Frequently Asked Questions (FAQ)

Transcription

1 Frequently Asked Questions (FAQ) Why screen your (therapeutic) antibody for cross-reactivity? Cross-reactivity of therapeutic antibodies leads to adverse effects and might render the antibody unsuitable for therapeutic use. A cross-reactivity screening at an earlier stage of the evaluation is an efficient way to obtain cross-reactivity profiles and avoid unnecessary costs for further evaluation in pre-clinical or clinical stages. Cross-reactivity of antibody reagents used for discovery and routine work is a serious drawback for applications such as biomarker discovery and may lead to false positive results. Therefore a complete cross reactivity profile ensures reliable results. What are the advantages of using HuProt TM arrays for antibody specificity profiling? HuProt TM arrays offer the world largest protein content on one microarray glass slide. They contain more than 17,000 full-length proteins representing at least human genes covering more than 60% of the annotated human genome. The main advantage of this large-scale array is the possibility to profile thousands of interactions in parallel rendering this approach very economical and efficient. This large number of full-length proteins, the optimal surface chemistry and optimised protocols as well as thoroughly tested secondary reagents enables high quality results. What is the protein content of the HuProt TM arrays? The HuProt TM arrays contain unique proteins representing at least 12,300 different full-length genes that cover more than 60% of the annotated human genome, and are continuing to be expanded. An overview table of the protein classes is given below (table 1). Please not that protein classes may overlap. Transcription factors 743 Protein kinases 796 Metabolism 819 Signal transduction 879 Cell death 1133 Cell communication 3216 Secreted proteins 4205 Nuclear proteins 6064 Membrane proteins 8318 Total number Table 1: Protein classes present on the HuProt TM arrays. Please note that the protein classes may overlap. The total number of unique proteins is P a g e

2 How does the antibody specificity profiling on HuProt TM arrays work? The profiling consists of two major steps (see workflow chart below) the actual probing with the antibody of interest and the data analysis and data QC. We will provide a written report on the study outcome. 1 P a g e

3 What microarray scanners are compatible with HuProt TM Arrays? In general most microarrays scanners compatible with 3x1 inch (75x25mm) microarray glass slides are suitable. Please see table 2 for details. We do not guarantee compatibility with any of the mentioned microarray scanners please refer to manufactures information. Compatible Table 2: Overview of HuProt TM Array compatible microarray scanner models. Not Compatible Molecular Devices GenePix 4000A Affymetrix GeneChip Scanner 3000 Molecular Devices GenePix 4000B Molecular Devices GenePix Professional 4200A Molecular Devices GenePix 4300A Molecular Devices GenePix 4400A Perkin Elmer ScanArray Lite Perkin Elmer ScanArray Express Perkin Elmer ScanArray Express HT Tecan LS Series Laser Scanner Tecan PowerScanner Agilent DNA Microarray Scanner What software can be used for data extraction and analysis? For data extraction there are many different options. The two most common options are: 1. Use in-build software of your microarray scanner to generate.gpr files (Genepix result files) or.txt files containing numerical values extracted from your.tif images (raw data file). Proceed with data analysis using a programme of your chioce. All kinds of programmes including MS-Excel or R-based programmes depending on your actual requirements can be used. 2. Use Genepix in the currently version 7 for data extraction and analysis. Genepix 7 is commercially available from Molecular Devices (Sunnyvale, California, United States) and suitable for most standard data extraction and analysis needs. In case you want to outsource data analysis Cambridge Protein Arrays Ltd. offer to carry it out and provide you with a detailed report or we can assist you with setting up your data analysis workflow. 2 P a g e

4 What amount of antibody do I have to provide and it what state? For one probing in one dilution we would recommend to send us 10 µg of the to-be-tested antibody in an aqueous solution. Please inquire for details and customised project planning. What antibody dilutions are most suitable for screening? We offer several different types of our screening service all tailored to your specific needs. To get an overview of antibody cross-reactivity we test the antibody at one concentration of e.g. 1 µg/ml to identify as much possible cross-reactions but avoiding the possibility of increased non-specific backgrounds due to high antibody concentrations. Other possibilities of testing are dilution series e.g. 0.1 µg/ml, 1.0 µg/ml and 10 µg/ml to estimate antibody affinities and to give a concentration depended overview of cross-reactivity profiles. The third possibility of testing is (mostly) for therapeutic antibodies which are screened at their respective concentrations of use (up to 50mg/ml depending on solubility). Are assay conditions optimised for the supplied materials? We offer a range of services to optimise our array system for specific reagents and conditions to your exact specifications. Please inquire about details and customisation options. Are protein concentrations on HuProt TM arrays known? Along with the probings of the HuProt TM arrays a positive control is carried out. This positive control is a staining of GST fused to the proteins on the array. The data obtained from this staining can be used to estimate protein amounts of individual proteins on the array. What might be the reasons for false-negative results? A false-negative result is the case then an antibody does not recognise the respective target or crossreactions are not detected. - the GST fusion might interfere with N-terminal epitope detection - proteins might be in a non-native or denatured conformation due to purification or printing on the array - antibodies raised against denatured proteins or peptides might not recognise their respective epitopes in the folded full-length protein on the array 3 P a g e

5 - the protein on the array might be a variation (e.g. different splice variant) of the target against the antibody was raised - the target epitope is inaccessible due to e.g. alternative folding - the target does not have the correct PTM-pattern for recognition How may further cross-reactions be found? Further cross-reactions might be found upon increasing the antibody concentration to enable even weak off-target interactions to be visualised. Cambridge Protein Arrays Ltd. offers dilution series experiments for in-depth screening of the provided sample and a large range of concentrations can be tested. How do antibody handling, shipping, storage conditions and storage buffer influence the reactivity? Antibodies as all proteins suffer from unsuitable handling, storage or shipping conditions. Our team of PhD-level scientist has extensive experience in the field of microarray technology and protein technology. We will use and handle all provided reagents as recommended by our clients and to our best knowledge to ensure the best and reproducible outcomes. Cambridge Protein Arrays Ltd. will immediately notify you in case there are any problems noticed such as unsuitable shipping conditions. 4 P a g e