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PlasmaScan™ 380    Antibody Microarrays


Arrayit offers the market’s only antibody microarray containing monoclonal antibodies generated against native human plasma proteins. The PlasmaScan antibodies react wth native human proteins with proper folding and post-translation modification to exhibit high antibody binding. The Arrayit microarray platform with the PlasmaScan™ monoclonal antibodies enables the exploration and identification of novel biomarkers in the human plasma proteome.

Contents

  • Introduction
  • Quality Control
  • Product Description
  • Frequently Asked Questions (FAQs)
  • Kit Contents
  • Short Protocol
  • Complete Protocol
  • Antibody Epitope Map and GAL file
  • Technical Assistance
  • Troubleshooting Tips
  • Recommended Equipment
  • Ordering Information
  • Storage Conditions
  • Warranty

Introduction
ArrayIt® PlasmaScan™ Antibody Microarrays will improve the precision, speed and rate of biomarker discovery for research, proteomic, clinical, pharmaceutical, and diagnostic applications. This handbook contains all the information required to take full advantage of our pioneering PlasmaScan™ Antibody Microarrays.

Quality Control
Arrayit uses the highest quality control (QC) and quality assurance (QA) measures to ensure the quality of Arrayit PlasmaScan™ Antibody Microarrays. The finest immunology and biochemistry was used to develop this product. The use of native human proteins, advanced monoclonal antibody production, superior surface chemistry and the world’s most advanced robotics, printing technology and microarray cleanroom manufacturing procedures guarantees that PlasmaScan™ outperforms the highest industry standards.

Product Description
Arrayit PlasmaScan™ Antibody Microarrays represent a first in the microarray field by offering the capacity to explore the human proteome using antibody binding elements with native human protein binding affinity and specificity. Each PlasmaScan™ spot reports to a single human plasma protein. The chip is so advanced that we are just now determining binding partners for each antibody. The combined attributes render PlasmaScan™ as the ultimate biomarker discovery tool.

Users will appreciate the following features:

  • Monoclonal antibodies raised against native human plasma proteins
  • Contains 80 monoclonal antibody binding elements
  • Novel one spot-one antibody design streamlines data analysis
  • Explore the function of the human proteome
  • Devoid of antibodies with weak binding affinity and cross-reactivity
  • Control antibodies add precision and reliability
  • Multiple uses include protein expression profiling, biomarker discovery, and others
  • Compare human, primate and rodent proteomes
  • Detect 80 human serum proteins in a single binding reaction
  • Purchase one or all of the 80 monoclonals
  • Milligram antibody quantities available for research and diagnostics
  • Future product releases will contain more antibodies for proteome-wide analysis
  • Manufactured using Arrayit’s patented printing technology (U.S. 6,101,946)
  • Antibodies purity exceeds 99.9% for each printed element
  • Content verified using advanced procedures to ensure correct antibody identity
  • Printed on atomically flat glass substrates for improved precision
  • Available in 1, 16 and 24 microarray per substrate slide formats
  • Compatible with an installed base of >10,000 fluorescent slide scanners
  • Use PlasmaScan™ to validate serum samples from thousands of patients
  • Printed elements occupy a compact 3 x 3 mm area
  • Teflon masks provide hydrophobic barriers between printed microarrays
  • 50 µl reaction volumes accelerate binding kinetics and minimize plasma consumption
  • Format compatible with all standard protein quantification and mining packages
  • Compatible with all fluorescent plasma protein labeling schemes
  • One, two, three and four color detection possible for multi-sample comparisons
  • Detectiong limit: 1 serum protein per 1,000,000 protein molecules
  • Dynamic range: 100,000-fold across multiple microarrays
  • Chip-to-chip CVs: ±20%
  • Corner chamfer provides unambiguous spatial orientation
  • Barcode assists in data tracking
  • Sophisticated anti-static packaging improves usability and storage
  • Produced in Arrayit’s high volume microarray manufacturing facility
  • Microarrays arrive processed and ready to use
  • Stable for 6 months from the date of purchase
  • Explore the human plasma proteome with unprecedented precision

Figure 1. Example of image from PlasmaScan™ 380 shown in pseudo color rainbow pallet. PlasmaScan microarrays are detected as 16-Bit gray scale tiff files using the Innoscan 710 or other suitable standard slide based fluorescent microarray scanner.

