INSULIN MOUSE SERUM KITS PROTOCOL Part # 62IN3PEF & 62IN3PEB Test size#: 200 tests (62IN3PEF), 5 x 200 tests (62IN3PEB) - assay volume: 20 µl Revision: 03-Jan.2018 Store at: 2-8 C (62IN3PEF); 2-8 C (62IN3PEB) For research use only. Not for use in diagnostic procedures. ASSAY PRINCIPLE Cisbio Bioassays Insulin Mouse Serum assay is only intended for quantitative measurement of Insulin in mouse plasma and serum using HTRF technology. The assay is not compatible with rat samples. Insulin is detected in a sandwich assay format using 2 different specific monoclonal antibodies, one labeled with Terbium Cryptate (donor) and the second with XL665 (acceptor). The detection principle is based on HTRF technology. When the dyes are in close proximity, the excitation of the donor with a light source (laser or flash lamp) triggers a Fluorescence Resonance Energy Transfer (FRET) towards the acceptor, which in turn fluoresces at a specific wavelength (665 nm). The two antibodies bind to the Insulin present in the sample, thereby generating FRET. Signal intensity is proportional to the number of antigenantibody complexes formed and therefore to the Insulin concentration (Fig. 1). mouse serum / plasma standard Insulin Insulin Anti-Insulin donor Antibody Anti-Insulin acceptor Antibody Insulin Figure 1: Principle of HTRF Insulin sandwich assay. PROTOCOL AT A GLANCE ADD READ ANALYSE Incubate overnight @ RT 5 µl Standard or Sample 10 µl Anti-Insulin acceptor Antibody 5 µl Anti-Insulin donor Antibody [Insulin] ng/ml or 15 µl of pre-mixed Anti-Insulin antibodies Make sure to use the set-up for Tb 3+ Cryptate. For more information about set-up and compatible HTRF readers, please visit our website at: http://www.cisbio.com/compatible-readers
2 MATERIALS PROVIDED: Kit components Insulin Standard Lyophilized Anti-Insulin-Tb 3+ Cryptate Antibody Anti-Insulin-XL665 Antibody Diluent #6 ** ready-to-use Detection buffer *** ready-to-use 200 tests * Cat # 62IN3PEF 1 vial Ref# 62INSCDA 1 vial Lyophilized 1 vial Lyophilized 1 vial 6 ml 1 vial 3 ml Detection Buffer #6 5 x 200 tests * Cat # 62IN3PEB 5 vials Ref# 62INSCDA 5 vials Lyophilized 5 vials Lyophilized 5 vials 6 ml 5 vials 3 ml Detection Buffer #6 * When used as advised, the two available kit sizes will provide sufficient reagents for 500 and 10,000 tests respectively in 20 µl final. Assay volumes can be adjusted proportionally to run the assay in 96 or 1536 well microplates. ** The Diluent is an alternative to medium to prepare working standard solutions. *** The Detection buffer is used to prepare working solutions of acceptor and donor reagents. PURCHASE SEPARATELY: HTRF 96-well low volume plate Ref# 66PL96001 white 384-well low volume plate * HTRF -Certified Reader **. Make sure the setup for Tb 3+ Cryptate is used. Use white plate only * For HTRF microplate recommendations, please visit http://www.cisbio.com/drug-discovery/htrf-microplate-recommendations ** For a list of HTRF-compatible readers and set-up recommendations, please visit http://www.cisbio.com/compatible-readers STORAGE AND STABILITY Store the kit at 2-8 C. Under proper storage conditions, reagents are stable until the expiry date indicated on the label. If lyophilized, reconstituted reagents, antibodies, and standard stock solutions may be frozen and thawed only once. To avoid freeze/thaw cycles, it is recommended to dispense remaining stock solutions into disposable plastic vials for storage at -60 C or below. Reagents Volume of Insulin Mouse Serum standard aliquots should not be under 20 μl. After first opening, store the diluent at -20 C. REAGENT PREPARATION BEFORE YOU BEGIN: It is very important to prepare reagents in the specified buffers. The use of an incorrect diluent may affect reagent stability and assay results. Allow the lyophilized reagents to warm up to room temperature for at least 30 mins before reconstitution Before use, allow Diluent and Detection buffer to warm up at room temperature and homogenize them with a vortex. It is recommended to filter buffers. Antibody solutions must be prepared in individual vials and can be mixed prior to dispensing. Insulin standards (for standard curve) must be prepared in diluent or in the same medium as the samples. TAKE CARE TO PREPARE STOCK AND WORKING SOLUTIONS ACCORDING TO THE DIRECTIONS FOR THE KIT SIZE YOU HAVE PURCHASED.
