molecules, complement components, signalling molecules) (4) immunological targets using disease modifying potential (i. g. cytokines, chemokines, linked receptors, co-stimulatory molecules, etc.) and (v) proteins without the need of known immune reactivity (since controls). In entire 797 proteins were picked for these initial trials.<br /><br />Human Protein Microarray Manufacture. Human gene clones were from the National Institutes associated with Health’s (NIH) Mammalian Gene Collection (MGC) since cDNA clones. Amplicons with the human genes were secured by PCR amplification associated with human genes from cDNA imitations. The primers (Sigma-Aldrich; Saint. Louis, MO) are made up of 20 base sets (bp) with gene-specific sequence and 20 bp with adapter sequences. The adapter sequences were that will be homologous to the cloning site with the linearized T7 expression vector pXT7 and allowed the PCR products to become cloned by homologous recombination within Escherichia coli DH5a cells. At the 5' end of the fusion protein, a polyhistidine (poly-His) fragment had been incorporated. The amplicons while using the flanking adapter sequences were raised for in vivo recombination cloning to a T7 promoter based plasmid expression vector. Once the expressible library was verified to support the correct inserts, the plasmids with human open reading frames (ORFs) were expressed using an in vitro transcription-translation system following the manufacturer's instructions (RTS 100 product; Roche; Indianapolis, IN). Microarrays have been printed onto nitrocellulose coated glass slides FAST slideshow (Whatman Inc., Piscataway, NJ) utilizing an OmniGrid Accent microarray lazer printer (DigiLab Inc., Holliston, MA). Protein expression levels were monitored in the microarrays by using anti-poly-His (clone His-1; Sigma-Aldrich; St. <a rel="nofollow" href="http://www.antigendiscovery.com/">Proteome Microarray</a></div>

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