ALPCO Diagnostics 1-2-3 UltraSensitive Mouse Insulin ELISA Prepared by: HS Date Prepared: January 14, 2002 Code: MP-010 Reviewed by: Randy Von Smith Revision #: V3 Date Revised: Jan. 31, 2005 0.0 Abstract: This test determines the amount of insulin in mouse plasma in units of ng/ml. Insulin levels are determined using plasma from blood. Blood is collected using 7.5ul Sodium Heparin 1000 units with Heparin coated capillary tubes. ELISA is a solid phase two-site enzyme immunoassay. 1.0 Experiment Layout: This test uses a Spectra Max 190 ELISA plate reader using Softmax software driven by a PC. 2.0 Supplied Materials and Materials Needed: 2.1 Supplied Materials: The ELISA Insulin kit is purchased from ALPCO Diagnostics, PO Box 451, Windham, NH 03087. Kit contains: (1) MicroPlates (twelve strips, eight wells each coated with mouse anti-insulin) (2) Standards of 0.025, 0.063, 0.25, 0.55, 1.40, 4.0, 7.5 ng/ml which are lyophilized and need to be reconstituted with 1ml of redistilled water. (3) Zero Standard, which is already reconstituted (4) Anti-Insulin Conjugate Stock solution is prepared by diluting 50ul Conjugate Stock Solution with 500ul Conjugate Buffer for each strip. (5) Washing solution is prepared by diluting 40ml of Washing Solution Concentrate into 800ml of redistilled water. (6) TMB Substrate and Stop solution are already prepared in kit. 2.2 Materials needed: (1) 5, 25, 50 ul micropipette, 50 and 250 repeating pipettes (2) containers for reagent preparation and Mammalian/Mouse Insulin Two level control (3) microplate rotator (4) redistilled water. 3.0 Setup: Make a template as to where your Blank, Standards, Controls and Samples are going to be placed within the microplate. Depending whether single, duplicate, or triplicate samples are run, the plate should be set up this way: 1 2 3 4 5 A STANDARD STANDARD SAMPLE SAMPLE SAMPLE B STANDARD STANDARD SAMPLE SAMPLE SAMPLE C STANDARD STANDARD SAMPLE SAMPLE SAMPLE D STANDARD STANDARD SAMPLE SAMPLE SAMPLE E STANDARD STANDARD SAMPLE SAMPLE SAMPLE F STANDARD STANDARD SAMPLE SAMPLE SAMPLE HIGH G STANDARD STANDARD SAMPLE SAMPLE CONTROL HIGH H BLANK BLANK SAMPLE SAMPLE CONTROL 4.0 Loading and Running: Referring to the template, Blanks, Standards, Controls, and Samples are loaded into the wells of the plate using the appropriate micropipettor in the following quantities. 15ul of Insulin Zero into all wells, an additional 10ul of insulin zero standard into appropriate wells that will be considered “Blank”, 10ul of each standard into appropriate wells. Corresponding to the template 10ul of each sample is also added. 50ul of Working Conjugate Buffer is added to all wells. Incubate the plate on a microplate rotator for two hours at 18-28°C at 800-1100 rpms. The plate can also be incubated overnight in a cold room. Once incubation is done the plate is washed six times by hand. (an Automatic plate washer can also be used.) Pipette 200ul of TMB Substrate Solution into each well. Incubate for 30 minutes at 18-28°C. It is very important that the plate is placed in a dark area. TMB is light sensitive; placing the plate in a drawer is good. After the thirty minute incubation is done add 50ul of Stop Solution to each well. It is advised that caution be practiced at this step. Stop Solution is made of Sulfuric Acid and you do not want this on your hands. Read optical density at 450nm and 640nm on a plate reader. The plate should be read within 30 minutes of adding the stop solution. Discard the plate in hazardous waste bin. 5.0 Clean Up: After the plate has been read, the plate is discarded into a hazardous waste bin. Place all leftover reagents back into refrigerator, place standards into the freezer and carefully place all equipment used, back to a location that will be easy to locate during the next test. 6.0 Data Reduction: After the results have been calculated by the plate reader, you can view the results for the Unknowns, Standards or the Standard Curve individually or as a whole depending on which set of data points you have open at any one time. To open any set of data simply click on the name of that data that you wish to view and the software opens the data requested. All data can be printed out by selecting print and then selecting the corresponding data set you have open. All data is reviewed, outliers are noted, so they can be repeated at a later time, and then data is sent to Mutajax to the Insulin folder on Mutaprd Peggy. 