Proinsulin (Total) Chemiluminescence ELISA
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1 Proinsulin (Total) Chemiluminescence ELISA For the quantitative determination of proinsulin in human serum, EDTA plasma, heparin plasma, and tissue culture supernatants For In Vitro Diagnostic use within the United States of America. This product is for Research Use Only outside of the United States of America. Catalog Number: Size: 80-PINHUT-CH01 96 Wells Version: March 16, G Keewaydin Drive, Salem, NH P: (800) F: (603) ts@alpco.com
2 INTENDED USE The Proinsulin (Total) Chemiluminescence ELISA is designed for the quantitative determination of proinsulin in human serum, plasma (EDTA and heparin), and tissue culture supernatants. PRINCIPLE OF THE ASSAY The Proinsulin (Total) Chemiluminescence ELISA is a sequential sandwich assay, which uses two mouse monoclonal antibodies: one as capture that is adsorbed to a 96-well black-well microplate and the other as a primary detector conjugated to biotin. Standards, sample, and controls are added to the plate and the capture-antigen is incubated at room temperature on a plate shaker set between rpm. Post standard/sample incubation, the plate is washed six times and the biotinylated detection antibody is added to each well. This capture-antigen-detector sandwich is incubated at room temperature on a plate shaker set between rpm. Post detection antibody incubation, the plate is washed six times, HRP (horseradish peroxidase)- conjugated streptavidin (SA) is added to each well, and incubated at room temperature on a plate shaker set between rpm. Post SA-HRP incubation, the plate is washed 6 times and the chemiluminescent substrate is added to each well. The microplate is analyzed on an appropriate plate reader 1 minute post substrate. The resulting relative light units (RLUs) are directly proportional to the amount of proinsulin present in the sample. MATERIALS SUPPLIED Component Quantity Preparation Proinsulin Microplate (96 wells) 12 x 8 strips Ready to use Zero Standard 5 ml Ready to use Standards (A-H) (5, 10, 25, 50, 100, 250, 1,000, 3,000 pg/ml) 8 x 1 ml Ready to use Controls 1, 2, and 3* 3 vials Lyophilized* Protease Inhibitor Stock 100 µl 100X Assay Buffer 18 ml Ready to use Detector Antibody 80 µl 201X SA-HRP 150 µl 101X SA-HRP Buffer 12 ml Ready to use Substrate A 6 ml Ready to use Substrate B 6 ml Ready to use Wash Buffer Concentrate 200 ml 21X Plate Sealers 3 Ready to use *Please refer to the Certificate of Analysis enclosed with each kit for more information. Proinsulin (Total) Chemiluminescence ELISA Page 2 of March 16, 2017
3 MATERIALS REQUIRED Precision pipettes for dispensing up to 100 µl (with disposable tips) Repeating or multi-channel pipette for dispensing up to 100 µl Volumetric containers and pipettes for reagent preparation Distilled/Deionized water for reagent preparation Microplate washer or wash bottle Microplate shaker capable of rpm Microplate reader capable of reading luminescence Centrifuge (1,000 xg to 2,000 xg), vortex, and ice for sample preparation PRECAUTIONS 1. The human blood products incorporated into this kit have been tested for the presence of HIV (Human Immunodeficiency Virus), HBV (Hepatitis B Virus), and HCV (Hepatitis C Virus). Test methods for these viruses do not guarantee the absence of a virus; therefore, all reagents should be treated as potentially infectious. Handling and disposal should be in accordance with all appropriate national and local regulations for the handling of potentially biohazardous materials. 2. All materials derived from animal sources are bovine spongiform encephalopathy (BSE) negative. However, all materials should be treated as potentially infectious. 3. Avoid direct contact with skin. 4. This product is not for internal use. 5. Avoid eating, drinking, or smoking when using this product. 6. Do not pipette any reagents by mouth. 7. Reagents from this kit are lot-specific and must not be substituted. 8. Do not use reagents beyond the expiration date. 9. Variations to the test procedure are not recommended and may influence the test results. 