Friday, February 15, 2013

Tech Note: Miniaturized, Multiplexed, Parallelized, and Simultaneous Auto-Antibody Measurements for Serodiagnosis of Disease

by: Todd Martinsky, Arrayit Corporation

Background: Arrayit patented microarray printing technology (6,101,946) enables the manufacturing of miniaturized arrays (microarrays) of all biomolecule types onto specially treated glass slides, microfluidic devices and other biochip substrates. We make protein microarrays by mechanically “nano” printing antigens into tiny ordered arrays to simultaneously quantify multiple immunoglobulin antibodies across many antigens in human sera. The miniaturization, multiplexing, parallelism and 1, 2 or 4-color platform increases the throughput and sensitivity of immunoassays, while at the same time lowers cost through miniaturization.

General Method: 24 sets of antigens are printed on specially prepared microscope slide size glass with a NanoPrint Microarrayer using Arrayit’s patented technology (6,101,946). Up to 4 slides of 24 microarrays, each microarray containing many antigens are installed into 96 well reaction cassettes that have a standard SBS footprint.  The microarrays are incubated first with serum samples or whole blood diluted in buffer and subsequently with fluorescently labeled secondary antibodies. Human immunoglobulin antibodies bind to the printed antigens are detected by a confocal microarray scanner, quantified and reported with specialized software.

Typical Results: The detection limit of Ig antibody bound to the antigens can be in the femtomolar or even attomolar range depending on the affinity of the antigen/antibody bound complex and the type of detection reagents used. Within-slide, between-slide, and between lot precision profiles CVs of less than 5% for all antigens are typical. Concordance between microarray assays and ELISAs in the quantification of Ig antibody in sera can be difficult to achieve only because our microarray methods are so much more sensitive, quantitative and have a much larger dynamic range. 

General Conclusions: Arrayit’s microarray platform is suitable assay format for the serodiagnosis of diseases and is suitable for clinical use. The miniaturization, multiplexing and parallelism along with the ability to simultaneously detect up to 4 Ig antibodies (i.e. IgG and IgM) in a single assay have important advantages in throughput, convenience, cost and sensitivity.

Applications:  Microarrays designed to detect in Ig antibodies with different specificities have a wide range of potential applications in health and disease research, vaccine development and the diagnosis of allergy, autoimmune and infectious diseases. Traditionally ELISA-based tests are used for these purposes, however, compared to microarray ELISA assays are more time-consuming, require large quantities of antigen, serum and reagents, thus limiting their utility for testing large populations.

The challenges to meet the needs of clinical utility are; stability of proteins, high quality surface chemistry, repeatable microarray manufacturing methods, elimination of background noise and weak detection signals. For these reasons sera diagnostics using protein microarrays pose unique technical challenges that Arrayit has overcome to meet the needs of clinical use.

Materials and Methods
Microarray Manufacturing

Antigens are diluted in proprietary printing buffers to stabilize the proteins for long term storage and improve manufacturing precision.  Whole molecule IgG, IgA, IgM or IgE depending on the assay are printed as marker spots, these spots directly bind to the detection reagents and act as good positive controls to show that the detection reagents are working properly.  Anti-Ig antibodies are printed as a dilution series for normalization. All samples are tracked with matching txt files and loaded into 384 well plates, using 5 go 8 ul of print ready material depending on the number of microarrays to be manufactured. Antigens and controls are spotted onto specially activated slides substrates using a NanoPrint Microarrayer and programming to spot 24 replicate identical microarrays on each slide in order to be processed in the 96 well AHC4x24 reaction tool.  Not all slide positions need to be loaded, so the microarrays on the slides can be used in a similar way to an ELISA strip plate format, only loading to tool the number of microarrays required to meet current testing needs.

Figure 1. NanoPrint Microarray Software Screen Shot. Click for larger image.

