Monday, October 26, 2009

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.