Issue 4 Summer 2002 - Applied Biosystems

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26 new product review Affinity Depletion Cartridges For Removal of Abundant Proteins from Human Serum Purpose Removal of abundant proteins from human serum using a highly specific immunoaffinity chromatography support. Overview One of the major difficulties in analysing the proteome of human serum is the dynamic range of the concentrations of the proteins present in the sample. Human serum albumin (HSA) constitutes 57-71% of total serum protein and gamma-immunoglobulin (IgG) ranges from 8-26%. Removal of these two proteins alone clears about 75% of the total protein present in serum, therefore allowing the detection of the remaining proteins that are present in far lower concentration. Here we describe the characterisation of anti-HSA and Protein G affinity chromatography cartridges, which remove HSA and IgG from human serum, with respect to their percentage depletion, non-specific binding and cross-reactivity. Key features ➜ Highly specific, immunoaffinity ligand, exhibiting very low to zero non-specific binding ➜ Proven POROS ® support for high throughput processing and cleanability ➜ Long lifetime and reusability ➜ Process samples by automated (VISION workstation) and manual modes using unique cartridge format Chromatography Media Development The new POROS anti-HSA support was developed using antibody ligand that has been optimised to specifically bind human albumin. Immobilisation of this immunoaffinity ligand was to POROS Perfusion chromatography media using polyethylene glycol spacers. Using the 0.2ml cartridge device, we have shown that the media will completely bind the albumin in a 10-70µl sample of human serum that has been diluted 1:10 (albumin concentration of 3.5mgs/ml). In combination with the Protein G cartridge, both albumin and IgG can be effectively removed in one step. Technology Application Experimental Goal In this work, a sample of human serum was passed over both a Protein G and Anti-HSA cartridge. The flow through (serum proteins) and eluted fractions (albumin and IgG) were analysed by 1 dimensional and 2 dimensional SDS PAGE gel, as well as a commercially available ELISA assay. This is in order to quantitate the removal of HSA and IgG from the sample, and examine the level of non specific binding. Bands from the 1D Gel were further analysed using peptide mass fingerprinting analysis on the Voyager workstation in order to identify visualised bands that did not correspond to the intact molecular weight of HSA. Comparisons of binding capacities of albumin from different species were also compared to determine cross reactivity. Experimental Conditions 70µL of human serum diluted 1:10 with Phosphate buffered saline (PBS) was passed through first to a 4mmDx15mmL (0.2ml) Protein G cartridge and then the flow-through FT fraction was diluted to 400µL. Then 100µL of the diluted protein G FT fraction was applied to a 4mmDx15mmL (0.2ml) anti-HSA cartridge at a flow rate of 0.5mls/min. Elution of each cartridge was done using 3 mls of 12mM HCL at 1ml/min and the cartridge was then cleaned with a 5ml step of 1M NaCl. All chromatographic steps were carried out using the VISION Workstation and the peak FL and eluted EL fractions were collected for further analysis. Protein concentration for each fraction was determined using Bradford assay. Equal amount of protein (6.5µg) from each fraction was analysed by SDS-PAGE. 2-D gel analysis was done using 200µg human serum, before and after affinity depletion by both cartridges and the proteins visualised by silver stain. MALDI-TOF peptide mass fingerprinting (PMF) was performed on bands from 1D Gel that did not correspond to the intact molecular weight of human Albumin using Voyager-DE STR Biospectrometry Workstation from in-gel digested peptides using 10µg/mL modified bovine trypsin in 25mM ammonium bicarbonate. Anti-HSA and anti-human IgG ELISA kits (Bethyl Laboratory) was performed according to the manufacture protocol. The FT fractions used for the ELISA were pooled from multiple runs (six runs from two anti-HSA cartridges from the same lot and four runs from two Protein G cartridges from the same lot). new product review Results and Discussion Figure 1 shows the chromatograph of affinity depletion of HSA from human serum using POROS and anti-HSA antibody affinity cartridge operated on the VISION Workstation. The sample was previously run over a POROS protein G cartridge in order to remove IgG from the solution. Figure 1. Chromatograph of affinity subtraction using POROS Anti-HSA cartridge Absorbance (A280) Figure 2 shows SDS-PAGE analysis of serum sample (lane 2) the FT and EL fractions and the enrichment of low intensity bands in the protein G and anti-HSA FT fraction (lane 5). The predominant proteins in the eluted fractions (lanes 4 and 6) are IgG heavy chain (55kDa) and light chain (25kDa) and HSA (66kDa) respectively. To further characterise the proteins eluting from the anti-HSA cartridge, peptide mass fingerprinting was performed on low intensity bands. That did not correspond to the intact molecular weight of Albumin. Most of these bands were identified as proteolytic fragments of Albumin. Figure 2. 4-20% SDS PAGE gel stained with coomassie blue. All lanes loaded with 6.5µg total protein. Bands A,B,D were identified as Albumin fragments, C was identified as Albumin fragments and Keratin. Lane 1 Molecular weight markers Lane 2 Serum sample diluted 1:10 with Phosphate buffered saline Lane 3 Flow through of POROS Protein G cartridge Lane 4 Eluted fraction of POROS Protein G cartridge Lane 5 Flow through of POROS Protein G and POROS Anti-HSA cartridges Lane 6 Eluted fraction of POROS Anti-HSA cartridge page 28 27