Antigens are mechanically “nano printed” from 384 micro titer plates, to glass slides by use of Arrayit patented microarray manufacturing technology, 6,101,946. Each data point prior to blocking and washing the microarrays contains 500 picoliters of antigen to the slide at 0.3 ug/ul final concentration or lower depending on the antigen’s manufacturing conditions.  Microarrays are printed in a matrix of spots that typically includes 3 technical replicates and measure 100 microns in diameter.  The type of internal calibration curve of IgG, IgM, IgE, IgA is assay dependent, but all 4 could be on the same microarray using a 4-color microarray scanner.

Figure 2.  One column of glass substrates with 24 microarrays on them as sit finished on the NanoPrint Microarrayer. Click for larger image.

Figure 3. Example IgE Raw Microarray Data with spot finding grid aligned on capture antigens, positive and negative controls, no dilution curve. 1 of 24 microarrays on a slide. Spots shown here are 100 um in diameter printed at 300 um center to center distance. Click for larger image.

The fluorophores or protein conjugated fluorescent dyes are not on the microarray so as not to cause background noise from unbound dye molecules to the surface chemistry.
Microarray manufacturing is performed in a class 100 clean room at 25 °C and 50% humidity. These conditions are constantly monitored with sensors and are not allowed to fluctuate during the microarray manufacturing. Printed slides are allowed to dry overnight and then blocked using 1.5 hour incubation in Arrayit Blockit Buffer BKTP followed by washing and drying. Slides are stored in boxes sealed in environmental proof bags dry at 2-8 °C until used.  Microarrays are allowed to come to room temperature before bags are opened.  Microarrays are stable for 12 months under these conditions.

Figure 4. Shown here is the numbering scheme for the 24 microarrays on each slide that the quantification and patient sample tracking software uses for reporting purposes. Click for larger image.

Processing Protocol
1.  Intended use
Serum based immunoassays using antigens as capture agents, designed to identify, measure and report levels of immunoglobulin proteins present in human serum, plasma or whole blood.

2.  Introduction
Antigens (biomarkers) are manufactured by Arrayit on a specially polished and treated glass microscope slide using US Patent 6,101,946. This slide contains biomarkers which can be incubated with either diluted patient serum, plasma or whole blood. Patient serum Ig will bind to recognized biomarkers printed on the microarray during the first incubation. After washing away unbound Ig antibodies, anti-human Ig secondary antibody conjugated to Cy3, Cy5 or equivalent dye compatible with the microarray scanner is added to the microarrays which bind to the antigen Ig antibody conjugate.  Alternatively biotin conjugated secondary antibodies can be used, followed by detection with streptavidin conjugated to an appropriate dye.  This can provide some signal amplification, but is limited to single color experiments.  Unbound secondary antibody is removed by washing. The slides are dried by Microarray Centrifuge and are then ready for scanning and quantification using a high resolution confocal microarray laser scanner (Innoscan 710) with the provided software (Mapix)

3.  List of Materials & Equipment Used
Microarrays 24 microarrays per slide substrate.  Microarray orientation is as follows.  There is a unique ID number outside the microarray areas on each slide.  Microarray 1 is in the upper left corner and the microarrays are numbered 1-24 left to right top down in a 3 x 8 pattern (see figure 3).

Figure 5. Microarrays are printed only on 1 side of the glass.  The side with the microarrays on them is the side with the notch in the glass in the upper right, with the slide in the portrait position.  The notch can only be in the upper right when the slide is in the portrait position.  Note that microarray spots are not visible with the naked eye after they have been processed. Click for larger image.

Figure 6. AHC4x24 reaction cassette pictured above. This tool holds up to 4 microarray slides of 24 microarrays each and is processed almost identical way as a typical ELISA plate.  1, 2 or 4 slides can be loaded to be used in a similar way to an ELISA plate strip system. Click for larger image.

Figure 7. Microarray Centrifuge is used to dry microarray slides after the final rinse prior to scanning. Click for larger image.

