Small-Molecule Microarrays

Drives me crazy when end users of our microarray manufacturing technology do not reference where it came from and don't talk about it using the proper terminology. That being said, the message is a good one....small molecule microarrays are just one of many applications that Arrayit can empower. Arrayit has never manufactured, sold, used nor endorsed the use of quill pins. Our microarray printing pins are covered by US Patent 6,101,946. For more information see www.arrayit.com

Microarray products for the detection, identification and quantification of autoantibodies in human serum

Objective: Run antigen microarrays to identify distinct serum antibody profiles.

Print protein antigens as purified extracts at a final concentration of 0.3 to 1 mg per ml as a final concentration in Protein Printing Buffer (Arrayit Cat. ID PPB) using an Arrayit NanoPrintTM or Arrayit SpotBot® Microarrayer equipped with 946MP3 Microarray Printing Pins. For colorimetric microarray detection it is recommended to use SuperPVDF (Arrayit Cat. ID SMPV) or SuperNitro (Arrayit Cat. ID SMN) microarray slide substrates and the SpotWare Colorimetric Microarray Scanner (Arrayit Cat. ID SPW110) for detection. For fluorescence detection print microarrays onto SuperEpoxy 2 Microarray slide substrates (Arrayit Cat. ID SME2) and detect with the Innoscan 710 Microarray scanner. Always have immunoglobulin positive control and blank printing buffer as a negative control spotted on the microarray. Consider immunoglobulin positive controls as a dilution series to create "dose response" curves. After microarray manufacturing at 50% humidity, let microarrays sit overnight on the worktable of the microarrayer to dry at 30% humidity or less. Store printed microarrays clean, dry and at room temperature. Protein printing buffer will stabilize the printed proteins for long periods of time.

Prior to using the microarrays they must be activated to remove unbound antigens printed on the microarray and to prepare the surface chemistry to prohibit binding of serum proteins to the surface chemistry. Incubate microarray(s) in 1X Protein Microarray Activation Buffer (Arrayit Cat. ID PMAB) for 1 hour. Activating for less than 1 hour can compromise results. After 1 hour wash microarray 3 times 5 minutes each in Protein Microarray Wash Buffer (Arrayit Cat. ID: PMWB).
React primary antibody to the microarray(s). Use diluted serum or other test sample diluted into 1X Protein Microarray Reaction Buffer (Arrayit Cat. PMRB). Typical dilutions are 1 to 300. Because kinetics of microarrays are fast, incubations can be as short as 15 minutes or as long as 1 hour. Agitation and increased temperature can be used to speed up kinetics but are typically not required. Wash off reaction 3 times 5 minutes each in Protein Microarray Wash Buffer (Arrayit Cat. ID PMWB).

React secondary immunoglobulin antibody to the microarray. For fluorescence detection, stain the microarrays with a fluorescent secondary reagent. For colorimetric detection, use alkaline Phosphatase conjugated secondary antibody. Prepare the secondary antibody reagent by diluting the conjugate to a final concentration of 1 µg/ml in Protein Microarray Reaction Buffer (Arrayit Cat. PMRB). A one thousand-fold (1:1,000) dilution of the 1 mg/ml works well. A 1.0 ml volume of secondary reagent is prepared by mixing 1.0 µl of conjugated secondary antibody (i.e. mouse monoclonal anti-human IgG) in 1 ml of Protein Microarray Reaction Buffer. Mix by inverting the tube 10 times. Stain the microarrays using 25-125 µl of diluted secondary reagent. The staining volumes will depend on whether a single or multi-well microarray format is being used. Stain for 60 minutes at room temperature with gentle agitation. Following the staining step, wash the microarrays three times for 1 min in Protein Microarray Wash Buffer (Arrayit Cat. PMWB) and one time for 1 sec in Protein Microarray Rinse Buffer (Arrayit 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 (Arrayit Cat. ID HTW) with gentle agitation. After washing, spin for 1 sec in a Microarray High-Speed Centrifuge (Arrayit Cat. ID MHC) to dry microarrays.
Scan the microarrays using an Arrayit SpotLight™ (Cat. SLMS) or Arrayit InnoScan® 710 (Cat. 710) microarray scanner. Arrayit protein microarrays can also be read with other brands of microarray scanners compatible with the open platform substrate slide 25 x 75 x 1mm mm format. Scanner settings should be adjusted to minimize saturated signals to 1% or less. All data files should be saved as 16-bit TIFF images for data analysis. To quantify and model the fluorescent data use Mapix Microarray Quantification Software or similar microarray quantification. Create a quantification grid or import the the spotmap GAL file created by the NanoPrintTM microarrayer. 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 the Mapix program. Minor adjustments to individual spot locations can be made using the “spots modification” command. Click on “quantification process” to quantify the spot intensities. Click on the “data viewer” in the “Window” menu to visualize the quantified data. Click on “Save results” to save the data as a text file (.txt). The .txt file can be imported into Microsoft Excel and other software programs such as ProMAT and for additional analysis. http://www.pnl.gov/statistics/ProMAT/