Frequently Asked Questions (FAQs)

Q: What are PlasmaScan™ Microarrays used for?
A: The PlasmaScan™ line of mAb Arrays were developed to permit open profiling of the plasma proteome similar to gene expression profiling microarrays. As such they can be used for the discovery of novel plasma biomarkers for multiple applications such as disease specificity, drug toxicity, drug response, and patient stratification as well as for fundamental research.

Q: Why use PlasmaScan™ Microarrays when Mass Spectrometry based methods are available?
A: Because of the lack of a standardized methodology, mass spectrometry-based profiling of complex biological mixtures presents a number of formidable technical challenges including problems of sensitivity and reproducibility. Furthermore,  mass spectrometry based proteomic profiling requires an extensive and costly infrastructure. Throughput and sensitivity of mass spectrometry based methods is insufficient as well as the translatability of the discoveries to antibody based ELISA or other assays. PlasmaScan™ microarrays provide a reliable and cost-efficient alternative as Arrayit’s microarray technology is well-established, standardized and robust.

Q: Who can use PlasmaScan™ Microarrays?
A: Any laboratory with a few inexpensive bench top tools and a standard fluorescent based microarray scanner can use PlasmaScan™ Microarrays.  Arrayit can provide quotations on any product that is needed.

Q: How do I conduct an experiment using PlasmaScan™ Microarrays?
A: For each plasma sample to be analyzed, biotinylate them using standardized approaches and reagents.  For example the EZ-Link Sulfo-NHS-LC-Biotin kit sold by Pierce can be used to biotinylate plasma. Following removal of free biotin, the microarrays are incubated with the biotinylated plasma proteins, washed, and protein binding measured by incubation of the arrays with fluorescently labeled streptavidin followed by scanning using any conventional fluorescent microarray scanner.

Q: How do I evaluate the data?
A: The data is quantified using the spot map and image analysis program provided with a typical fluorescent microarray scanner using standard microarray scanning procedures.

Q: How do I obtain more antibody to follow up an interesting result?
A: Contact Arrayit (or BSI) and we will provide the necessary antibody(s) for further characterization. In most cases, the antibodies will be associated with an epitope ID. As the natural antigens for the antibodies are identified, this information will be updated to this web page on a regular basis. If by chance, the antigen ID for an antibody of interest has not yet been determined, contact us and BSI will put your antibody to the front of the pipeline for the antigen ID process.

As additional mAbs become available PlasmaScan™ arrays of increasing complexity will be released. At the same time previous versions of the arrays will remain available.

Kit Contents

  • PlasmaScan™ 380 Antibody Microarray Kit
  • PlasmaScan™ 380 Antibody Microarray (6 microarrays per substrate slide)
  • Protein Microarray Activation Buffer (250 ml of 1X solution)
  • Protein Microarray Wash Buffer (1000 ml of 1X solution)
  • Protein Microarray Reaction Buffer (250 ml of 1X solution)
  • Protein Microarray Rinse Buffer (500 ml of 1X solution)
  • Buffers Provided per 5 Kits Purchased
  • 4 x Protein Microarray Activation Buffer (250 ml of 1X solution)
  • 8 x Protein Microarray Wash Buffer (1000 ml of 1X solution)
  • 4 x Protein Microarray Reaction Buffer (250 ml of 1X solution)
  • 4 x Protein Microarray Rinse Buffer (500 ml of 1X solution)

Short Protocol (Steps 1-7)
1. Obtain human plasma samples.
2. Label the human plasma protein samples by biotinylation.
3. Activate the PlasmaScan™ Antibody Microarrays.
4. React the activated PlasmaScan™ microarrays with labeled plasma samples.
5. Stain the PlasmaScan™ microarrays with a fluorescent streptavidin secondary reagent.
6. Scan the PlasmaScan™ microarrays for fluorescence emission.
7. Quantify and model the fluorescent data.