Figure 8. Hybridization Station that controls temperature and mixing speed for all processing steps of the experiment. Click for larger mage.

4.  Required

Arrayit Item Number
Components /details
Custom Made
Manufactured by Arrayit
Wash buffer 1, 
aqueous solution
Wash buffer 2, 
aqueous solution
Shipped 10X
used as 1X, 
Proprietary aqueous solution
Blockit Plus
250 ml use to block gasket 
prior to use. 
Proprietary aqueous solution
Microarray Rinse
1x, PMNB
Use as a 1x Buffer to rinse
 slides for 1 second, this is 
the last step prior to drying.
Various Vendors
Example: Goat anti-human 
 IgG Cy5 secondary antibody.
96 Well 
Reaction Tool
Reaction cassette holds up to
 4 slides of 24 microarrays.
Small bench top instrument 
 used to dry microarray slides
 prior to scanning.
Array Plate
High throughput digital hybridization
 instrument enables mixing for 
microplate-formatted hybridization
cassettes containing AHC4x8L and
AHC4x24 well configurations. 
110 volt or 220V version.

5.  Protocol

lUse a robust method to acquire/dilute sample serum and control.  Consider using Arrayit Blood Serum Collection Cards.
 lOnce you start the test, run it all the way through to scanning (or shipping it to Arrayit to be scanned).  It is possible to take necessary human breaks at the incubation steps.
lPrior to use, the AHC4X24 reaction tool gasket must be incubated using the 1X Blockit Plus for minimum of 30 minutes or longer in blocking period then rinsed with distilled or deionized water, and dried.
l The microarrays are often made ready for testing purposes by Arrayit, meaning that no pre-processing of the microarray slides is required by the end user.  If microarrays are stored cold, allow them to come to room temperature before opening the environmental proof bag.
l After the completion of each test, please sonicate the AHC4X24 reaction tool with distilled or deionized water for 10 min, and let it air dry in a clean place or dry with clean room wipe.  Avoid dust and particles from paper towels and low quality wipes.  NEVER use air from an air canister, the propellants that force the air out of the container will contaminate the gasket. Consider an Arrayit Microarray Air Jet.
l Only experienced laboratory personnel should operate this test
l When installing the microarray slides into the AHC4x24 reaction tool, never touch the top surface of the microarray slide. Handle by the edges only. Before assembling microarray slides into the AHC4x24 reaction tool, check that gasket is seated properly into the lid first.  The thick side of the gasket goes against the lid and the thinner side touches the glass.   Make sure the gasket is seated flat. Then once slides are in and flat in the base, do not over tighten the slide holder screws.  Do tighten the top screws that compress the gasket to the slides until the tool “bottoms out” to the base of the slide.  Then invert the tool and look through the glass of each slide to make sure the gasket is making a sealed contact with the glass. This can be seen as a dark line against the glass.  Always document which slide number is at each location in the tool and what serum sample is being applied to which microarray before starting. View a video of this procedure here:
l Warm up hybridization station to 37 °C while serum preparations are being done.

5.1.                    Then react each microarray with a 75ul total reaction volume for each well of the AHC4x24 cassette.  Use the appropriate amount of sample serum or control serum with 1X Reaction Buffer (PMRBP) in 96 well polypropylene plates, matching the serum sample number on plate with serum sample number on the reaction tool.  A proper mix of serum, plasma or whole blood and reaction buffer and accurate pipetting is critical at this stage of the test.  Ratio of serum to reaction buffer depends on the assay, but as high as whole blood undiluted or as dilute as 1 to 5,000 has been used.  Generally a dilution of 1 to 100 is a good place to start; reaction times are serum concentration dependent with the highest concentrations needing less reaction time.  Once serum is mixed in the 96 well plate(s), use multichannel pipette to dispense to the AHC4x24 tool(s) containing the microarrays and reaction buffer to the appropriate wells for a total reaction volume of 75ul.  Note: wells of AHC4x24 can accommodate up to 150 ul reaction volumes.

