Friday, July 29, 2011

Protein Arrays

Protein Arrays, Microarray ELISA by Todd Martinsky, Arrayit Corporation, todd@arrayit.com

Enzyme-linked immunosorbent assays {(ELISAs or enzyme immunoassay (EIA)} are traditionally plate-based assays designed for detecting and quantifying capture analytes such as peptides, proteins, antibodies, antigens and hormones.  Here at Arrayit we miniaturize and multiplex these types of assays using our suite of microarray instruments, tools, kits and reagents (see www.arrayit.com).  I like to call our suite of products that make up a full platform ImmunoPlex.

Microarray “ELISA” assays we enable do not require enzymes for detection, but can use them.  In a traditional assay, a capture antigen is absorbed to a polystyrene surface with an antibody that is linked to an enzyme for detection. The conjugated enzyme activity is measured via a signal generated by incubation with a substrate to produce a measurable product.  These assays are done in 96 and 384 well polystyrene plates.   In our microarray assays, enzymes are often replaced with fluorescent dyes for more sensitive and quantitative results, but label free detection techniques are also possible using SPR and planar waveguide detection instruments.

So technically it is not correct to use the acronym ELISA when reporter enzymes are not involved, but it is still routine for many people in our industry to do so.  But the most important difference with our platform is the miniaturization, multiplexing and parallelism we provide.  Instead of detecting a single analyte in the bottom of the well of a plate, dozens, hundreds, thousand and even tens of thousands of capture analytes are covalently linked through specific chemistry to a microarray surface with can be measured in a single assay with very low amounts of input sample.


25,000 spot microarray raw data, click to see larger image.

Our Microarrays are most often run on microscope slide size glass slides with special coatings on them to immobilize the capture analytes of interest for the assay.  Microarray slides have also been called microarray substrates to differentiate them from standard microscope slides since they are of much higher quality. This can be confusing because the term substrate is also used to define reagents used to generate colorimetric signals in traditional ELISA assays.  The most robust microarray substrate surface types covalently immobilize the capture proteins to the surface. Good examples are our SuperEpoxy 2 surface chemistry and our SuperNHS Surface chemistry.

These same reactive surfaces can be provided in 96-well and 384 well plates based formats with many capture proteins printed into an array at the bottom of each well.  It is this binding and immobilization of antibodies, antigens, peptides and other biomarkers makes immunoassays easy to design and perform on the Arrayit ImmunoPlex Platform. Having the multiplexed panel of biomarkers of the assay immobilized to a 2 dimensional planar surface makes it easy to separate bound from non-bound material during the assay much more specifically than bead based assays. We also use lower amounts of sample input than bead based assays.  Our ability to wash away nonspecifically bound materials in a complex mixture of proteins such as serum makes the Arrayit microarray platform a powerful tool for measuring specific analytes from a crude preparation such as serum, a cell or tissue lysate.

A detection enzyme, a biotin tag or fluorescent dye can be linked directly to the primary antibody or introduced through a secondary antibody that recognizes the primary antibody. The most popular fluorescent dyes used in microarray are “Green” (~550 nm excitation, ~570 nm emission and therefore appear green), and “Red” is fluorescent in the red region (~650/670 nm) but absorbs in the orange region (~649 nm). Dyes in these excitation and emission spectrum are supported by a variety of vendors, including Arrayit.  They are standard for all good slide based microarray scanners and work perfectly in our ArrayPix and Innoscan systems a standard deliverable for any platform we put together.  These dyes are usually synthesized with reactive groups on either one or both of the nitrogen side chains so that they can be chemically linked to proteins. The most commonly used enzyme labels are horseradish peroxidase (HRP) and alkaline phosphatase (AP). A large selection of substrates is available for performing the ELISA with an HRP or AP conjugate, Arrayit sells suitable substrate for AP conjugates and compatible high resolution microarray scanners for colorimetric detection (see the SpotWare and ArrayPix Colorimetric systems at Arrayit.com). The choice depends upon the required assay sensitivity and the throughput required.

Our microarray formats are very flexible and include: slides, plates, plate size glass and microfluidic devices.  One key aspect of the platform, regardless of the format is the low amount of sample input required.  With our microarrays, hundreds of ELISA assays can be simultaneously run with just 1 ul of serum.  One of the most popular tools for performing more microarrays in parallel is our AHC4x24 reaction tool that turns 4 slides into a 96 well plate. 





This enables 4 slides of 24 microarrays each, each microarray containing from dozens to thousands of capture proteins, compatible with standard 96 well processing instruments and ELISA plate washers and incubators.  Slides in the base can be removed to scan with standard slide based microarray scanners such as our Innoscan system or stay in the base and scanned with our ArrayPix Microarray scanner.

As we discussed, one of the key steps in the process of a microarray ELISA is the immobilization of the antigens of interest.  This is best accomplished by directly covalently binding the antigens to our microarray surface in a miniaturized and planar array. . Antigen microarrays can be used to measure immunoglobulin levels (IgG, IgE, IgA, IgM) in serum.  Alternatively the assays can be done via capture antibodies that have been made into a microarray.  Sandwich assays can also be done. In this case the analyte to be measured is bound between two primary antibodies – the capture antibody and the detection antibody. The sandwich format is often used because it is sensitive, however, in microarray formats cross reactivity for the antibody pairs must be tested and careful selection of antibodies must be made.  Serum, cells, cell lysates and tissue lysate proteins can also be directly labeled and run on our platform. These sample types can also be printed into microarrays and run in what is often called the reverse phase protein microarray format.


