TITLE: Potency Test: Glucose Stimulated Insulin Release Assay
AUTHORS: Integrated Islet Distribution Program

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The goal of the Integrated Islet Distribution Program (IIDP) is to develop a uniform standardized shipping method among all the subcontracted IIDP centers that will result in minimal loss of quality or quantity of shipped islets (as compared to control islets remaining at the IIDP center). The objective of this Standard Operating Procedure (SOP) is to be a model for site-specific SOPs that define the method for quantitative determination of insulin released after glucose stimulation for proving the potency of the human islet preparation shipped by the IIDP.</div><div class = "text-block">GSIR Overview:</div><div class = "text-block">After overnight culture at 37°C (12 – 24 hrs), human islets are incubated with media containing a relatively low</div><div class = "text-block">concentration of glucose (2.8 mM) and a sample of the supernatant is taken. Then the same islets are incubated</div><div class = "text-block">with media containing a higher glucose concentration (28 mM) and a sample of the supernatant is taken. The</div><div class = "text-block">amount of insulin present in both the supernatant samples is measured using a commercially available Human</div><div class = "text-block">Insulin ELISA kit. The stimulation index is calculated by dividing insulin concentration of the supernatant</div><div class = "text-block">from the 28 mM glucose incubation by the insulin concentration of the supernatant from the 2.8 mM glucose</div><div class = "text-block">incubation.</div><div class = "text-block"><span style = "font-style:italic;">Reference:</span></div><div class = "text-block">Purified Human Pancreatic Islets, Glucose Stimulated Insulin Release Determination by ELISA; DAIT, NIAID, NIH SOP Appendix, Document No. 3104, A03, Revision Number 03; Effective Date: 25 Oct 2010; Supersedes Date: 08 June 2010.</div></div>

[STEPS]
?. [Culture Media Preparation]
Culture media that is to be used for overnight culturing is prepared as described below.The Prodo Labs PIM(R) should be stored between 2° and 8°C upon receipt.The (heat inactivated) Gemini AB serum  and the PIM(G) vials should be stored at -5° to -20°C.
?. [Culture Media Preparation]
The  Ciprofloxacin powder can be stored on the shelf but filter sterilized suspension aliquots should be stored at -5° to -20°C.
Record preparation on Attachment 1: Solutions Preparation Sheet, of this SOP.
?. [Culture Media Preparation]
Prepare 1 bottle of PIM(R) media prior to the collection by adding the following:Thaw and add 5 mL of PIM(G).Add 25 mL of AB serum (5%v/v).Add 0.5 mL of prepared sterile ciprofloxacin aliquot. Once all additives have been added to the bottle of PIM(R), it is now referred to as PIM(R) complete.
Note: If a prepared media bottle is to be used from a previous isolation, it must have been filter sterilized at the end of the previous use. The media will expire within 30 days, once it has been fully supplemented.
Record preparation on Attachment 1: Solutions Preparation Sheet, of this SOP.
?. [Islet Sample Preparation]
On the day of islet shipment, remove at least 400 IEQ (calculate using islet count per volume) from the purest layer using sterile technique.The formula will be:Total  IEQ/(Total  mL * 1000 μL)= x  IEQ/μL then 400 IEQ/x IEQ/μL = y μL
?. [Islet Sample Preparation]
Place islets in a 100 mm Petri dish, add , label, and cover.
[PIM(R) Complete]
?. [Islet Sample Preparation]
Place the dish in a 37°C, 5 % CO2 incubator.
?. [Islet Sample Preparation]
Culture islets for  – .
?. [Glucose Stimulation Reagent Preparation: Krebs Buffer Stock Solution]
Combine in a 1 liter volumetric flask:  (final concentration 25 mM)  (final concentration 115 mM)  (final concentration 24 mM)  (final concentration 5 mM)  (final concentration 1 mM) (final concentration 0.1 %)
[HEPES powder]
[NaCl]
[NaHCO3]
[KCl]
[MgCl2•6 H2O]
[ BSA]
Record preparation on Attachment 1: Solutions Preparation Sheet, of this SOP.
?. [Glucose Stimulation Reagent Preparation: Krebs Buffer Stock Solution]
QS to   with distilled water and mix until dissolved.
1 L
?. [Glucose Stimulation Reagent Preparation: Krebs Buffer Stock Solution]
Add   (final concentration 2.5 mM) and stir the solution (the CaCl2•2 H2O may not completely dissolve until the solution pH is adjusted).