7.0 Safety: This test should be conducted wearing safety gloves. Although it is unlikely to be infected by mouse plasma, caution should be taken as though it were possible. This test also uses Sulfuric Acid in the stop procedure, and therefore it is hazardous to humans should skin contact occur. Caution is practiced, and all materials are dispensed into hazardous waste bins. 8.0 Time and Capacity: 26 samples bench time= 1 hour 35 minutes 26 samples calendar time= 4-5 hours Loading of all standards (in duplicate), samples (in duplicate or triplicate) and reagents for the first reaction into a 96 well plate usually takes approximately 45 minutes, which is then followed by a two hour incubation. The wash immediately following the incubation takes about 15 minutes by hand for one plate. Loading of TMB takes approximately 2 to 5 minutes with a final incubation of 30 minutes. The loading of stop solution is about 2 minutes per plate. Reading the plate with the plate reader takes approximately 20 minutes, loading the sample names as well as standards takes the longest for they are entered by hand. Actual time for plate reader to calculate results is about 30 seconds. Total bench time equals 1 hour 35 minutes; total time equals 4 hours 5 minutes. 9.0 Protocols and Quality Control: 9.1 Protocols: Thaw reagents day before. Standards: (when new) Add 1ml of distilled H2O to each, run standards in the first two strips of each plate (unless doing more than one plate). Store at -20°C. for storage over 1 week. Washing Solution: For every 800ml of dH2O add 40ml of Washing Solution Concentrate. Make Plate Sheet: Label the template with the corresponding sample numbers, standards and controls. Pipette 15ul of Insulin Zero Standard into each well, add another 10ul of Insulin Zero Standard for the Blanks. Add 10ul of each standard into representing wells. Add 10ul of each Sample into representing wells on sheet. Add 50ul per well of Working Conjugate Buffer (Blue) Working Conjugate Buffer: For every strip you will need 50ul of Anti Insulin Conjugate stock plus 500ul of Conjugate Buffer. Example: 2 strips = 100ul of stock + 1ml of Conjugate Buffer or for 12 strips = 600ul of stock + 6ml of Conjugate Buffer. Incubate for 2 hours at 18 - 28°C (room temp) on plate rotor at 800-1100rpms. Wash six times with Washing Solution: by placing washing solution into each well, discarding liquid completely, tapping firmly on paper towel, and repeat five more times. (Using an automatic plate washer can also be used) Pipette 200ul of TMB Substrate Solution into each well. Incubate for 30 minutes at 18-28°C (room temp)***PROTECT PLATE FROM SUNLIGHT*** Add 50ul of Stop Solution to each well *** Wear Gloves, Stop Solution is Sulfuric Acid*** Read optical density at 450nm and 640nm. READ THEM WITHIN 30 MINUTES. 9.2 Quality Control: Mouse insulin of a high and low concentration is tested each time for quality assurance. Data for all controls, standards and unknowns are kept on file. Standards are checked for quality assurance by checking if the controls and standards fall in range of the test. All data is reviewed, outliers are excluded and is then sent to Mutajax by exporting the file in a text format to the Insulin folder under “Mutaprd Peggy”. Rejected samples are repeated when possible, and are stored at - 20°C until they can be repeated. When samples are above the standard curve, plasma will be diluted 1:10 with Zero Standard and then retested when possible. Sample data is rejected when two or more data points do not fall within the standard curve. If only one out of three data points, for any sample, is out of range, that data point is rejected and the two other data points for that sample remain and are sent to Muta Jax.