10. An appendix has been included with examples of instrument settings for analyzing a chemiluminescent assay. Each lab should optimize their instrument settings according to the manufacturer s instructions. Please contact the technical services department of the manufacturer of the microplate reader for optimal instrument settings. STORAGE CONDITIONS The kit should be stored at 2-8 C. The kit is stable until the expiration date on the box label. If desired, the reconstituted controls can be stored at -20 C in aliquots for up to 6 months. The controls should not be repeatedly frozen and thawed. SAMPLE HANDLING Serum, plasma (EDTA and heparin), and tissue culture supernatant samples are appropriate for use in this assay. For serum samples, allow collected blood to clot on ice for 20 minutes and then subject the samples to 1,000 to 2,000 x g centrifugation for 20 minutes at 2-8 o C, because proinsulin is not stable in blood at room temperature. If testing will occur within 2 hours after collection, keep the serum samples on ice. If the assay is to be performed at a later time, store the serum samples at -20 C. Proinsulin (Total) Chemiluminescence ELISA Page 3 of March 16, 2017
4 If EDTA or heparin plasma samples are to be assayed within 2 hours after collection, keep the samples on ice. If the assay is to be performed at a later time, store the samples at -20 C. Upon thawing samples, keep the samples on ice until performing the assay. Avoid repeated freeze/thaw cycles. It is recommended that all samples be centrifuged at 13,000 RPM at 2-8 C for 10 minutes before loading them on the plate. For samples with a concentration of intact proinsulin above the assay measurable range (AMR), dilute the sample in Zero Standard and repeat the analysis. REAGENT PREPARATION All reagents must be equilibrated to room temperature prior to preparation and subsequent use in the assay. Controls (Levels 1, 2, and 3) are provided in a lyophilized form. Refer to the Certificate of Analysis enclosed with each kit for the appropriate volume of deionized water for reconstitution. Close the vial with the rubber stopper and cap, gently swirl the vial, and allow it to stand for 30 minutes prior to use. The contents of the vial should be in solution with no visible particulates. If desired, the controls can be stored at -20 C in aliquots for up to 6 months. The controls should not be repeatedly frozen and thawed. Protease Inhibitor Concentrate (100X) is to be diluted with Assay Buffer for the Working Strength Protease Inhibitor. Number of plates Amount of Concentrate Amount of Assay Buffer 0.5 (6 strips) 30 µl ml 1 (12 strips) 60 µl ml µl ml µl ml µl ml Detector Antibody (201X) is to be diluted with 200 parts Assay Buffer for the Working Strength Detector Antibody. Number of plates Amount of Concentrate Amount of Assay Buffer 0.5 (6 strips) 30 µl 6 ml 1 (12 strips) 60 µl 12 ml µl 20 ml µl 50 ml µl 100 ml Proinsulin (Total) Chemiluminescence ELISA Page 4 of March 16, 2017
5 SA-HRP (101X) is to be diluted with SA-HRP Buffer for the Working Strength SA-HRP. Number of plates Amount of Concentrate Amount of SA-HRP Buffer 0.5 (6 strips) 60 µl 6 ml 1 (12 strips) 120 µl 12 ml µl 24 ml µl 60 ml µl 120 ml Wash Buffer Concentrate (21X) is to be diluted with 20 parts distilled water for the Working Strength Wash Buffer. NOTE: All labs should account for plate washer void volume to prime the machine. The values below do not account for priming automated plate washers. Amount of Concentrate Amount of dh2o Number of plates (manual wash) (manual wash) 0.5 (6 strips) 20 ml 400 ml 1 (12 strips) 35 ml 700 ml 2 70 ml 1400 ml ml 3200 ml ml 6400 ml Chemiluminescent Substrates A & B are provided individually and should be combined in equal parts to create the Working Strength Chemiluminescent Substrate prior to use. The working chemiluminescent substrate is stable for 1 hour. Number of plates Amount of Substrate A Amount of Substrate B 0.5 (6 strips) 3 ml 3 ml 1 (12 strips) 6 ml 6 ml 2 12 ml 12 ml 5 25 ml 25 ml ml 50 ml QUALITY CONTROL It is recommended that the controls provided with the Proinsulin (Total) Chemiluminescence ELISA be included in every assay. The concentration ranges of the controls are provided on the certificate of analysis provided with each kit; however, it is recommended that each laboratory establishes its own acceptable ranges. Proinsulin (Total) Chemiluminescence ELISA Page 5 of March 16, 2017