Complete Protocol (Steps 1-7)
1. Obtain human plasma samples. The first step is to use a robust method to acquire serum plasma protein samples. There are many commercial products available to perform this step. For best results and to allow comparisons across many microarrays, the same serum plasma collection procedure should be used for each microarray.  PlasmaScan 380 uses 3 µl of depleted biotinylated serum plasma or 5 µl of non-depleted biotinylated serum plasma in a 300 µl reaction.

2. Label the human plasma protein samples by biotinylation. There are a number of commercial kits that can be used for biotinylation. Custom protocols can also be developed and used for this step. The same biotinylation procedure should be used for all microarrays subject to comparison. Following biotinylation, the labeled plasma proteins should be purified to remove free biotin and other labeling contaminants. It is also possible to label the serum proteins directly using Arrayit Green540 and Red640 reactive fluorescent dyes (Cat. AR540 and AR640). Dye-labeled serum proteins should be purified prior to use on PlasmaScan™.

3. Activate the PlasmaScan™ Antibody Microarrays. PlasmaScan™ requires activation prior to use to remove unbound monoclonal antibody molecules and to shield the surface against non-specific binding. Activate PlasmaScan™ by mixing for 60 min in Protein Microarray Activation Buffer (Cat. PMAB). The activation step can be performed with gentle mixing in 15 ml of Protein Microarray Activation Buffer in a petri dish or 450 ml of Protein Microarray Activation Buffer in a High-Throughput Wash Station. Skipping this step will result in very high background.  Following activation, wash the PlasmaScan™ microarrays two times for 5 min in Protein Microarray Wash Buffer (Cat. PMWB) and one time for 1 sec in Protein Microarray Rinse Buffer (Cat. PMRB). All washes can be performed using 25 ml volumes in a petri dish or 450 ml volumes in a High-Throughput Wash Station (Cat. HTW) with gentle agitation. After washing, spin for 1 sec in a Microarray High-Speed Centrifuge to dry the PlasmaScan™ microarrays.

4. React the activated PlasmaScan™ microarrays with labeled plasma samples. Samples are prepared by mixing 5-25 µg of labeled plasma protein with Protein Microarray Reaction Buffer (Cat. PMRB), making sure not to dilute the Protein Microarray Reaction Buffer by more than 1.5-fold. The same volume of protein sample should be used for all reactions in which comparative information is sought. Reactions are performed using an AHC1x24 or AHC4x24 with a modified gasket making a perfect tool to process 6 microarrays per slide substrate, each with a 300 µl reaction volume .  Undiluted or diluted biotinylated plasma samples can be used depending on the sample concentration and plasma protein expression level. Typically 5 µg of labeled protein is sufficient to obtain strong signals. Up to 25 µg of labeled protein can be used, though saturating signals and non-linearity can be observed at high sample protein concentrations. Fluorescent samples can be reacted in the same manner as biotinylated samples. Binding reactions should be allowed to proceed for 60 min at room temperature with gentle mixing, but shorter and longer reaction times may be suitable for some applications.  Following the reaction step, wash the PlasmaScan™ microarrays three times for 3 min in Protein Microarray Wash Buffer (Cat. PMWB) with gentle agitation. Washes can be performed using 25 ml volumes in a petri dish or 450 ml volumes in a High-Throughput Wash Station with gentle agitation. After washing, spin for 1 sec in a Microarray High-Speed Centrifuge to dry the PlasmaScan™ microarrays.