5.2.                    [1st incubation]
Immediately proceed to incubate for5 to 90 min depending on the concentration of serum at the 37°C.  Use oscillation speed set to 350 rpm on Arrayit Hybridization Station.  An example of typical concentrations and times are 1 to 100 dilution of serum in reaction buffer for 30 minutes

5.3.                    [1st Washing, for serum removing & preparing for secondary antibody]
                    i.          Add 50ul Protein Microarray Wash 1 into each well, and incubate for 1 min at 37°C with gentle mixing, oscillation speed set to 300 rpm. Expel the sample.
                  ii.          Add 75ul Protein Microarray Wash 1 into each well, and incubate for 1 min at 37°C with gentle mixing and oscillation speed at 300rpm. Expel out the sample.
                iii.          Repeat this process (ii) again before proceeding
                iv.          Add 75ul Wash 2 into each well, and incubate for 1 min at 37°C with gentle mixing and oscillation speed at 300rpm. Expel the sample.
                  v.          Repeat this process (iv) again before proceeding. But note, if the secondary antibody is not ready, then do not expel.  Never let the microarray dry out while it is being processed through the test.
                vi.          NOTE: An extra wash or 2 is not going to negatively effect results. Mixing up Wash 1 and 2, doing them in reverse will have a bad effect on the test. Always start with wash 1 and end with wash 2.  Last wash must be done with wash 2.  Do not let microarrays dry out between reactions!

5.4.                     [2nd incubation – secondary antibody]
Make sure Wash 2 buffer is expelled and add 75ul mAb-Cy3 conjugate with the protein chip to initiate the reaction, incubate together for 1 hour at 37°C with gentle mixing, oscillation speed at 500rpm.  Dilution of secondary conjugate is 1 to 10,000 in 1X Reaction Buffer.
5.5.                    [3rd Washing, for removing mAb-Cy5 conjugate]
                    i.          Add 50ul Wash 1 into each well using multichannel pipette, incubate for 1 min at 37°C with gentle mixing, oscillation speed at 300rpm. Expel the sample.
                  ii.          Add 75ul Wash 1 into each well, incubate for 1 min at 37°C incubator with gentle mixing, speed is 300.
                iii.          Repeat this process (ii) again before proceeding. Expel the sample
                iv.          Add 75ul Wash 2 (PBS) into each well, incubate for 1 min at 37°C with gentle mixing, oscillation speed set to 300rpm
           v.        Repeat this process (iv) again before proceeding. Expel the sample and proceed immediately to the final 1 second dip in Protein Microarray Rinse Buffer and dry with a microarray centrifuge (see section 5.6)
         vi.        NOTE: An extra wash or 2 is not going to negatively affect results. Mixing up Wash 1 and 2, doing them in reverse will have a bad effect on the test. Always start with wash 1 and end with wash 2.  Last wash must be done with wash 2.  Do not let microarrays dry out between reactions.
5.6.                    [4th Quick rinse, for optimal signal to noise ratio, preparing to scan]
                      i.       Have ready a petri dish filled with Protein Microarray Rinse buffer.  Dip slide in and out of liquid 1 time quickly, straight in and straight out.  Never touch the top surface of a microarray slide.
5.7.                    [Drying, slides must be completely dry to load into scanner ]
Dry the slide using the Microarray High-Speed Centrifuge. Insert microarray slide bar code side facing up and make sure slide is all the way in past the safety rail.

5.8.                    [Scanning ]
Scan the microarrays using the 532nm laser with the laser power set to high, the PMT set to 25, speed set 35, resolution set to 5 um.  We will be optimizing the laser/pmt settings for best results.

5.9.                    [Data Analysis ]
Quantify and calculate the values of the collected fluorescent data.  Watch computer movie for details.