Exosomes Immunocapture and Isolation Tools: Immunoplates

Transcription

1 Product Insert Exosomes Immunocapture and Isolation Tools: Immunoplates HBM-POF-##/## HBM-POC-##/## Overall Exosome capture from Biological fluids (plasma, urine) Overall Exosome capture from Cell culture supernatant HBM-PTF-##/## Tumor-derived Exosome capture and enrichment from Biological fluids (plasma, urine ) HBM-PNF-##/## HBM-PGF-##/## HBM-PPP-##/## HBM-POS-##/## Neural-derived Exosome capture and enrichment from Biological fluids (plasma, urine) Glial-dervied Exosome capture and enrichment from Biological fluids (plasma, urine) Monocytes- and Platelets-derived Exosome capture and enrichment from plasma samples Overall Exosome capture from Human serum This product is for research use only. It is highly recommended to read this users guide in its entirety prior to using this product. Do not use this kit or its components beyond the indicated expiration date.

2 Table of Contents PRODUCT INFORMATION PRODUCT DESCRIPTION 4 PRODUCT CONTENT 4 PROTOCOL PROCEDURE 4 SPECIFICITY 5 RNA ANALYSIS 5 PROTEOMIC ANALYSIS 6 APPENDIX A SAMPLES PREPARATION (Plasma, serum, urine, cell culture supernatant) 8 APPENDIX B 9 Related products 9 TECHNICAL SUPPORT 10 DISTRIBUTORS 11 ELISA Plates Product Insert HansaBioMed 2

3 PRODUCT DESCRIPTION Overview PRODUCT INFORMATION HansaBiomed ExoTEST platform features multiwell plates pre-coated with specific antibodies that capture pan and tissue-specific exosomes from biological fluids, such as plasma, serum, urine, and cell culture supernatant. About Exosomes About the plate Exosomes are small endosome derived lipid nanoparticles ( nm) actively secreted by exocytosis by most living cells. Exosome release occurs either constitutively or upon induction, under both normal and pathological conditions, in a dynamic, regulated and functionally relevant manner. Both amount and molecular composition of released exosomes depend on the state of a parent cell. Exosomes have been isolated from diverse cell lines (hematopoietic cells, tumor lines, primary cultures, virus infected cells) as well as from biological fluids in particular blood (e.g. serum and plasma from cancer patients) and other body fluids (bronchoalveolar lavage fluid, pleural effusions, synovial fluid, urine, amniotic fluid, semen, saliva etc). Exosomes have pleiotropic physiological and pathological functions and an emerging role in diverse pathological conditions such as cancer, infectious and neurodegenerative diseases. Our multiwell plates are standard 96-well format where assays can be conducted as singletons and/or multiple wells. This enables optimization of a wide range of sandwich ELISA assays or other downstream applications, such as RNA extraction and proteomic analyses. Transparent, white and black plates are available depending on the downstream detection approach (colorimetric, luminometric and fluorimetric). Uncoated and/or covalently-coated plates are available for ordering. PRODUCT CONTENT Type of Immunoplates available: Immunoplate for Overall Exosome capture from Biological fluids (plasma and urine) Immunoplate for Overall Exosome capture from Cell culture supernatant Immunoplate for Tumor-derived Exosome capture from Biological fluids (plasma and urine) Immunoplate for Neural-derived Exosome capture from Biological fluids (plasma and urine) Immunoplate for Monocytes- and Platelets-derived Exosome capture from Plasma sample Immunoplate for Overall Exosome capture from Human serum Transparent, white and black plates are available depending on the downstream detection approach (colorimetric, luminometric and fluorimetric). Content Expiration date Standard 96-well format pre-coated plate 24 months, store at 4 C 6 month after opening HansaBioMed 3 ELISA Plates Product Insert

4 IMMUNOPLATES PROTOCOL PROCEDURE Overview The Immunoplate is designed to capture exosomes from any biological sample up to 100 μl per well, if necessary adjust the well volume with 1x PBS. Hereby proposed protocol is applicable to all immunoplates presented in the catalog. Plates are precoated with anti-rabbit or anti-mouse antibodies. The type of antibody used for coating is indicated on the label of the plate. To avoid crossreactivity in detection, do not use the same type of antibody used for coating. Products are for research use only and is not intended for diagnostic use. Binding Exosomes onto the plate for further analysis NOTE: Make sure to never touch the bottom or sides of the wells or you will scrape off your samples/standards. As a reminder No Touch is placed on that line. 1. The plate is ready to use, no pre-washing is required. 2. Add up to 100 μl of sample per well, adjust with 1X PBS up to 100 μl if necessary. 3. Seal the plate with parafilm and incubate at room temperature while shaking for 20 minutes. 4. Depending on your sample, incubate overnight: Plasma and serum samples at 4 C. Cell culture supernatant and urine samples at 37 C. 5. Wash the plate (washing buffer suggested: PBS + 0,05 % Tween20) i. Add 200 μl/well of Washing Buffer and discard plate contents by pouring out. No Touch. ii. Wash 3 times with 300 μl/well of Washing Buffer. After each addition, pour off wash. No Touch. 6. Thus prepared plate can be used for captured exosome characterization in an ELISA assay, or captured exosomes can be further recovered for mrna extraction and analysis (see related products code). ELISA Plates Product Insert HansaBioMed 4