TO PREPARE STANDARD, DILUENT & ANTIBODY STOCK SOLUTIONS: 3 200 TESTS KIT - 62IN3PEF 5 X 200 TESTS KIT - 62IN3PEB Reconstitute the Anti-Insulin-Tb 3+ Cryptate antibody with 200 µl distilled water. Mix gently. This 5 X stock solution can be frozen and stored at -60 C or below. Anti-Insulin-Tb3+ Cryptate antibody Reconstitute the Anti-Insulin-Tb 3+ Cryptate antibody with 200 µl distilled water. Mix gently. This 5 X stock solution can be frozen and stored at -60 C or below. Reconstitute the Anti-Insulin-XL665 antibody with 400 µl distilled water. Mix gently. This 5 X stock solution can be frozen and stored at -60 C or below. Anti-Insulin-XL665 antibody Reconstitute the Anti-Insulin-XL665 antibody with 400 µl distilled water. Mix gently. This 5 X stock solution can be frozen and stored at -60 C or below. Reconstitute the Insulin Standard with distilled water in order to obtain a 500 ng/ml stock solution. See instructions on vial label for reconstitution volume. Mix gently after reconstitution. This stock solution can be frozen and stored at -60 C or below. The diluent is ready-to-use. Insulin Standard Diluent Reconstitute the Insulin Standard with distilled water in order to obtain a 500 ng/ml stock solution. See instructions on vial label for reconstitution volume. Mix gently after reconstitution. This stock solution can be frozen and stored at -60 C or below. The diluent is ready-to-use. TO PREPARE ANTIBODY WORKING SOLUTIONS: Each well requires 5 µl of Anti-Insulin-Tb 3+ Cryptate Antibody and 10 µl of Anti-Insulin-XL665 Antibody. Prepare the two antibody solutions in separate vials. 200 TESTS KIT - 62IN3PEF 5 X 200 TESTS KIT - 62IN3PEB Dilute 5-fold the 5 X stock solution (reconstituted reagent) of Anti Insulin Tb3+ -Cryptate-antibody with the Detection buffer #6: add 1 volume of Cryptateantibody stock solution in 4 volumes of Detection buffer #6 (e.g., 200 µl of reconstituted Cryptateantibody stock solution + 800 µl of Detection Buffer #6). Dilute 5-fold the 5 X stock solution (reconstituted reagent) of Anti-Insulin-XL665 antibody with the Detection buffer #6: add 1 volume of XL665-antibody stock solution in 4 volumes of Detection buffer #6 (e.g., 400 µl of reconstituted Anti Insulin XL665 + 1600 µl of Detection Buffer #6). It is possible to pre-mix the two ready-to-use antibody solutions just prior to dispensing the reagents by adding 2 volumes of XL665-antibody solution to 1 volume of Cryptate-antibody solution (e.g. 1 ml of XL665-antibody + 0.5 ml of Cryptateantibody). Anti-Insulin- Cryptate antibody 1 vol 4 vol 1 vol 4 vol Dilute 5-fold the 5 X stock solution (reconstituted reagent) of Anti-Insulin-Tb3+ Cryptate antibody with the Detection buffer #6: add 1 volume of Cryptate antibody stock solution in 4 volumes of Detection buffer #6 (e.g., 200 µl of reconstituted Cryptate antibody stock solution + 800 µl of Detection Buffer #6). Anti-Insulin-XL665 antibody 1 vol 4 vol 1 vol 4 vol Dilute 5-fold the 5 X stock solution (reconstituted reagent) of Anti Insulin XL665-antibody stock solution with the Detection buffer #6: add 1 volume of XL665-antibody stock solution in 4 volumes of Detection buffer #6 (e.g., 400 µl of reconstituted XL665-antibody stock solution + 1600 µl of Detection Buffer #6). Antibody mix It is possible to pre-mix the two ready-to-use antibody solutions just prior to dispensing the reagents by adding 2 volumes of XL665-antibody solution to 1 volume of Cryptate-antibody solution (e.g. 10 ml of XL665-antibody + 5 ml of Cryptateantibody).