Microarray versus Luminex, Bioplex,

There are many advantages of the Arrayit microarray platform over Luminex and Bioplex and Xmap type equipment, herein after referred to as "bead arrays". This blog entry does not discuss the "bead array" approach used by Illumina. Let's examine the information from Luminex's website and compare it to the ArrayIt microarray platform. Advantages listed as follows:

1. Reduced cost and labor by multiplexing.
This is true, but microarrays also multiplex, miniaturize and parallelize many more assay types. Ex-situ microarrays made at Arrayit have the ability to multiplex and miniaturize to a much greater degree. Bead arrays read by flow cytometry have a maximum is 500 different capture analytes (specialized instrumentation and beads required to increase to 500). A spotted microarray platform provided by ArrayIt can simultaneously capture data from single analytes to tens of thousands of capture analytes in a single binding reaction using a microscope slide size piece of glass. These microarrays can be processed manually or with the use of automation. The 2-color fluorescent microarray scanner can be used to read the results, these instruments are all over the world. Single colorimetric systems cost less than $4K, SPR, Planar Wave Guides, Chemiluminesence and other detection methods can also be used. Bead array platforms cannot use label free detection methods.

2. Shortened time-to-results by favorable reaction kinetics, with smaller sample requirements.
Also true for spotted microarray...with even smaller volumes than bead assays run in 96 or 384 well plates and run through a flow cytometer detection instrument. In miniaturized "array of arrays" format on activated glass slides, 192 microarrays of 25 capture analytes each can be run with reaction volumes of approximately 1 ul. Every spot in the microarray is subjected to the same processing conditions. Common bead array plate based protocols use 50-100 ul reactions, each well is under separate processing and detection conditions. Smaller spots, less analyte and less detection reagents make microarray analysis less expensive to run than bead arrays.

3. Liquid reaction kinetics give faster, more reproducible results.
In reviewing protocols, in general we do not see binding reaction times shorter than with microarray protocols. Capture analytes in both cases...beads and planar microarrays...the capture analytes are bound to a solid surface. Beads, like the surfaces of microarrays, are insoluble. However in the case of spotted planar microarrays, the capture analytes are covalently bound to the surface of the microarray. They cannot interact with each other in solution, as can capture analytes attached to beads in solution. All planar microarrays are reacted with liquids. The miniaturization of printed microarrays provide a platform where the detection proteins in solution are in far excess concentration than the capture analytes bound to the microarray. Increased concentration means faster binding kinetics and increased sensitivity. A properly executed microarray provide more sensitive and reproducible results than bead arrays.

4. Focused, flexible multiplexing in the range of 1 to 500 analytes meets the needs of a wide variety of applications.
True in both cases, with spotted microarray having the ability for a higher degree miniaturization, number of capture analytes and faster data collection time (Full slide scans of 25,000 array elements can be scanned in 3.5 minutes). Microarrays have used antibodies, antigens, peptides, serum, amplified DNA, oligonucleotides, small molecules, carbohydrates, oligosaccarides, hybridomas, phage and others as capture analytes...let's see bead arrays do that! The microarray platform by ArrayIt is clearly a universal biochemistry assay platform for an almost infinite number of assay types.


