| ```markdown | |
| # Goal/Experiment: | |
| The goal of this experiment is to quantify the insulin secretion and content from human pancreatic islets after glucose stimulation to assess islet function. This protocol provides a detailed methodology for performing Insulin Enzyme-linked Immunosorbent Assay (ELISA) to measure insulin concentrations. | |
| # Analysis of Islet Function by Insulin Enzyme-linked Immunosorbent Assay (ELISA) | |
| **Date of Publication:** December 02, 2021 | |
| **DOI:** [dx.doi.org/10.17504/protocols.io.bz7bp9in](https://dx.doi.org/10.17504/protocols.io.bz7bp9in) | |
| **Cited by:** IIDP-HIPP | |
| This Standard Operating Procedure (SOP) is based on the Vanderbilt University Medical Center Human Islet Phenotyping Program (HIPP) Islet Functional Analysis. This SOP provides the HIPP procedure for measuring islet insulin content and secretion to assess islet function. It defines the assay method used by the Human Islet Phenotyping Program (HIPP) for the qualitative determination of Purified Human Pancreatic Islet product, post-shipment, for use in National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)-sponsored research in the Integrated Islet Distribution Program (IIDP). | |
| **SOP #: HIPP-09-v01** | |
| ## Organizations & Key Terms | |
| ### Integrated Islet Distribution Program (IIDP) | |
| The IIDP is a grant-funded program commissioned and funded by the NIDDK to provide quality human islets to the diabetes research community to advance scientific discoveries and translational medicine. | |
| ### IIDP Coordinating Center (CC) | |
| The IIDP CC integrates an interactive group of academic laboratories including the subcontracted IIDP centers. | |
| ### Human Islet Phenotyping Program (HIPP) | |
| The HIPP is a subcontracted entity of the IIDP through the COH and Vanderbilt University. | |
| ### Islet Equivalent (IEQ) | |
| An islet with a diameter of 150 μm determined mathematically by compensating for islet shape. | |
| ### Islet Perfusion Assay | |
| A functional assay that acquires dynamic hormone secretory profiles simultaneously from islet cell types. | |
| ### Enzyme-linked immunosorbent assay (ELISA) | |
| A sensitive in vitro assay technique used to measure concentrations of antigens by making use of an enzyme conjugated to an antibody recognizing an antigen of interest. | |
| ## Equipment | |
| 1. The following equipment is necessary to assess human islet function by Insulin ELISA: | |
| - **1.1 Micropipettes:** (10-100 µL, 20-200 µL, and 100-1000 µL ranges) | |
| - **1.2 Multi-channel micropipette:** (20-200 µL range) | |
| - **1.3 Computer:** with Excel (Microsoft) and Prism (Graphpad) software | |
| - **1.4 epMotion Liquid Handling Workstation:** (Eppendorf 5075) | |
| - **1.5 Benchmark Orbi shaker:** (BT1502) | |
| - **1.6 Fisherbrand accuWash microplate washer:** (5165100) or similar plate washer (14-377-577 or 14-377-578) | |
| - **1.7 BMG Labtech CLARIOstar microplate reader:** (Plus Model) | |
| - **1.7.1 CLARIOstar software** | |
| - **1.7.2 MARS data analysis software** | |
| - **1.8 Vortex mixer** | |
| ## Supplies and Materials | |
| 2. The following supplies and materials are necessary to assess human islet function by Insulin ELISA: | |
| - **2.1 Human Insulin ELISA Kit, Mercodia** | |
| - **Catalog #10-1113-10** | |
| - **2.1.1 Coated 96-well plates:** (20-2622) | |
| - **2.1.2 Calibrators 0, 1, 2, 3, 4, 5:** Insulin concentrations are known values provided by manufacturer (20-2615, 20-2616, 20-2617, 20-2618, 20-2619, 20-2620) | |
| - **2.1.3 Enzyme Conjugate 11X:** (20-2631) | |
| - **2.1.4 Enzyme Conjugate Buffer:** (20-2630) | |
| - **2.1.5 Washer Buffer 21X:** (20-3194) | |
| - **2.1.6 Substrate TMB:** (20-3136) | |
| - **2.1.7 Stop Solution:** (20-2694) | |
| - **2.2 Buffer, Mercodia** | |
| - **Catalog #10-1195-01** | |
| - **2.3 Human Diabetes Antigen Controls** | |
| - **Catalog #10-1134-01** | |
| - **2.4 50 µL epMotion pipette tip, Eppendorf** | |
| - **Catalog #30014421** | |
| - **2.5 10 mL (Fisher Scientific 13-678-11E) and 25 mL (Fisher Scientific 13-678-11) serological pipets** | |
| - **2.6 2 mL microcentrifuge tube, Sarstedt** | |
| - **Catalog #72.695.500** | |
| - **2.7 200 µL (ART P-200) and 1000 µL (ART P-1250) pipette tips** | |
| ## Procedures | |
| ### 1. Preparation of Samples, Standards, and Internal Quality Controls | |