5 SPECIFICITY Immunoplate captures specifically exosomes in your sample due to interaction of coating antibodies with exosome associated surface antigens, while no significant crossreactivity observed with soluble antigens or vesicle-associated proteins. Exosomes capture efficiency and specificity are presented in figure 1 and 2. OD at 450nm OD at 450nm OD at 450nm G Pellet G Pellet G and Filtered Pellet ml of plasma µg of exosomes Figure 1: Specific exosome immunocapture without other microvesicles. Comparison of exosome capture efficiency (pellet after centrifugation g) and microvesicles (pellet 1200g and g) Exosomes specific HansaBioMed proprietary antibody Exosomes specific Commercial antibody Figure 2: Exosome immunocapture ability of HansaBioMed exosomes specific proprietary antibody. Comparison of exosome capture ability of HansaBioMed proprietary antibody vs other exosomes specifics commercial antibodies on ELISA plate RNA ANALYSIS Plate allows total RNA extraction from purified exosomes and/or exosomes directly captured from unfractioned plasma, serum and urine samples. Obtained RNA is suitable for downstream analysis of mrna or small RNA markers. Blank β-actin 200 bp Figure 3: RT-PCR analysis of mrna mrna extracted from human unfractionated plasma derived exosomes captured on plate and detection of β-actin transcript. Figure 4: Comparison of RNA yield obtained with exosomes ultracentrifuged and with exosomes immunocaptured on ELISA plate. Each time the starting material was 0,8 ml of unfractionated human plasma. HansaBioMed 5 ELISA Plates Product Insert

6 Figure 5: Agilent bioanalyzer analysis of total RNA extracted by Total exosome RNA extraction immunoplates. Aliquots of extracted RNA were loaded on chips for both total RNA and small RNA Agilent analysis. Although total RNA analysis revealed a low profile in mrna (RIN 2,5), small RNA analysis showed an enrichment in small RNA. PROTEOMIC ANALYSIS Exosomes incorporate a wide range of membranes and cytosolic proteins involved in many cellular functions and reflecting both their endosomal origin and a protein fingerprint unique for parent cell type and condition. Starting with exosomes capture on immunoplates is an ideal starting point for further studies on exosome biomarkers. Immunoplates for Overall Exosome capture from Biological fluids Figure 6: Comparison of purified exosomes and unfractionated samples in a set of healthy donors. Comparison of CD9 detection on purified (ultracentrifuged, UC) plasma exosomes and on correspondent unfractionated samples that were precleared by microfiltration and concentrated by spin concentrators. All results are representative of 0,5 ml of original plasma samples. Figure 7: Common exosomal biomarkers analysis. Analysis of several common exosomal biomarkers in the same healthy donor plasma sample Immunoplates for Overall Exosome capture from Cell culture supernatant Figure 8: ELISA exosome capture from different cell cultures supernatant. Samples are concentrated 30 times by using centrifugal concentrator before immunocapture. ELISA Plates Product Insert HansaBioMed 6

7 Immunoplates for Tumor-derived Exosome capture from Biological fluids Figure 9: Comparison of signal obtained in different cancer samples using Immunoplates for Overall and Tumor-derived exosome capture. Enrichment of tumoral derived exosomes in early and advanced stages of melanoma ( Mel I; Mel adv) and ovary carcinoma (Ova I; Ova adv). Immunoplates for Neural-derived Exosome capture from Biological fluids 1 0,8 0,6 0,4 0,2 0 neuroblastoma Neural-dervied exosomes exosomes in human plasma in human plasma HD Healthy plasma exosomes donors exosomes in human plasma micrograms of exosomes diluted in plasma Figure 10: Signal comparison observed with Immunoplates for Neural-derived Exosome capture from Biological fluids between lyophilized neural-derived and healthy donors exosomes diluted in plasma. Different quantities of lyophilized exosomes are diluted in human plasma. The signal obtained with neural-derived exosomes increase with the original quantity, healthy donors exosomes behaved like a blank. The figure shows that this immunoplate capture specifically and selectively neural-derived exosomes. Immunoplates for Glial-derived Exosome capture from Biological fluids 0,7 0,6 0,5 glial Glial-dervied exosomes in human exosomes plasma in human plasma HD Healthy plasma exosomes donors exosomes in human plasma 0,4 0,3 0, micrograms of exosomes diluted in plasma Figure 11: Signal comparison with Immunoplates for Glial-derived Exosome capture from Biological fluids between lyophilized glialderived and healthy donors exosomes diluted in plasma observed. Different quantities of lyophilized exosomes are diluted in human plasma. The signal obtained with neural-derived exosomes increases with the original quantity, healthy donors exosomes behaved like a blank. The figure shows that this immunoplate capture specifically and selectively glial-derived exosomes. Immunoplates for Monocytes- and Platelets-derived Exosome capture from plasma samples 1,6 1,4 1,2 1 0,8 0,6 0,4 0,2 0 UF plasma UF plasma BLANK UF urine UF urine BLANK UF plasma UF plasma BLANK UF urine UF urine BLANK Figure 12: Comparison of signal obtained in different unfractionated (UF) samples with the Immunoplate for Monocytes- and Platelets-derived Exosome capture from Biological fluids. Relevant signal was observed in UF plasma samples and none in urine. This shows that the plate capture specifically exosomes only present in plasma like monocytes- and platelets-derived exosomes. HansaBioMed 7 ELISA Plates Product Insert

