The Jackson Laboratory

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.

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 5 to 10 minutes with a  final incubation of 30 minutes.  The loading of stop solution is about 5 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.

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. 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.