5. Open architecture allows market leading companies to provide compatible kits and software.
Printed microarray platform is an open architecture with microscope slide glass being the default substrate size used in microarray detection and processing instruments. Microarray detection is routinely done with fluorescent, colorimetric and chemiluminescent detection..providing a much more flexible and open operating environment. Both CCD and laser/PMT microarray scanner architectures are available based on the end users need for quantitation, cost and sensitivity. Very inexpensive colorimetric microarray detection can also be used. Bead arrays are limited to fluorescent detection and specialized equipment to read only specific bead types. Colorimetric detection, considered accurate and the least expensive of all types of detection, cannot be done with existing bead array technology.

What else is nice about microarray that bead array vendors don't tell you?

1. Microarray data quality is checked by careful examination of the raw data of the microarray scan. Bead array flow cytometer dectection instruments do not provide any raw data. The raw data of flow cytometry bead array experiment cannot be examined and checked for quality. Bead array systems rely on the redundancy of the system to verify the quality of the data. With microarray the quality of the data can be physically examined by the end user with as much redundancy and controls built in as needed. Additionally, unlike beads, microarrays can be check for quality prior to their use.

2. Colorimetric microarray detectors, that can capture 16bit quantitative data from analyte spots as small at 50 microns in diameter cost less than $4K. (see SpotWare colorimetric system)

3. Microarrays are manufactured at very low cost, pennies per data point. 100 um data size data points are made with a volume of little more than 500 picoliters of capture analyte.

4. Microarrays do not need internal dyes to identify different capture analytes. Simple spot location identifies the capture analytes.

5. Microarrays do not need many readings of beads to validate data, only 1 image scan is required.

6. Microarray elisa based assays are easy to implement and process, requiring no specialized equipment for processing. Microarrays in the bottoms of 96 well plates and on slides are amenable to existing IFA and ELISA type automation equipment.

7. The proteins coated on beads stored in solution are not stable for long periods of time. Printed microarrays are stable for years at room temperature.

8. Beads in solution can aggregate together over time, rendering them useless. Microarray spots by their nature cannot aggregate.

In conclusion it's my opinion that the spotted microarray platform is superior to flow cytometry based bead platforms. This is a blog entry that will be added to over time.

Quantitative Microarray Data

Just wanted to show off the quantitative abilities of our colorimetric microarray platform. This is a 5 um resolution scan done with our SpotWare Colorimetric Microarray Scanner. Spots in the microarray are approximately 100 microns in diameter. Microarrays manufactured with the NanoPrint Microarrayer using 946MP3 Micro Spotting Pins, 2-fold serial dilution colorimetric detection, gray scale data presented in pseudo-color rainbow pallet to make it easy to see the differences in signal from each row.

Microarray Patent News

Arrayit Lands Patent for Population Scale Diagnostics in World's Most Populous Country

China Is Expected to Be a 'Diagnostics Business Bonanza' for Arrayit

SUNNYVALE, CA--(Marketwire - September 2, 2009) - Arrayit Corporation (OTCBB: ARYC), a proprietary life sciences technology leader, announces that on August 31st, 2009 the company received a patent certificate from the most populous country on the planet, the People's Republic of China. The patent covers Dr. Mark Schena's microarray methods of genotyping multiple patient samples, commonly referred to as Arrayit's "Variation Identification Platform™" (VIP) technology.
Arrayit VIP is a universal platform for deoxyribonucleic acid (DNA)-based genetic screening, testing, diagnostics, genotyping and single nucleotide polymorphism (SNP) analysis. In simple terms, VIP allows clinical laboratories to examine DNA samples from as many as 80,000 individuals with a single test rather than relying on limited "one test per patient" techniques. China is the world's largest marketplace, and Arrayit sees nearly unlimited diagnostics sales potential in this emerging region.
"VIP is well suited for analyzing DNA from large populations in a rapid, affordable and highly accurate manner," states Rene Schena, CEO and co-founder of Arrayit Corp. "We look forward to introducing VIP technology to the People's Republic."
VIP is a versatile testing platform that allows scientists and medical doctors to screen mass numbers of humans for the presence of genetic markers, as well as for viral and bacterial infections. VIP also empowers the analysis of livestock, poultry and crop plants. This is a very critical aspect of VIP, as it allows the tracing of disease origins from agents such as the H1N1 flu strain. VIP could clearly become the gold standard for genetic and infectious disease detection and management.
Arrayit has obtained VIP patents in a long list of countries, and the China issuance represents a significant milestone for the company.