5. Stain the PlasmaScan™ microarrays with a fluorescent streptavidin secondary reagent. Prepare the secondary antibody reagent by diluting the Cy3™- or Cy5™-streptavidin conjugate to a final concentration of 1 µg/ml in Protein Microarray Reaction Buffer (Cat. PMRB). A one thousand-fold (1:1,000) dilution of the 1 mg/ml Cy3™-Streptavidin staining reagent from Jackson ImmunoResearch (Cat. 109-165-088) works well for this application. A 1.0 ml volume of secondary reagent is prepared by mixing 1.0 µl Cy3™-streptavidin in 1 ml of Protein Microarray Reaction Buffer. Mix by inverting the tube 10 times. Stain the PlasmaScan™ microarrays using 300 µl of diluted secondary reagent in each reaction area of AHC1x24 or AHC4x24 with the modified gasket. Stain for 60 min at room temperature with gentle agitation. Following the staining step, wash the PlasmaScan™ microarrays three times for 3 min in Protein Microarray Wash Buffer (Cat. PMWB) and one time for 1 sec in Protein Microarray Rinse Buffer (Cat. PMNB) with gentle agitation. Washes can be performed using 25 ml volumes in a petri dish or 450 ml volumes in a High-Throughput Wash Station with gentle agitation. After washing, spin for 1 sec in a Microarray High-Speed Centrifuge to dry the PlasmaScan™ microarrays.

6. Scan the microarrays for fluorescence emission. Scan the PlasmaScan™ Antibody Microarrays using an Arrayit SpotLight™ (Cat. SLMS) microarray scanner set to detect Cy3™ or Cy5™. PlasmaScan™ can also be read with other brands of microarray scanner compatible with the open platform substrate slide 25 x 76 mm format. Scanner settings should be adjusted to minimize saturated signals to 1% or less. Multiple scans can be taken to capture data at different sensitivity levels. All data files should be saved as 16-bit TIFF images for data analysis.

7. Quantify and model the fluorescent data. Launch Arrayit Mapix Microarray Quantification Software and open the PlasmaScan™ Antibody Microarray TIFF. Create a quantification grid and import the PlasmaScan™ GAL file. Adjust the grid so that it fits approximately around each printed element. Once the grid is aligned, click “find spots automatically” to allow automated spot finding by Mapix. Minor adjustments to individual spot locations can be made using the “spots modification” command. Click on “quantification process” to quantify the PlasmaScan™ spot intensities. Click on the “data viewer” in the “Window” menu to visualize the quantified data. Click on “Save results” to save the PlasmaScan™ data as a text file (.txt). The .txt file can be imported into Microsoft Excel and other software programs for additional analysis.

Antibody Epitope Map
The novel monoclonal antibodies present on Arrayit PlasmaScan™ 80 Antibody Microarrays were interrogated using phage display with quasi-random 12 residue peptide sequences to determine antibody binding affinity and specificity. Epitope sequences provide unique identification tags for each antibody. Epitopes represent either linear amino acid binding sequences or mimotopes of the natural antibody binding site, the latter reflective of biochemical attributes of the cognate antigen including topology, shape, rigidity, hydrophobicity and charge distribution. Epitopes and mimotopes correspond to human serum proteins of both known and unknown identity. A total of 12-24 epitopes for each antibody were identified and aligned using BioEdit software. Additional epitope information and protein IDs are available for each antibody upon customer request.
Click here to download the PlasmaScan™ 80 Epitope Map.
Click here to download the PlasmaScan™ 80 GAL file content map.

Troubleshooting Tips
1. Weak signals

  • Degraded protein samples.  Check plasma sample preparation procedure.
  • Poor plasma labeling efficiency. Try biotin labeling procedure.
  • Wash stringency too high.

2. High background fluorescence

  • Probe mixture drying out during the binding step. Keep hydrated at all times.
  • Probe drying after hybridization.  Transfer quickly to wash steps.
  • Probe volume too low.  Try 50.0 µl+ for multi-well formats.
  • Purify probe mixture to remove contaminating unincorporated label.
  • Poor washing procedure.  Increase time or stringency.