4 TO PREPARE STANDARD WORKING SOLUTIONS: Each well requires 5 µl of standard. Dilute the standard stock solution serially with diluent #6 In order to check for a potential interference effect from your assay buffer when using the assay for the first time, we highly recommend the parallel preparation of a standard curve in your own supplemented cell culture medium and in diluent #6. In order to counteract any standard sticking, we recommend changing tips between each dilution. A recommended standard dilution procedure is listed and illustrated below: Dilute the standard stock solution 62.5-fold with diluent #6 to prepare high standard ( 7) for the top of the curve: e.g. take 8 µl of standard stock solution and add it to 492 µl of diluent #6. Mix gently. Use the high standard ( 7) to prepare the standard curve using 1/1.8 serial dilutions as follows: Dispense 80 µl of diluent #6 in each vial from 6 to 0. Add 100 µl of standard to 80 µl of diluent #6, mix gently and repeat the 1/1.8 serial dilution to make standard solutions: std6, std5, std4, std3, std2, std1. This will create 7 standards for the analyte. 0 (Negative control) is diluent #6 or appropriate culture medium alone. 8 µl 100 µl 100 µl 100 µl 100 µl 7 6.... 1 0 Insulin 492 µl diluent #6 or appropriate medium 80 µl diluent #6 or appropriate medium * Standard curve can be prepared in other appropriate medium
TO PREPARE SAMPLES: 5 Each well requires 5 µl of sample. Just after their collection, put the samples at 4 C and test them immediately. For later use, samples should be dispensed into disposable plastic vials and stored at -60 C or below. Avoid multiple freeze/thaw cycles. In order to prepare plasma samples, we recommend collecting blood in EDTA-tubes. Samples with a concentration above the highest standard ( 7) must be diluted in diluent #6 or in your appropriate sample medium. Avoid the use of samples with a high degree of hemolysis ASSAY PROTOCOL Standard ( 0-7) Samples Step 1 Dispense 5 µl of each Insulin standard ( 0-7) into each standard well Dispense 5 µl of each sample into each sample well Step 2 Add 10 µl of Anti-Insulin-XL665 working solution to all wells Step 3 Add 5 µl of Anti Insulin-Tb 3+ Cryptate working solution to all wells Step 4 Seal the plate and incubate overnight @ RT at room temperature Step 5 Remove the plate sealer and read on an HTRF compatible reader
6 A B C D E F G H 5 µl 0 (Negative control) 5 µl 1 5 µl 2 5 µl... 5 µl... 5 µl... 5 µl... 5 µl... 1 2 3 4 5 6 Repeat Well A1 Repeat Well B1 Repeat Well C1 Repeat Well D1 Repeat Well E1 Repeat Well F1 Repeat Well G1 Repeat Well H1 Repeat Well A1 Repeat Well B1 Repeat Well C1 Repeat Well D1 Repeat Well E1 Repeat Well F1 Repeat Well G1 Repeat Well H1 5 µl Sample 1 5 µl Anti Insulin-Tb3+ Cryptate 5 µl Sample 2 5 µl Sample 3 5 µl Sample... 5 µl Sample... 5 µl Sample... 5 µl Sample... Repeat Well A4 Repeat Well B4 Repeat Well C4 Repeat Well D4 Repeat Well E4 Repeat Well F4 Repeat Well G4 Repeat Well A4 Repeat Well B4 Repeat Well C4 Repeat Well D4 Repeat Well E4 Repeat Well F4 Repeat Well G4 5 µl Sample 1 2... 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 A B Repeat Well H4 Repeat Well H4 C 5 µl Anti D Insulin-Tb 3+ Cryptate E F G E H I J K L M N O P
7 DATA REDUCTION & INTERPRETATION 1. Calculate the ratio of the acceptor and donor emission signals for each individual well. Ratio = Signal 665 nm Signal 620 nm x 10 4 2. Calculate the % CVs. The mean and standard deviation can then be worked out from ratio replicates. CV (%)= Standard deviation Mean Ratio x 100 3. Calculate the % delta F which reflects the signal to background of the assay. The negative control (Standard 0) plays the role of an internal assay control. Delta F is used for the comparison of day to day runs of the same assay. delta F (%) = Ratio Standard or sample - Ratio Negative Control Ratio Negative Control x 100 For more information about data reduction, please visit http://www.cisbio.com/htrf-ratio-and-data-reduction RESULTS This data must not be substituted for the data obtained in the laboratory and should be considered only as an example (readouts on PHERAstar Plus with a flash lamp). Results may vary from one HTRF compatible reader to another. Standard curve fitting with the 4 Parameter Logistic (4PL) model:
This product contains material of biologic origin. Use for research purposes only. Do not use in humans or for diagnostic purposes. The purchaser assumes all risk and responsibility concerning reception, handling and storage. The use of the cell line will be done with appropriate safety and handling precautions to minimize health and environmental impact. Remaining disclaimer. Copyright 2016 Cisbio Bioassays. All rights reserved. HTRF and and the HTRF logo are trademarks or registered trademarks of Cisbio Bioassays. FOR MORE INFORMATION Europe and other countries +33(0)466-796-705 U.S. and Canada 1-888-963-4567 China +86-21-5018-9880 Japan +81-(0)43-306-8712 Visit www.cisbio.com to find a list of our regional distributors
INSULIN MOUSE SERUM KITS SHORT PROTOCOL: Part # 62IN3PEB (5 x 200 tests) & 62IN3PEF (200 tests) - assay volume: 20µL Allow the reagents to warm up to room temperature for at least 30 mins before use. 1 Reconstitute antibodies & Insulin standard with distilled water following table on next page. Mix gently. 2 Prepare conjugate working solutions 1X (from 5 X see table on next page) using detection buffer by diluting 5-fold the stock solutions. Prepare standard curve (7-1) by making 1:1.8 serial dilutions from 7 in diluent #6 or other appropriate medium. Mix between each dilution. 7 is obtained by 3diluting 62.5-fold the insulin stock solution. Dispense standard curve and sample in their 4dedicated well*: Add 5 µl of each standard ( 7 to 0) and 5 µl of sample. 5 Dispense the antibody solutions in all wells 5 µl of anti Insulin-cryptate-antibody and 10 µl of anti Insulin-XL665-antibody or 15 µl of the 2 pre-mixed antibodies. Seal the plate and incubate overnight at RT. 6 7 Remove the plate sealer and read on an HTRF compatible reader. See assay recommendations on next page
KIT SIZE ANTI-INSULIN DONOR ANTIBODY Reconstitution volumes Concentrations after reconstitution ANTI-INSULIN ACCEPTOR ANTIBODY Reconstitution volumes Concentrations after reconstitution 200 TESTS 200 µl 5 X 400 µl 5 X 5 X 200 TESTS 200 µl 5 X 400 µl 5 X 200 TESTS & 5 X 200 TESTS INSULIN STANDARD Reconstitute Insulin standard with distilled water following instruction indicated on vial label SHORT PROTOCOL - RECOMMENDATIONS Store the kit at 2-8 C until the expiration date indicated on the labels. It is very important to prepare reagents in the specified buffers. The use of an incorrect diluent may affect reagent stability and assay results. Allow the lyophilized reagents to warm up to room temperature for at least 30 mins before reconstitution. It is recommended to filter buffers (diluent & detection buffer). After first opening, store the diluent at -20 C. Once reconstituted, antibodies and standard stock solutions may be frozen and thawed only once: to avoid freeze/thaw cycles, it is recommended to dispense remaining stock solutions into disposable plastic vials for storage at -60 C or below. After first opening, store the diluent at -20 C. Volume of Insulin Mouse Serum aliquots should not be under 20 μl. Standard curve must be prepared in diluent or in the same medium as the samples. For standard curve preparation, in order to prevent any standard carry-over we recommend changing tips between each dilution. In order to prepare plasma samples, we recommend collecting blood in EDTA-tubes. Avoid the use of samples with a high degree of hemolysis. This product contains material of biologic origin. Use for research purposes only. Do not use in humans or for diagnostic purposes. The purchaser assumes all risk and responsibility concerning reception, handling and storage. Copyright 2017 Cisbio, France. All rights reserved. HTRF, Tag-lite, and EPIgeneous are trademarks or registered trademarks of Cisbio All other trademarks are the property of their respective owners. FOR MORE INFORMATION Europe and other countries +33(0)466-796-705 U.S. and Canada +1-888-963-4567 China +86-21-5018-9880 Japan +81(0)43-306-8712 Visit www.cisbio.com to find a list of our regional distributors