About Arrayit Corporation
Arrayit Corporation, headquartered in Sunnyvale, California, leads and empowers the genetic, research, pharmaceutical, and diagnostic communities through the discovery, development and manufacture of proprietary life science technologies and consumables for disease prevention, treatment and cure. It now offers over 650 products to a customer base of more than 2,500 laboratories worldwide, including most every major university, pharmaceutical and biotech company, major agricultural and chemical company, government agency, national research foundation and many private sector enterprises. Please visit www.arrayit.com for more information.

Safe Harbor Statement
Except for historical information contained herein, statements made in this release that would constitute forward-looking statements may involve certain risks and uncertainties. All forward-looking statements made in this release are based on currently available information and the Company assumes no responsibility to update any such forward-looking statement. The following factors, among others, may cause actual results to differ materially from the results suggested in the forward-looking statements. The factors include, but are not limited to, risks that may result from changes in the Company's business operations; our ability to keep pace with technological advances; significant competition in the biomedical business; our relationships with key suppliers and customers; quality and consumer acceptance of newly introduced products; market volatility; non-availability of product; excess inventory; price and product competition; new product introductions, the outcome of our legal disputes; the possibility that the review of our prior filings by the SEC may result in changes to our financial statements; and the possibility that stockholders or regulatory authorities may initiate proceedings against Arrayit and/or our officers and directors as a result of any restatements. Risk factors associated with our business, including some of the facts set forth herein, are detailed in the Company's Form 10-K for the fiscal year ended December 31, 2008 and Form 10-Q/A for the fiscal first quarter ended March 31, 2008 and Form 10-Q for the fiscal third quarter ended September 30, 2008.

Peptide Microarrays

The data above was generated shows that the FLAG peptide epitopes were recognized by the ANTI-FLAG antibodies, but not by the ANTI-CD28 antibody used as an isotype matched control (negative control). In addition, the scrambled FLAG sequence that was used as another negative control was also not recognized by the ANTI-FLAG antibodies. These results indicate the specificity of the peptide-antibody binding. The data also indicate that peptides containing additional sequences from both PILL and GST demonstrated enhanced reactivity with the ANTI-FLAG antibody. Adding PILL or GST sequences to the peptides did not enhance reactivity to the irrelevant antibody. Data courtesy of Dr. Jonathan Graf and Dr. John Imboden, UCSF. Peptides courtesy of Sigma-Aldrich.
For more information go to:
http://arrayit.com/Services/Peptide_Microarrays/peptide_microarrays.html

biomarkers

A biomarker can be defined as measuring tool of a biological state that is used to objectively measure a biological process or condition. This can be a classification, score or index of various types of measurements. Biomarkers are also used to predict a response to specific therapies, used to group patients as responders and non-responders. Historically biomarkers are single whole molecules, such as the prostate-specific antigen (PSA), used in prostate cancer testing.
Arrayit's microarray technology, in used since 1997, is transforming biomarker testing techniques from single analyte tests to a more reliable multi-analyte microarray based tests. Microarrays are miniturized devices sort of like computer processors, only biological.
Just like the old saying, "two heads are better than one", we use multiple biomarkers in a single test using a microarray to make more reliable "biological decisions" (see video below for more information). We make the tests miniturized to make them inexpensive and higher throughput. All different types of molecules, including RNA, miRNA, DNA, proteins, lipids and carbohydrates are being used (see http://www.arrayit.com/). Microarray assays fall into the FDA regulated catagory of In Vitro Diagnostic Multivariate Index Assays. Microarrays are a driving force behind the movement towards personalized medicine.