3. Lower than expected reproducibility between PlasmaScan microarrays

  • Use the same isolation procedure for all plasma samples.
  • Perform parallel assays in batch mode.
  • Utilize 16-well, 24-well and 96-well formats.

4. Artifacts observed in two-color experiments

  • Utilize “dye swap” labeling procedure.

Recommended Equipment

Use AHC4x24 or AHC1x24 with “ribs” cut from the gasket to form 6 x 300 µl reactions.

Microarray High-Speed Centrifuge
High-Throughput Wash Station
Arrayit Green540 and Red640 Fluorescent Dyes
Arrayit SpotLight Personal Fluorescence Microarray Scanners
Hybridization Cassettes
Multi-Well Hybridization Cassettes
Microarray Cleanroom Gloves
Microarray Cleanroom Wipes

Ordering Information

Product

Description

Catalog ID

PlasmaScan™ 380 Antibody Microarrays

PlasmaScan™ 380 Antibody Microarrays, human plasma proteome discovery tool, monoclonals raised against native human serum plasma proteins, standard 25 x 76 mm glass substrate slide format, 6 replicate PS380 microarrays on a single substrate slide, Protein Microarray Activation Buffer (250 ml of 1X solution), Protein Microarray Wash Buffer (1000 ml of 1X solution), Protein Microarray Reaction Buffer (250 ml of 1X solution), Protein Microarray Rinse Buffer (500 ml of 1X solution).

PS380

Protein Microarray Activation Buffer

Protein Microarray Activation Buffer for activating PlasmaScan™ 80 Antibody Microarrays prior to use, 250 ml of 1X solution, 0.1 µm filtered, arrives ready to use, store at 4°C.

PMAB

Protein Microarray Wash Buffer

Protein Microarray Wash Buffer for washing PlasmaScan™ 80 Antibody Microarrays after activation and reaction steps, 1000 ml of 1X solution, 0.1 µm filtered, arrives ready to use, store at 20-25°C.

PMWB

Protein Microarray Reaction Buffer

Protein Microarray Reaction Buffer for reacting PlasmaScan™ 80 Antibody Microarrays with plasma protein samples and secondary staining reagents, 250 ml of 1X solution, 0.1 µm filtered, arrives ready to use, store at 20-25°C.

PMRB

Protein Microarray Rinse Buffer

Protein Microarray Rinse Buffer for rinsing PlasmaScan™ 80 Antibody Microarrays prior to scanning, 500 ml of 1X solution, 0.1 µm filtered, arrives ready to use, store at 20-25°C.

PMNB

Storage Conditions
Arrayit PlasmaScan™ Antibody Microarrays are manufactured and packaged in state-of-the-art class 100 cleanroom facilities and are stable at 4°C for 6 months from the date of receipt. Store in the sealed anti-static shipping envelope and avoid exposure to elevated temperature, humidity, and dust until use.

Warranty
Arrayit PlasmaScan™ Antibody Microarrays were scientifically developed and are manufactured in a state-of-the-art class 100 cleanrooms. Extreme care and exact attention should be practiced in the use of the materials described herein.  All Arrayit products are subject to extensive quality control and are guaranteed to perform as described when used properly. Any problems with these products should be reported to Arrayit immediately. Our liability is limited to the replacement of the product, or a full refund. Any misuse of this product is the full responsibility of the user, and Arrayit makes no warranty or guarantee under such circumstances. PlasmaScan™ was developed in collaboration with BioSystems International, is sold for research purposes only, and is not for use in human diagnostics or for drug purposes, and should not be administered to humans. Diagnostic use of PlasmaScan™ microarrays and antibodies requires a license from both Arrayit and Biosystems International. Please contact Arrayit directly for diagnostic uses of this product.