[CaCl2•2 H2O]
?. [Glucose Stimulation Reagent Preparation: Krebs Buffer Stock Solution]
Check the pH of the solution and adjust to  to  using either   or  as necessary.
[NaOH]
[HCl]
?. [Glucose Stimulation Reagent Preparation: Krebs Buffer Stock Solution]
Filter sterilize into a sterile 1 liter bottle using a 0.22 μm bottle top filter.
?. [Glucose Stimulation Reagent Preparation: Krebs Buffer Stock Solution]
Divide into 18 X 50-ml aliquots in sterile 50 ml tubes.
NOTE:  Reserve remaining Krebs Buffer for 280 mM stock glucose below.
?. [Glucose Stimulation Reagent Preparation: Krebs Buffer Stock Solution]
Label as Krebs Buffer Stock Solution with preparation date, expiration date (4 weeks after preparation), and store at  – .
2 °C
8 °C
?. [Glucose Stimulation Reagent Preparation Stock Solutions: 280 mM Glucose Solution]
Add   to .
[D-(+)-Glucose]
[Krebs Buffer Stock Solution]
Record preparation on Attachment 1:  Solutions Preparation Sheet, of this SOP.
?. [Glucose Stimulation Reagent Preparation Stock Solutions: 280 mM Glucose Solution]
Filter sterilize into a sterile 100 ml bottle, using a 0.22 μm bottle top filter.
?. [Glucose Stimulation Reagent Preparation Stock Solutions: 280 mM Glucose Solution]
Divide into 10 X 6-ml aliquots, using sterile 15 ml tubes. Label with preparation date, expiration date (4 weeks after preparation), and store at  – .
2 °C
8 °C
?. [Glucose Stimulation Reagent Preparation Stock Solutions: High Glucose (28 mM) Solution]
Prepare High Glucose () Solution by making a 1:10 dilution of , using Krebs Buffer Stock Solution. Prepare as much solution as needed.
[Glucose stock]
Record preparation on Attachment 1: Solutions Preparation Sheet, of this SOP.
?. [Glucose Stimulation Reagent Preparation Stock Solutions: High Glucose (28 mM) Solution]
Label with preparation date, expiration date (1 week after preparation), and store at  – .
2 °C
8 °C
?. [Glucose Stimulation Reagent Preparation Stock Solutions: Low Glucose (2.8 mM) Solution]
Prepare Low Glucose () Solution by making a 1:10 dilution of , using Krebs Buffer Stock Solution. Prepare as much solution as needed.
[High Glucose Solution]
Record preparation on Attachment 1: Solutions Preparation Sheet, of this SOP.
?. [Glucose Stimulation Reagent Preparation Stock Solutions: Low Glucose (2.8 mM) Solution]
Label with preparation date, expiration date (1 week after preparation), and store at  – .
2 °C
8 °C
?. [Glucose Stimulation:  Plate preparation]
Label a 24-well plate with sample ID and date.
?. [Glucose Stimulation:  Plate preparation]
Add  per well to wells A1, A2, A3.
1 mL
[Low Glucose Solution]
?. [Glucose Stimulation:  Plate preparation]
Add  of  per well to wells B1, B2, B3.
1.3 mL
[Low Glucose Solution]
?. [Glucose Stimulation:  Plate preparation]
Add  of  per well to wells C1, C2 and C3.
1.3 mL
[High Glucose Solution]
?. [Glucose Stimulation:  Plate preparation]
Using forceps, place one Millicell Cell Culture Plate Insert (insert) into well A1, A2 and A3.
?. [Glucose Stimulation:  Plate preparation]
Cover plate with lid and incubate the plate to  in 5 % CO2 incubator for  (to equilibrate pH and temperature of media).
37 °C
?. [Glucose Stimulation:  Plate preparation]
Transfer  of each glucose solution into a marked tube (High Glucose Control and Low Glucose Control) and store at  –  until assay completion.
1 mL
4 °C
8 °C
?. [Glucose Stimulation: Preparation of islets]
Remove the Petri dish containing islets from the incubator.
?. [Glucose Stimulation: Preparation of islets]
Collect the islets from the Petri dish by centering the tissue in the dish and removing the islets in , using a wide bore micropipette.
[PIM(R) complete media]
?. [Glucose Stimulation: Preparation of islets]
Transfer the islets to a 5 ml conical tube.