Because our microarray scanners have two fluorescent channels, simultaneous measurements of test and controls can be done at the same time.  Additionally 2 immunoglobulin levels such as IgG and IgM measurements can be done simultaneously against an array of antigens. See stoke shift dye information of the two most common wavelengths below.


Antigens, monoclonal and polyclonal antibodies can be used as the capture and detection antibodies. Monoclonal antibodies have an inherent mono-specificity toward a single epitope that enables detection and quantitation of small differences in antigen. A polyclonal when used as a capture antibody can pull down more antigen from a complex mixture reaction than a monoclonal antibody, but has less specificity. Monoclonal antibodies can be used as the detecting secondary antibody in the sandwich assay to provide improved specificity.  Good antibody pairs must be tested for specificity in multiplexed assays.  If a good antibody pair does not exist, then indirectly or directly label the complex mixture of proteins to apply to an antibody microarray surface or consider a label free detection method. The most important thing to remember in designing a sandwich antibody microarray is that the capture and detection antibodies should recognize two different non-overlapping epitopes. When the antigen binds to the capture antibody, the epitope recognized by the detection antibody must not be obscured or altered. Capture and detection antibodies that do not interfere with one another and can bind simultaneously are called "matched pairs" and are suitable for developing sandwich assays.  As the microarray field matures, antibody suppliers will provide more detailed information about epitopes and indicate pairs of antibodies that have been validated in multiplexed assays. They will be able to do this because they will use microarray technology to screen hybridomas in a higher throughput more specific manner.


Coating plates and printing microarrays are two very different things.  When coating plastic ELISA plates, the binding capacity of the microplate wells are typically higher than the amount of protein coated in each well. In microarray manufacturing the opposite is true; a saturating amount of protein is “printed” to each spot to the binding sites on the microarray surface chemistry.  In ELISA plates, the proteins are absorbed to the plate, in microarray we covalently bind proteins to the glass through specific covalent bonds (see picture below).


What both traditional ELISA and our microarray platform have in common is the remaining surface area must be blocked to prevent antibodies or other proteins from adsorbing to the surface during binding and subsequent processing and detection steps. So a blocking buffer is needed.  A blocking buffer is a solution typically made up of irrelevant protein(s) or other compound that attaches to all locations around printed microarray spots to inactivate the binding sites of the surface chemistry.  Blocking is critical to the sensitivity of an assay.  Good buffer and processing technique at this step lowers background and improves signal-to-noise ratio. The ideal blocking buffer attaches itself to all the areas around the spots of a microarray where a nonspecific interaction could take place.  The goal is to eliminate background without changing any epitope activity on the microarray.  Here at Arrayit we have developed several different blocking buffers for a variety of DNA, peptide and protein microarray assays. See raw data presented in pseudo color rainbow scale below, all microarray data is 16-bit gray scale where the values range from 0 to 65,535.
Since an excess of protein is printed in each spot of the microarray, excess material must be washed away prior to the reaction in addition to the washes between each binding (reaction) step.  Washing steps are necessary to remove non-bound proteins to keep background signals low.  Insufficient washing is a major cause of high background, so consider using a good tool for washing slides if processing manually.  Click here to view some useful manual tools.  

Note that standard ELISA plate washers can be used with our AHC4x24 and other processing tools that hold microarray slide substrates, these tools have the same footprint as 96 and 384 well plates.  Also when washing, keep in mind that all protein binding reactions have specific on and off rates.  Excessive washing or washing in the wrong buffers will comprise results.  Microarray ELISA washes can be performed with phosphate-buffered saline (PBS) without any additives. A detergent such as 0.05% Tween-20 is added to the buffer to help remove non specific binding.  The planar array we provides offers very high specificity. The proteins bound to the surface of the microarray can only interact with what is in solution.  Like all things miniaturized, our microarray assays require a higher level of cleanliness and purity than macro based ELISAs assays in microplates.  WE have developed separate buffers for blocking, washing, reactions and rinse just prior to scanning.  Please see the buffer information here: Buffers link.


As we have mentioned, fluorescent detection dominates microarray applications with its superior sensitivity, linear dynamic range.  Additionally fluorescent detection enables 2 or more labels in the same experiment providing more data with less microarrays.  With HRP and AP conjugates the enzyme converts a recognition substrate to a detectable signal for a single color assay. If a microarray has been properly executed, the intensity of signal produced when the detection substrate is added will by directly proportional to the amount of antigen captured in the plate and bound by the detection reagents. Enzyme-conjugated antibodies offer a lot of flexibility because of the wide variety of detection reagents available for chromogenic, chemifluorescent and chemiluminescent imaging. Chemiluminescent signals can be detected by various means including digital camera systems.  However, chemiluminescent signal intensity has more inherent variability than other detection method.  It is also very difficult to run in a high throughput environment because the signal begins to decay before tests can be detected. Though not as sensitive as fluorescent or chemiluminescent substrates, chromogenic ELISA substrates allow direct visualization and enable kinetic studies to be performed. Furthermore, chromogenic ELISA substrates are detected with standard absorbance plate readers common to many laboratories. Fluorescent ELISA substrates are not as common and require a fluorometer that produces the correct excitation beam to cause signal emission to be generated from the fluorescent tag.