28 new product review Figure 3 shows an expanded region of a 2 dimensional gel the enrichment of low intensity protein spots before and after the affinity depletion using 2-D gel. In many cases, protein spots that were barely visible in a normal sample became much more intense upon depletion of the albumin and IgG. Figure 3. Expanded section of 2-Dimensional Silver stained gels. Top gel is serum sample before removal of IgG and HSA. Bottom gel is serum sample after processing on POROS Protein G and Anti-Hsa cartridges. Circled spots represent increase in concentration of some low abundant proteins Figure 4 shows results of measuring the removal of IgG and albumin from the flow through fraction of both cartridges. Removal of both IgG and HSA was shown to be greater than 99%. Figure 4. Results from measurement of IgG and HSA in the serum sample using an ELISA assay, before and after affinity subtraction using the POROS Protein and Anti-HSA cartridges. The cross reactivity of the Anti-HSA antibody was also examined to determine binding capacity for various species of Albumin. Table 1 shows the capacity measurements made on the media using purified forms of albumin from various species. Although albumin from other species do bind with some specificity, this antibody demonstrates the highest specificity for Human Serum Albumin. Table 1. Cross reactivity of Anti-HSA media for albumin from various species. Capacity measurements are based on 10% breakthrough capacity measured on a 2.1mmDx100mmL column. Species mg Bound mMoles Bound per ml media per ml media Bovine 0.24 0.004 Goat 0.01 0.000 Human 2.04 0.031 Mouse 0.70 0.011 Porcine 0.52 0.008 Rabbit 0.31 0.005 Rat 0.75 0.011 Sheep 0.01 0.000 Conclusions POROS Protein G and Anti-HSA cartridges can quickly and efficiently remove both IgG and HSA, which are the two most abundant proteins in human serum. Using this immunoaffinity technique greater than 99% of both proteins can be removed from serum with very low to zero non-specific binding of other proteins in the sample. Using a convenient cartridge format, samples can be processed manually or using an automated LC system to increase throughput. Capacity for human serum Albumin was measured at around 2.5mgs/ml. For more information on: Affinity Depletion Cartridges enter: No. 420 Moving forward in Genomic Research Assays-by-Design service Assays-by-Design service is a custom assay design service dedicated to providing quality controlled assay products for gene expression and SNP genotyping needs. You simply submit your target DNA sequence, and Applied Biosystems returns a QC-verified, 5' nuclease MGB assay that is ready to use with TaqMan ® Universal PCR Master Mix and to load on your sequence detection system platform of choice. Assays for any DNA sequence of any species can be generated and delivered in a convenient single tube format. Assays-by-Design service; ➜ shortens your path to results ➜ lets you focus on research questions, not on designing assays ➜ eliminates the manual task of designing primers and probes ➜ eliminates assay optimisation, saves time and labour ➜ is designed to ensure a successful experiment See also pages 13 & 46 for related articles For more information on: Assays-by-Design Service and Assays-on-Demand Products enter: No. 421 new product review Applied Biosystems Introduces the New Assays-by-Design SM Service and Assays-on-Demand Products Assays-on-Demand products The introduction of the Assays-on-Demand products for SNP genotyping and gene expression studies for all human genes, signals Applied Biosystems’ next major innovation to move genome analysis to the next level by changing the point of departure for genetic research from gene sequence to gene information content. Off-the-shelf products for 30,000 genes respectively 200,000 SNPs; researchers will be able to order these validated assays by gene name or attribute without the requirement of knowing the gene’s sequence or oligonucleotide design techniques. Just add your sample and go! Assays-on-Demand products; ➜ provide rapid, quantitative gene expression or SNP genotyping results ➜ eliminate manual design, optimisation and functional testing ➜ allow direct and accurate comparison of data between labs ➜ ensure high quality, high performance and reliability For more information on Assays-on-Demand please visit our web store at https://store.appliedbiosystems.com or contact your local Sales Office. Registered customers can order assays on-line from this website*. Feel free to re-visit this site periodically as new assays are continually being added, fully released by summer 2002. * Please contact your local Applied Biosystems office if you don’t find your country in the drop down menu on the registration page 29
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