?. [Glucose Stimulation: Preparation of islets]
Add  to the tube.
[PIM(R) complete media]
NOTE: If 100 % of the 400 IEQ are cultured and there is overnight loss, there are 80 IEQ/300 μl in this suspension of islets. There will be a loss of islets during the culture, so the recommended target of 50 – 75 IEQ per well is likely to be achieved. (A 300 μl aliquot containing ~80 IEQ will be added to each insert in the next step).  400 IEQ ÷ 1500 μl (100 μl islets in PIM(R) Complete Media + 1400 μl  PIM(R) Complete media) = 0.26 IEQ/ μl 0.26 IEQ/μl X 300 μl = 79.9 IEQ per well (with no islet loss).
?. [Glucose Stimulation: Basal Equilibration (Row A)]
Place an insert in each of the empty wells D1, D2, D3.
?. [Glucose Stimulation: Basal Equilibration (Row A)]
Slowly pipette  of well-mixed islet suspension into each insert in row D.
300 µl
?. [Glucose Stimulation: Basal Equilibration (Row A)]
Place insert on sterile gauze to drain liquid out completely. Transfer insert with islets to the wells A1, A2, A3.
?. [Glucose Stimulation: Basal Equilibration (Row A)]
Replace the lid on the plate and incubate for  at  in 5 % CO2.
37 °C
?. [Glucose Stimulation: Low Glucose Stimulation (Row B)]
Remove plate from incubator.
?. [Glucose Stimulation: Low Glucose Stimulation (Row B)]
Transfer the inserts from row A to corresponding wells in row B (B1, B2, B3) as described below:
Row A allows islets to equilibrate from the culture media to Krebs basal media. Samples from these wells in Row A are discarded at the end of the procedure.
?. [Glucose Stimulation: Low Glucose Stimulation (Row B)]
Slowly and gently, lift the insert up from its corresponding well, using forceps.
NOTE:  Bumping insert will cause the islets to release insulin resulting in false stimulation results.
?. [Glucose Stimulation: Low Glucose Stimulation (Row B)]
Allow liquid to drain from each insert back into the well by “wicking” the liquid from the mesh bottom of the insert against the side of the well.
?. [Glucose Stimulation: Low Glucose Stimulation (Row B)]
When the insert looks well-drained, gently blot the bottom of the insert on sterile gauze before gently setting the insert in the corresponding well in row B.
?. [Glucose Stimulation: Low Glucose Stimulation (Row B)]
Repeat the above steps from 38.1-38.3 for all inserts from row A to row B.
?. [Glucose Stimulation: Low Glucose Stimulation (Row B)]
Take Zero Time media samples for Low Glucose media from wells B1, B2, B3:
?. [Glucose Stimulation: Low Glucose Stimulation (Row B)]
Allow the level of liquid to equilibrate between inside and outside of each insert (now in its corresponding well).
?. [Glucose Stimulation: Low Glucose Stimulation (Row B)]
Lift each insert and drain the liquid back into its corresponding well. Do not blot the insert on sterile gauze.
?. [Glucose Stimulation: Low Glucose Stimulation (Row B)]
Remove  of media from each well (from the well, not the insert). Transfer media to corresponding microcentrifuge tubes labeled “Zero time – Low Glucose”, B1, B2, or B3, Lot#, Date, Tech.
300 µl
?. [Glucose Stimulation: Low Glucose Stimulation (Row B)]
As soon as the sample is collected, immediately replace the insert in its corresponding well. Repeat this for each of the inserts in row B.
?. [Glucose Stimulation: Low Glucose Stimulation (Row B)]
Replace the lid on the plate and incubate for  at  in 5 % CO2.
37 °C
?. [Glucose Stimulation: Low Glucose Stimulation (Row B)]
After 1 hour, carefully remove the plate from incubator.
?. [Glucose Stimulation: Low Glucose Stimulation (Row B)]
Remove inserts from row B wells and transfer each into the corresponding well in row C as described above. Collect all media from each well in row B to corresponding microcentrifuge tubes labeled “Low Glucose”, B1, B2, or B3 and store at  –  until assay completion.
4 °C
8 °C
?. [Glucose Stimulation: Low Glucose Stimulation (Row B)]
Take Zero Time media samples for High Glucose media as described in steps 39.1 through 39.4 above but transfer media to tubes labeled “Zero time - High Glucose”.
