Microsoft word - glp-1 _7-36_- active elisa_v4.doc

L i S t a r F i s h S . r . l . V i a C a v o u r , 3 5 - 2 0 0 6 3 C e r n u s c o S / N ( M I ) , I t a l y T e l . + 3 9 - 0 2 - 9 2 1 5 0 7 9 4 - F a x . + 3 9 - 0 2 - 9 2 1 5 7 2 8 5 i n f o @ l i s t a r f i s h . i t - w w w . l i s t a r f i s h . i t EDI™ Active GLP-1 (7-36) Specific ELISA Kit Enzyme Linked ImmunoSorbent Assay (ELISA) for the measurement of active Glucagon-like peptide-1 (7-36) Level in Test Samples Catalog Number: EPI-KT 871 Store at 2 – 8ºC Upon Receipt One vial containing 0.6 mL of biotinylated GLP-1 (7-36) specific This high sensitive ELISA (enzyme-linked immunosorbent assay) kit antibody. It should be used only after mixed with GLP-1 Tracer is produced for the exclusively quantitative determination of Antibody and the tracer antibody diluent according to the assay glucagon-like peptide-1 (7-36) [GLP-1 (7-36)] level in serum and procedures. This reagent should be stored at 2 – 8°C and is EDTA-plasma sample among human, rat, mouse, goat, etc. stable until the expiration date on the kit box. This ELISA is designed, developed and produced for the quantitative One bottle contains 20 mL of 30 fold concentrate. Before use measurement of bioactive GLP-1 (7-36) in plasma sample. The the contents must be diluted with 580 mL of distilled water and assay utilizes the two-site “sandwich” technique with two selected mixed well. Upon dilution this yields a working wash solution containing a surfactant in phosphate buffered saline with a non- azide and non-mercury based preservative. The diluted wash Assay standards, controls and test samples are directly added to buffer should be stored at room temperature and is stable until wells of a microplate that is coated with streptavidin. Subsequently, a mixture of biotinylated GLP-1 (7-36) specific antibody and a horseradish peroxidate (HRP) conjugated GLP-1 (7-36) specific antibody is added to each well. After the first incubation period, a One bottle contains 22 mL of tetramethylbenzidine (TMB) with “sandwich” immunocomplex of “Streptavidin – Biotin-Antibody – stabilized hydrogen peroxide. This reagent should be stored at GLP-1(7-36) – HRP conjugated antibody” is formed and attached to 2 – 8°C and is stable until the expiration date on the kit box. the wall of the plate. The unbound HRP conjugated antibody is removed in a subsequent washing step. For the detection of this immunocomplex, each well is then incubated with a substrate One bottle contains 12 mL of sulfuric acid. This reagent should solution in a timed reaction and then measured in a be stored at 2 – 8°C or room temperature and is stable until the spectrophotometric microplate reader. The enzymatic activity of the immunocomplex bound to GLP-1 (7-36) on the wall of the microtiter well is directly proportional to the amount of GLP-1 (7-36) in the GLP-1 Standards (Cat. No. 30261 – 30265) Five vials containing different levels of lyophilized GLP-1 (7-36) in a liquid protein matrix with a non-azide, non-mercury based preservative. Refer to vial for exact concentration for each standard. These reagents should be stored at 2 – 8°C and are This test kit must be stored at 2 – 8°C upon receipt. For the expiration date of the kit refer to the label on the kit box. All stable until the expiration date on the kit box. components are stable until this expiration date. GLP-1 Controls (Cat. No. 30266 – 30267) Prior to use allow all reagents to come to room temperature. Two vials containing different levels of lyophilized GLP-1 (7-36) Regents from different kit lot numbers should not be combined or in a liquid protein matrix with a non-azide, non-mercury based preservative. Refer to vials for exact concentration range for each control. Both controls should be stored at 2 – 8°C and Streptavidin Coated Microplate (Cat. No. 10040) are stable until the expiration date on the kit box. One well-breakable microplate with 12 x eight strips (96 wells total) coated with streptavidin. The plate is framed and sealed in Tracer Antibody Diluent (Cat. No. 30017) a foil zipper bag with a desiccant. This reagent should be stored One vial containing 12 mL ready to use buffer. It should be at 2 – 8°C and is stable until the expiration date on the kit box. used only for tracer antibody dilution according to the assay procedures. This reagent should be stored at 2 – 8°C and is stable until the expiration date on the kit box. One vial containing 0.6 mL HRP labeled Anti-GLP-1 specific antibody in a stabilized protein matrix. This reagent must be mixed with GLP-1 (7-36) Capture Antibody and the tracer antibody diluent before use (for details see Assay Procedure). The reagents must be used in a professional laboratory environment This reagent should be stored at 2 – 8°C and is stable until the and are for research use only. Source material (e.g. highly purified bovine serum albumin) of bovine serum was derived in the contiguous 48 United States. It was obtained only from donor healthy GLP-1 (7-36) Capture Antibody (Cat. No. 30230) animals maintained under veterinary supervision and found free of contagious diseases. Wear gloves while performing this assay and EDI Kit insert: Active GLP-1 (7-36) Specific ELISA / version 4 /2008-12 handle these reagents as if they are