| 1.1 Thaw archived samples intended for analysis in room temperature water. Once thawed, invert capped samples ten times to thoroughly mix. | |
| 1.2 Retrieve the islet hormone extracts and keep on ice. | |
| 1.3 Prepare serial dilutions of hormone extract (1:100, 1:1000, 1:2000, 1:5000, and 1:10000) in 2 mL tubes using the Calibrator 0 media from the ELISA Kit or Diabetes Sample Buffer (See Figure 1). Vortex each tube to mix contents before generating subsequent dilutions. | |
| 1.4 Generate 1:3 dilutions for perfusion fractions #23, #24, #25, and #43 by adding 40 µL sample to 80 µL of Calibrator 0 or Diabetes Sample Buffer in 2 mL tubes. | |
| 1.5 Transfer all Calibrators and Antigen Controls from original bottles to 2 mL tubes. | |
| ### 2. Preparation of Enzyme Conjugate and Wash Buffer Solutions | |
| 2.1 Prepare Enzyme Conjugate 1X solution by diluting Enzyme Conjugate 11X in Enzyme Conjugate Buffer. Mix gently. Prepare a volume sufficient to add 100 µL to each well (see step 3.2). | |
| 2.2 Prepare Wash Buffer 1X solution by diluting Wash Buffer 21X in redistilled water. Mix thoroughly. Prepare a volume sufficient to add 4.2 mL to each well (see step 3.4). | |
| ### 3. Performing Insulin Assay | |
| 3.1 By using epMotion 5075 or hand-pipetting, pipette 25 µL each of Calibrators and Antigen Controls (in duplicate), samples, extract dilutions, and sample dilutions into appropriate wells of ELISA 96-well plate. | |
| 3.2 Add 100 µL of enzyme conjugate 1X solution to each well. | |
| 3.3 Incubate ELISA 96-well plate on a microplate shaker (900 rpm, orbital movement) for 1 hour at room temperature (18-25°C). | |
| 3.4 Using the plate washer, wash 6 times with 700 µL wash buffer 1X solution. After final wash, invert and tap the plate firmly against absorbent paper. Do not include soak step in washing procedure. | |
| 3.5 Add 200 µL Substrate TMB into each well. | |
| 3.6 Incubate on the bench for 15 minutes at room temperature (18-25°C). | |
| 3.7 Add 50 µL Stop Solution to each well. Mix thoroughly for 5 seconds by tapping gently on all sides of the plate without dispersing liquid in wells. | |
| 3.8 Using the microplate reader, determine the optical density and insulin concentration of each well within 30 minutes of adding stop solution. Set to 450 nm. | |
| ### 4. Data Analysis | |
| 4.1 Values for all standards must be within ±15% of their expected values and replicate values of each standard must have a Coefficient of Variation (CV) ≤20%. If standards vary beyond these limits, the assay must be repeated. | |
| 4.2 Values for quality control samples, corresponding to lower and upper assay detection ranges, must be within their known ranges. If QCs vary beyond these limits, the assay must be repeated. | |
| 4.3 Calculate the average of the insulin concentrations from the 4 extract dilutions to determine insulin content, expressed as ng/mL. | |
| 4.4 Normalize secreted insulin concentrations per islet volume (IEQs), expressed as ng/100 IEQs/min and islet insulin content, expressed as % content/min. | |
| 4.5 Use Prism software to create graphs and to calculate stimulation index (SI) and area under curve (AUC) values. | |
| - 4.5.1 **Stimulation index (SI):** A ratio calculated as maximum response to a given stimulus relative to baseline. | |
| - 4.5.2 **Area under curve (AUC):** Calculated by integrating islet secretory response to a given stimulus over time. | |
| ## Data Storage and Reporting | |
| ### 5. Data Storage and Reporting | |
| 5.1 To facilitate data management and ensure data security, the VUMC HIPP uses an institutional server-based platform for data storage and analysis. | |
| 5.2 Upon analysis completion, the VUMC HIPP uploads raw data, including hormone levels, data analysis, and graphical representations of each human islet perfusion into the IIDP HIPP database. Example of human islet perfusion results performed in HIPP is shown in **Figure 1**. | |
| 5.3 Functional data on islet insulin and glucagon secretion will be uploaded within 3 business days to the HIPP database built by IIDP programming team and immediately available to IIDP-affiliated investigators and islet isolation centers. | |
| ## Deviations and Resolutions | |
| ### 6. Deviations and Resolutions | |
| Document any deviations that occurred during this protocol that affect the final results and report with the analysis of the assay. | |
| endofoutput | |
| ``` |