?. [Glucose Stimulation: High Glucose Stimulation (Row C)]
Replace lid on plate and incubate for  at  in 5 % CO2.
37 °C
?. [Glucose Stimulation: High Glucose Stimulation (Row C)]
After 1 hour, carefully remove the plate from incubator.
?. [Glucose Stimulation: High Glucose Stimulation (Row C)]
Remove inserts from row C  wells and transfer each into the corresponding well in row D as described above. Collect all media from each well in row C  to corresponding microcentrifuge tubes labeled “High Glucose”, C1, C2, or C3.
?. [Sample Storage]
According to Mercodia (kit manufacturer), samples stored at 4 – 8ºC must be assayed within 24 hours, while samples stored at stable -20ºC can be assayed within 3 months. Place all samples in appropriate storage.
Note, if RIA is the assay to be used for insulin determination, store as the RIA protocol recommends.
?. [Assay Insulin Samples]
Assay insulin samples using an ELISA assay kit (such as Mercodia) within 10 days of broadcast.
If RIA is the assay to be used for insulin determination, results should be available and entered into the database within 10 days of broadcast.
?. [Assay Results]
Results should be reported on the website under Potency Assay as Stimulation Index (SI);Stimulation\; Index (SI) =  \left( \frac{Insulin\; concentration\; after\; high\; glucose\; concentration\; stimulation}{Insulin\; concentration\; after\; low\; glucose\; concentration\; stimulation} \right)Results from each of three samples are averaged and reported within 10 days of broadcast.
?. [Preparation of Human Insulin ELISA Solutions]
Human Insulin ELISA KitThe following reagents come in the Human Insulin ELISA kit, ready for use:Insulin Standards (S0-S5) Microplate strips coated with murine anti-insulinSubstrate TMB Colorless Solution (light sensitive)Stop SolutionMicroplate strips coated with murine anti-insulinSubstrate TMB Colorless Solution (light sensitive)Stop Solution
Note: It is not required to use the ELISA for measuring insulin content of media. If using an RIA however, standards should be established and dilutions should be made in accordance with the assay’s recommendations.
?. [Preparation of Human Insulin ELISA Solutions: Wash Solution]
Prepare Wash Solution by adding   and   to a clean 1 l bottle. Mix the solution well.
[distilled water]
[Concentrate Wash Solution]
Record preparation on Attachment 1: Solutions Preparation Sheet, of this SOP.
?. [Preparation of Human Insulin ELISA Solutions: Wash Solution]
Label the bottle: Wash Solution with Preparation Date, Expiration Date (4 weeks after preparation), Lot #, and preparer’s initials.
?. [Preparation of Human Insulin ELISA Solutions: Wash Solution]
Store at  – .
2 °C
8 °C
?. [Preparation of Human Insulin ELISA Solutions: Anti-insulin Conjugate Solution]
For each microstrip used, mix  with . Examples:  ABC1# of StripsConjugate Stock SolutionConjugate Buffer21100 μl1 ml36600 μl6 ml4121.2 ml12 ml
Prepare immediately prior to use.
Record preparation on Attachment 1: Solutions Preparation Sheet, of this SOP.
[Conjugate Stock Solution]
[Conjugate Buffer]
ABC1# of StripsConjugate Stock SolutionConjugate Buffer21100 μl1 ml36600 μl6 ml4121.2 ml12 ml
?. [Preparation of Human Insulin ELISA Solutions: Anti-insulin Conjugate Solution]
Label the bottle: Conjugate Solution with Preparation Date, Expiration Date (1 day after preparation), Lot #, and preparer’s initials.
?. [Preparation of Human Insulin ELISA Solutions: Anti-insulin Conjugate Solution]
Store at  – .
2 °C
8 °C
?. [Preparation of Human Insulin ELISA Solutions: Preparation of Controls]
Remove the Diabetes Ag Control vials from the refrigerator and allow them to equilibrate to  for .
8 Room temperature
Record preparation on Attachment 1:  Solutions Preparation Sheet, of this SOP.
?. [Preparation of Human Insulin ELISA Solutions: Preparation of Controls]
Reconstitute Low and High Insulin controls as follows:Add  of  to each vial.Replace cap and let sit for .
[distilled water]
?. [Preparation of Human Insulin ELISA Solutions: Preparation of Controls]
Aliquot  of “Low Control” into 5 1-ml microcentrifuge tubes.Store at .