potential infectious. Avoid and controls to sit undisturbed for 10 minutes, and then contact with reagents containing TMB, hydrogen peroxide, or sulfuric mix well by gentle vortexing. These reconstituted acid. TMB may cause irritation to skin and mucous membranes and standards and controls must be stored at - 20°C or below. cause an allergic skin reaction. TMB is a suspected carcinogen. Sulfuric acid may cause sever irritation on contact with skin. Do not get in eyes, on skin, or on clothing. Do not ingest or inhale fumes. On contact, flush with copious amounts of water for at least 15 minutes. Although EDTA-plasma or serum samples can be directly measured for the Active GLP-1 (7-36) concentration, some studies using this assay by pharmaceutical companies indicated that sample pre- treatment with a column extraction is necessary to obtain an Precision single channel pipettes capable of delivering 25 accurate and clinical meaningful test results. Repeating dispenser suitable for delivering 100 µL. Epitope Diagnostics provides a validated and user friendly column Disposable pipette tips suitable for above volume extraction procedures and reagents packed as a GLP-1 sample extraction kit (Catalog No. KT-910). This kit provides all the reagents Disposable 12 x 75 mm or 13 x 100 glass/plastic tubes. ready to use. This extraction procedure does not require any special Disposable plastic 100 mL and 1000 mL bottle with caps. equipment, such as vacuum centrifuge, etc. Plastic microtiter well cover or polyethylene film. (1) Place a sufficient number of streptavidin coated microwell 10. ELISA multichannel wash bottle or automatic (semi- strips/wells in a holder to run GLP-1 (7-36) standards, controls and unknown samples in duplicate. 11. Spectrophotometric microplate reader capable of reading Although both serum and EDTA-plasma samples can be used for measuring Active GLP-1 (7-36) using this assay, It is recommend to use EDTA-plasma, because the plasma sample showed a better stability than serum sample. (1) Only 200 µL of EDTA-plasma is required for bioactive GLP-1 (7-36) measurement in duplicate. More sample volume is needed if a sample extraction procedure is (3) Prepare GLP-1 (7-36) Antibody Mixture: mixing GLP-1 (2) No special preparation of individual is necessary prior to Tracer Antibody and Capture Antibody by 1:21 fold dilution specimen collection. However, fasting sample and non- of the Tracer Antibody (30229) and by 1:21 fold dilution of fasting/glucose induced sample may present great the biotinylated Capture Antibody (30230) with the Tracer significance for bioactive GLP-1 (7-36) level. antibody Diluent. For each strip, it is required to mix 1 mL (3) Whole blood should be collected into a lavender top of the Tracer Antibody Diluent (30017) with 50 µL the Vacutainer EDTA-plasma tube. Invert tube to mix well Capture Antibody and 50 µL of the Tracer Antibody in a and place the tube on ice. Centrifuge the tube within an hour at 1000 g for 10 minutes in a refrigerated centrifuge. (4) Add 100 µL of standards, controls and test samples into (4) EDTA-plasma samples should be stored at 2 – 8°C if they will be tested within 3 hours of collection. For longer (5) Add 100 µL of GLP-1 (7-36) Antibody Mixture to each well storage, it is recommended to store the plasma sample at - (6) Cover the plate with one plate sealer and incubate plate at 70°C. Avoid more than three times repeated freezing and thawing cycles. Aliquot samples before freezing if (7) Remove plate sealer. Aspirate the contents of each well. Wash each well 5 times by dispensing 350 µL of working (5) It is very important to add appropriate amount of DPP-4 wash solution into each well and then completely inhibitor to the collected specimen right after the aspirating the contents. Alternatively, an automated separation of plasma from the blood cells. Refer to DPP-4 (8) Add 200 µL of ELISA HRP Substrate into each of the (6) BD™ P700 Blood Collection and Preservation System (contains a DPP-4 protease inhibitor cocktail) is (9) Cover the plate with one plate sealer and also with recommended. However, each institute should verify the aluminum foil to avoid exposure to light. collection system for their specific research and study. (10) Incubate plate at room temperature, static for 20 min. (11) Remove the aluminum foil and plate sealer. Add 50 µL of ELISA Stop Solution into each of the wells. Mix gently. (12) Read the absorbance at 450nm/620 nm within 10 minutes (1) Prior to use allow all reagents to come to room temperature. Regents from different kit lot numbers should NOTE: to reduce the background, one can set the instrument to dual wavelength measurement at 450 nm with background wavelength correction set at 595 nm or (2) ELISA Wash Concentrate must be diluted to working solution prior use. Please see REAGENTS section for (3) Reconstitute all standards and controls by adding 1.0 mL of deminerialized water to each vial. Allow the standards EDI Kit insert: Active GLP-1 (7-36) Specific ELISA / version 4 /2008-12 It is recommended that all standards, controls and unknown samples be assayed in duplicate. The average absorbance reading of each duplicate should be used for data reduction and the calculation of results. For samples with higher than level 5 