Headquarters & Europe Office Cisbio Bioassays Phone: +33 (0)4 66 79 67 05 Fax: +33 (0)4 66 79 19 20 bioassays@cisbio.com cisbio_dd_pi_62in3pef-2µl USA Office Cisbio US, Inc. Phone: +1 888 963 4567 Fax: +1 781 687 1500 htrfinfo@cisbio.us China Office Cisbio China Phone: +86-21-5018-9880 Fax: +86-21-5020-3055 htrfinfoasia@cisbio.cn Japan Office Cisbio Japan Phone: +81-(0)43-306-8712 Fax: +81-(0)43-306-8713 htrf@cisbio.jp www.cisbio.com Product information: Document reference : 62IN3PEF-2µL rev 01 (Jan 2016) Mouse serum Insulin 200 tests For in vitro research use only Storage temperature: 4-8 C Packaging details: white 384-well low volume plate (20 µl) 200 tests 1. Assay description This assay is intended for the quantitative measurement of Insulin in mouse plasma and serum using HTRF technology. The assay is not compatible with rat samples. As shown on the right, 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). 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 antigen present in the sample, thereby generating FRET. Signal intensity is proportional to the number of antigen complexes formed and therefore to the Insulin concentration. 2. Protocol at a glance Anti-Insulin-Tb3+Cryptate Insulin Anti-Insulin-XL665 FRET Incubate overnight at room temperature Read 665 nm & 620 nm signals on an HTRF compatible reader 2 µl of Insulin standard or sample + 18 µl of pre-mixed HTRF antibodies white 384-well low volume plate Caution! A white plate must be used for the assay. Plates are not provided in the kit. For more information on compatible microplates, please visit our website at http://www.cisbio.com/drug-discovery/htrfmicroplate-recommendations
3. HTRF reagents Insulin standard Anti-Insulin- XL665 Anti-Insulin- Tb3+Cryptate Diluent # 6 Reconstitution buffer # 6 green cap blue cap red cap transparent cap transparent cap 1 vial lyophilized 1 vial lyophilized 1 vial lyophilized 1 vial of 6 ml 1 vial of 7 ml Supplied reagent See label indications for reconstitution volume Reconstitute with 400 µl H2O Reconstitute with 200 µl H2O ready-to-use ready-to-use Storage ref# (when available separately) All supplied reagents are stable at 2-8 C until the expiry date indicated on the label 62INSCDA n/a n/a n/a n/a 4. Reagent preparation HTRF reagent concentrations have been set for optimal assay performance. Note that any dilution or improper use of the Inulin standard and antibodies will result in impaired assay quality. All reagents should be stored at 2-8 C until reconstituted. Under proper storage conditions, they are stable until the expiry date indicated on the label. Once reconstituted, 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 -20 C or below. Volume of insulin standard aliquots should not be under 20 µl. Warning: before use, it is recommended to filter buffers. Before use, allow all reagents to warm up to room temperature. Prepare the working solutions from stock solutions by following the instructions below. 4.1. Preparation of diluent # 6 Diluent #6 is ready-to-use. After first opening, store the diluent at -20 C. 4.2. Preparation of Insulin standard solutions When preparing insulin standard solutions, in order to prevent any standard carry-over we recommend changing tips between each dilution.. Reconstitute the lyophilized material with distilled water in order to obtain a 500 ng/ml Insulin stock solution that may be frozen at - 20 C or below for longer storage. See label indications for the reconstitution volume, mix gently. Determine how many standard replicates will be tested. Each well requires 2 µl of standard. Dilute the standard stock solution serially in diluent # 6
A recommended procedure for standard dilution is listed and illustrated below. Dilute the standard stock solution 62.5-fold with Diluent # 6 to prepare the standard 7 (8 ng/ml). e.g. take 8 µl of standard stock solution and add it to 492 µl of Diluent # 6. Mix gently. Starting with standard 7 (8 ng/ml), prepare 1/1.8 serial dilutions as follows: Dispense 80 µl of Diluent # 6 into each vial from standard 6 to 0. Add 100 µl of standard 7 (8 ng/ml) to 80 µl of Diluent # 6, mix gently and repeat the 1/1.8 serial dilutions to make standard solutions: 4.44, 2.47, 1.37, 0.76, 0.42 and 0.24 ng/ml. 0 (Negative control) is Diluent # 6 alone. Dispense Diluent # 6 in each tube 492 µl 80 µl Make standard serial dilutions 8 µl 100 µl 100 µl Diluent # 6 Standard stock solution max 0 max 4.3. Preparation of samples If you suspect your samples contain insulin levels above the highest standard (8 ng/ml), use diluent # 6 to dilute them. Just after their collection, put the samples at 4 C and test them immediately. For later use, they 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. Avoid the use of samples with a high degree of hemolysis.. 