95 µl
-20 °C
?. [Preparation of Human Insulin ELISA Solutions: Preparation of Controls]
Aliquot  of “High Control” into 5 1-ml microcentrifuge tubes.Store at .
95 µl
-20 °C
?. [Preparation of Human Insulin ELISA Solutions: Preparation of Controls]
Establishment of acceptable ranges for a new lot of controls using manufacturer’s procedures.
?. [Sample Dilution]
Supernatants collected from the glucose challenge wells are diluted using DPBS for the ELISA. The commonly used dilution factors are 1:50 and 1:100. To select an optimal dilution factor, the insulin concentration detected by ELISA should be in the range of 31.8 to 191 mU/l.
?. [Human Insulin ELISA Procedure ]
Bring all reagents and samples to .
See Attachment 3:  Glucose Stimulated Insulin Release Worksheet
0 Room temperature
?. [Human Insulin ELISA Procedure ]
Prepare sufficient microplate wells to accommodate calibrators, controls and samples in triplicate. Record the plate location of samples on Attachment 2: Microtiter Plate Layout.
?. [Human Insulin ELISA Procedure ]
Pipette  each, calibrators, controls and samples into appropriate wells. Pipette directly onto the bottom of the well. Change tips between each standard, control and sample.
25 µl
?. [Human Insulin ELISA Procedure ]
Add  to each well.
[Enzyme Conjugate]
?. [Human Insulin ELISA Procedure ]
Cover the plate and incubate on a plate shaker at  at  – .
Centrifuge: 800 33, 60 min
18 °C
25 °C
?. [Human Insulin ELISA Procedure ]
Wash manually: fill each well completely with Wash Buffer () using a squeeze bottle. Turn the plate upside down over a sink and shake to discard liquid completely. Repeat 5 times. After the final wash, invert and tap the plate firmly against absorbent paper. Or, wash the plate six times with an automatic plate washer.
350 µl
?. [Human Insulin ELISA Procedure ]
Fill each well completely with Wash Buffer () using a squeeze bottle. Turn the plate upside down over a sink and shake to discard liquid completely. (1/6)
350 µl
?. [Human Insulin ELISA Procedure ]
Fill each well completely with Wash Buffer () using a squeeze bottle. Turn the plate upside down over a sink and shake to discard liquid completely. (2/6)
350 µl
?. [Human Insulin ELISA Procedure ]
Fill each well completely with Wash Buffer () using a squeeze bottle. Turn the plate upside down over a sink and shake to discard liquid completely. (3/6)
350 µl
?. [Human Insulin ELISA Procedure ]
Fill each well completely with Wash Buffer () using a squeeze bottle. Turn the plate upside down over a sink and shake to discard liquid completely. (4/6)
350 µl
?. [Human Insulin ELISA Procedure ]
Fill each well completely with Wash Buffer () using a squeeze bottle. Turn the plate upside down over a sink and shake to discard liquid completely. (5/6)
350 µl
?. [Human Insulin ELISA Procedure ]
Fill each well completely with Wash Buffer () using a squeeze bottle. Turn the plate upside down over a sink and shake to discard liquid completely. (6/6)
350 µl
?. [Human Insulin ELISA Procedure ]
Invert and tap the plate firmly against absorbent paper.
?. [Human Insulin ELISA Procedure ]
Add   into each well.
[TMB Substrate Solution]
?. [Human Insulin ELISA Procedure ]
Cover plate with aluminum foil  to protect it from direct light and incubate for  at  ( –).
25 Room temperature
18 °C
25 °C
?. [Human Insulin ELISA Procedure ]
Add  to each well. Place plate on a shaker for approximately  to ensure mixing.
[Stop Solution]
?. [Human Insulin ELISA Procedure ]
Read optical density at 450 nm using bi-chromatic measurement with reference at 600 – 690 nm within .
?. [Human Insulin ELISA Procedure ]
Record the data on Attachment 3: Glucose Stimulated Insulin Release Worksheet, of this SOP, and perform calculations required.
?. [Quality Control, Interpretation and Release Criteria]
For assay acceptance:All control values must be within their 2 SD established rangesStandard values must be within ± 15 % of their nominal valuesTriplicates of standards, controls and samples must have Coefficients of Variation (CV) ≤ 20 %
For an unacceptable assay: If the assay is unacceptable for any of these reasons, sample values may not be reported. The assay should be investigated and repeated.