standard, it is recommended to measure diluted the specimen with an appropriate GLP-1 free human serum matrix for a more accurate report. Keep light sensitive reagents in the original amber bottles. Store any unused streptavidin coated strips in the foil zipper bag with desiccant to protect from moisture. Careful technique and use of properly calibrated pipetting devices are necessary to ensure reproducibility of the test. Incubation times or temperatures other than those stated Avoid air bubbles in the microwell as this could result in lower binding efficiency and higher CV% of duplicate All reagents should be mixed gently and thoroughly prior Calculate the average absorbance for each pair of Subtract the average absorbance of the STD 1 (0 ng/mL) from the average absorbance of all other readings to The standard curve is generated by the corrected absorbances of all standard levels on the ordinate against the standard concentration on the abscissa using point-to- point or log-log paper. Appropriate computer assisted data Each laboratory should establish it own normal range by using reduction programs may also be used for the calculation of samples collected from normal healthy people. Please be note that results. We recommend using Point-to-Point or the normal range may be variable by using fasting samples vs. non- The GLP-1 (7-36) concentrations for the controls and test samples GLP-1(7-36) pg/ml = GLP-1 (7-36) pmol/l x 3.298 are read directly from the standard curve using their respective corrected absorbance. If log-log graphic paper or computer assisted data reduction program utilizing logarithmic transformation are used, sample having corrected absorbance between the 2nd standard and Since there is no Gold Standard concentration or the next highest standard should be calculated by the formula: international standard available for GLP-1 (7-36) measurement, the values of assay standards were established using a highly purified GLP-1 (7-36) peptide and validated by Epitope Diagnostics. Results obtained with different assay methods or kits cannot be used For unknown sample value read directly from the assay that is greater than assay standard level-5, it is recommend measuring a diluted sample for more accurate A typical absorbance data and the resulting standard curve from this GLP-1 ELISA are represented. This curve should not be used in Bacterial or fungal contamination of serum specimens or lieu of standard curve run with each assay. reagents, or cross contamination between reagents may Water deionized with polyester resins may inactivate the To assure the validity of the results each assay should include adequate controls with known GLP-1 (7-36) levels. The sensitivity of this High Sensitive GLP-1 (7-36) ELISA as determined by the 95% confidence limit on 12 duplicate determination of zero standard is about 0.3 pmol/L. By linear dilution of assay standard level 2 (1.82 pmol/L) to 0.91 pmol/L and 0.46 pmol/L, the assay can clearly detect both of them. Therefore, a realistic detection limit of 0.5 pmol/L is easily achieved by this assay. EDI Kit insert: Active GLP-1 (7-36) Specific ELISA / version 4 /2008-12 3. Nauck MA, Weber I, Bach I, Richter S, Orskov C, Holst JJ, This Bioactive GLP-1 (7-36) assay is specific measure GLP-1 (7-36). Schmiegel W. Normalization of fasting glycaemia by intravenous It is expected that this assay does not detect following peptides. GLP-1 ([7-36 amide] or [7-37]) in type 2 diabetic patients. 4. Byrne MM, Göke B. Human studies with glucagon-like-peptide-1: potential of the gut hormone for clinical use. 5. Mannucci E, Tesi F, Bardini G, Ognibene A, Petracca MG, Ciani S, Pezzatini A, Brogi M, Dicembrini I, Cremasco F, Messeri G, Rotella CM. Effects of metformin on glucagon-like peptide-1 levels in Two samples were diluted with GLP-1 (7-36) zero human serum obese patients with and without Type 2 diabetes. Diabetes Nutr matrix. These diluted samples are measured in this assay and the - Add 100 µl/well of standards, control and patient sample - Incubate 20 - 24 hour at 2-8°C, static Spike Recovery Patient samples were spiked each other in the sample volume (200 µl + 200 µl) and measured with this assay. The spike recovery are calculated. WARRANTY This product is warranted to perform as described in its labeling and literature when used in accordance with all instructions. Epitope Diagnostics, Inc. DISCLAIMS ANY IMPLIED WARRANTY OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE, and in no event shall Epitope Diagnostics, Inc. be liable for consequential damages. Replacement of the product or refund of the purchase price is the exclusive remedy for the purchaser. This warranty gives you specific legal rights and you may have other rights, which vary from state to state. REFERENCES 1.Levy JC. Therapeutic intervention in the GLP-1 pathway in Type 2 diabetes. Diabet Med. 2006 Mar;23 Suppl 1:14-9. 2. Mannucci E, Ognibene A, Cremasco F, Bardini G, Mencucci A, Pierazzuoli E, Ciani S, Fanelli A, Messeri G, Rotella CM. Glucagon-like peptide (GLP)-1 and leptin concentrations in obese patients with Type 2 diabetes mellitus. Diabet Med. 2000 Oct;17(10):713-9. EDI Kit insert: Active GLP-1 (7-36) Specific ELISA / version 4 /2008-12

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