8 APPENDIX A SAMPLE PREPARATION (Plasma, Serum, Urine, Cell Culture Supernatant) Protocol Plasma and serum samples preparation Prepare samples by 3 centrifugation steps to eliminate red blood cells and cellular debris: 10 at 300 g 20 at g 30 at g After each step save the supernatant. Processed plasma is ready to be loaded onto the plate (100 μl/well) or incubated with beads. Urine samples preparation Preclear urine samples by centrifugation at g for 20 at RT Filter by using 0.45 um filter Concentrate urine samples by spin concentrator for times for proteomic and for nucleic acid studies*. Cell culture supernatant samples preparation Prepare cell supernatants by 3 centrifugation steps: 10 at 300 g 20 at g 30 at g Concentrate cell supernatant times in spin concentrator* *The quantity of exosomes could vary between samples. Concentration factors are given for information purposes only, a larger starting amount of sample should be used if the signal is weak. ELISA Plates Product Insert HansaBioMed 8

9 APPENDIX B RELATED PRODUCTS Overview As the first company entirely dedicated to the field of exosomes, we offer a panel of kits and reagents for exosome research. Ordering information on a variety of reagents and apparatus available from HansaBioMed is provided below. For more information, visit our website at Products Quantity Catalog Number ExoTEST Ready To Use Kit for Overall Exosome capture and quantification from Biological fluids HBM-RTK-POF/## ExoTEST Ready To Use Kit for Overall Exosome capture and quantification from human Serum HBM-RTK-POS/## ExoTEST Ready To Use Kit for Overall Exosome capture and quantification from Cell culture supernatant HBM-RTK-POC/## ExoTEST Ready To Use Kit for Tumor-derived Exosome enrichment and quantification from Biological fluids HBM-RTK-PTF/## Custommade ExoTEST for Specific Exosome capture and quantification HBM-RTK-CMK Exosome Total RNA Extraction Kit (Immunobeads), 10 or 20 Reactions HBM-RNA-BOF-##/# Exosome Total RNA Extraction Kit (Immunoplate) HBM-RNA-POF/# Tumor-derived Exosome Total RNA Extraction Kit (Immunobeads), 10 or 20 Reactions HBM-RNA-BTF-##/## Tumor-derived Exosome Total RNA Extraction Kit (Immunoplate) HBM-RNA-PTF/# Lyophilized Exosomes from plasma (healthy donor), from serum (healthy donor), from urine (healthy donor) and from cell culture supernatant (well known cell lines) Exosome Binding Antibodies 2/4 or 6X 100 μg 2/4 or 6X 30 μg Check Exosome Standards available on Check Ab available on HansaBioMed 9 ELISA Plates Product Insert

10 TECHNICAL SUPPORT Get support For the latest services and support information for all locations, go to At the website, you can: Search for user documents, handbooks, certificates of analysis, citations, and other product support documents Access telephone and fax numbers to contact Technical Support and Sales facilities Or contact us at Material Safety Data Sheet (MSDS) Material Safety Data Sheets (MSDSs) are available at Trademarks ExoTEST and HansaBioMed are trademarks of Hansabiomed OÜ..Other brands or product names are trademarks of their respective holders. Research Use All products are sold for research or laboratory use only and are not intended to be administered to humans or used for medical diagnostics. ELISA Plates Product Insert HansaBioMed 10

11 HansaBioMed 11 ELISA Plates Product Insert

12 For the latest product and technical information, visit hansabiomed.eu and exotest.eu HansaBiomed Homepage Online Shop HansaBioMed LLC Akadeemia tee 15, Tallinn, ESTONIA Contact in US Galen Laboratory Supplies P.O. Box 682 Middletown, CT Tel: Fax: Tel: Fax: For support visit or Visit our website and