4.4. Preparation of working solutions Determine the amount of XL665- needed for the experiment. Each well requires 12 µl of XL665. Reconstitute with 400 µl distilled water. Mix gently. Once reconstituted, stock solution may be frozen and thawed only once. To avoid freeze/thaw cycles, it is recommended to dispense remaining stock solution into disposable plastic vials for storage at -20 C or below. Then dilute the stock solution 6-fold with reconstitution buffer # 6. Anti-Insulin-XL665 Step 1: Reconstitute the XL665 with distilled water Step 2: Dilute 6-fold with reconstitution buffer # 6 400 µl 1 volume 5 volumes XL665- Distilled water XL665 reconstituted Reconstitution Buffer # 6 Reconstitute the vial with 400 µl of distilled water Dilute the XL665 stock solution 6-fold with the reconstitution buffer: e.g. take 0.2 ml of XL665 stock solution and add it to 1 ml of reconstitution buffer. Determine the amount of Cryptate- needed for the experiment. Each well requires 6 µl of Cryptate. Reconstitute with 200 µl distilled water. Mix gently. Once reconstituted, stock solution may be frozen and thawed only once. To avoid freeze/thaw cycles, it is recommended to dispense remaining stock solution into disposable plastic vials for storage at -20 C or below. Then dilute the stock solution 6-fold with reconstitution buffer # 6. Anti-Insulin-Cryptate Step 1: Reconstitute the Cryptate with distilled water Step 2: Dilute 6-fold with reconstitution buffer # 6 200 µl 1 volume 5 volumes Cryptate- Distilled water Cryptate reconstituted Reconstitution Buffer # 6 Reconstitute the vial with 200 µl of distilled water Dilute the Cryptate stock solution 6-fold with the reconstitution buffer: e.g. take 0.1 ml of Cryptate stock solution and add it to 0.5 ml of reconstitution buffer. It is possible to pre-mix the two ready-to-use solutions just before dispensing the reagents: 2 volumes of XL665- for 1 volume of cryptate solution, e.g. pre-mix 1.2 ml of XL665- solution with 0.6 ml of cryptate- solution.
5. Assay protocol Dispense the reagents into a white 384-well low volume plate in the following order: 2 µl of standard or sample + 12 µl of anti-insulin-xl665 + 6 µl of anti-insulin-cryptate or 18 µl of pre-mixed detection antibodies Cover the plate with a plate sealer. Incubate overnight* at room temperature. Remove the plate sealer and read the fluorescence emission at two different wavelengths (665nm and 620nm) on an HTRF compatible reader. *Following incubation, the HTRF signal is stable up to 48H Caution! Make sure the setup for Tb3+Cryptate is used. For more information about HTRF compatible readers, please visit our website at: www.cisbio.com/drug-discovery/htrf-compatible-readers/ Buffer control Used to check background fluorescence Negative control Used to calculate Delta F% Insulin standard / Sample Insulin standard or sample - - 2 µl Diluent # 6 2 µl 2 µl - Anti-Insulin-XL665-12 µl 12 µl Anti-Insulin-Tb3+Cryptate - 6 µl 6 µl Reconstitution buffer # 6 18 µl - - Final volume 20 µl 20 µl 20 µl 6. Data reduction This data must not be substituted for the data obtained in the laboratory and should be considered only as an example (readouts on PHERAstar FS with a flash lamp). Results may vary from one HTRF compatible reader to another. The assay standard curve is drawn by plotting Delta F% versus the analyte concentration. Standard curve fitting with the 4 Parameter Logistic (4PL) model: Ratio (1) CV% (2) Signal 665nm --------------------- x 10 4 Signal 620nm Standard deviation --------------------- x 100 Mean ratio Ratio must be calculated for each individual well. The mean and standard deviation can then be worked out from ratio replicates. Delta F% (3) Ratio standard or sample Ratio Negative control --------------------- x 100 Ratio Negative control Reflects the signal to background of the assay. The negative control plays the role of an internal assay control. For more information about data reduction, please visit our website at: cisbio.com/drug-discovery/htrf-ratio-and-data-reduction To obtain additional information or support, please contact your technical support team (htrfservices@cisbio.com).
7. Analytical characteristics 7.1. Assay range and sensitivity Assay range Limit of Detection (LoD)* (= dose of mean zero + 2SD) Incubation time 0.24-8 ng/ml 0.123 ng/ml overnight at RT *The LoD was calculated from data obtained with the PHERAstar Plus reader (flash lamp excitation) after overnight incubation. This may vary from one HTRF compatible reader to another. The HTRF Insulin assay was calibrated against the NIBSC international standard 66/304. The following equivalence was found: 1 ng Insulin HTRF 1 ng Insulin NIBSC 66/304 (i.e.: 2.3x10-05 IU). Copyright 2016 Cisbio Bioassays, France HTRF, Tag-lite, EPIgeneous and the HTRF logo are trademarks belonging to Cisbio Bioassays.