6 ASSAY PROCEDURE All reagents and microplate strips (while sealed in foil pouch) should be equilibrated to room temperature prior to use. Gently mix all reagents before use. A standard curve must be performed with each assay run and with each microplate if more than one is used at a time. All standards, controls, and samples should be run in duplicate. 1. The microplate should be equilibrated to room temperature prior to opening the foil pouch. It is recommended to designate enough microplate strips for duplicate determinations of the standards, controls, and samples. Any remaining microplate strips should be stored at 2-8 C in the tightly sealed foil pouch containing the desiccant. 2. Pipette 50 µl of each standard, control, and sample into their respective wells. See Reagent Preparation for control reconstitution instructions. A suggested plate layout is included on page Pipette 50 µl of Working Strength Protease Inhibitor (see Reagent Preparation) into each well. 4. Cover microplate with a plate sealer and incubate for 1 hour at room temperature, shaking at rpm on a microplate shaker. 5. Decant the contents of the wells and wash the microplate 6 times with 350 µl of Working Strength Wash Buffer per well (see Reagent Preparation) using an automated microplate washer. a. Manual Wash: Alternatively, fill the wells with Working Strength Wash Buffer using a wash bottle equipped with a wash nozzle or manual washer. (It is not recommended to use a multichannel pipette. Wash buffer must be dispensed with adequate and equal force in order to properly wash the wells.) Soak the wells for 1 minute. Invert the microplate to discard the liquid and firmly tap the inverted microplate on absorbent paper towels. Repeat the wash and soak procedure 2 more times, for a total of 3 washes. After the final wash, (automated or manual), remove any residual wash buffer and bubbles from the wells by inverting and firmly tapping the microplate on absorbent paper towels. 6. Pipette 100 µl of Working Strength Detector Antibody (see Reagent Preparation) into each well. 7. Cover microplate with a plate sealer and incubate for 1 hour at room temperature, shaking at rpm on a microplate shaker. 8. Decant the contents of the wells and wash the microplate 6 times as in Step 5 above. 9. Pipette 100 µl of Working Strength SA-HRP (see Reagent Preparation) into each well. 10. Cover microplate with a plate sealer and incubate for 30 minutes at room temperature, shaking at rpm on a microplate shaker. Proinsulin (Total) Chemiluminescence ELISA Page 6 of March 16, 2017
7 11. Decant the contents of the wells and wash the microplate 6 times as in Step 5 above. 12. Pipette 100 µl of Working Strength Chemiluminescent Substrate (see Reagent Preparation) into each well. 13. Place the microplate in a microplate reader capable of analyzing the luminosity of the wells. The microplate should be analyzed 1 minute after the addition of the chemiluminescent substrate, and no later than 10 minutes after substrate addition. Analyze the plate using a 1 second integration time. CALCULATION OF RESULTS Construct a standard curve from the standards. It is recommended to use a software program to calculate the standard curve and to determine the concentration of the samples. A 5 pl curve fit with 1/y 2 weighting is recommended for data analysis. The Proinsulin (Total) Chemiluminescence ELISA is a ligand binding assay, with responses exhibiting a sigmoidal relationship to the analyte concentration. Currently accepted reference models for such curves use a 4 or 5 parameter logistic (PL) fit, as these models optimize the accuracy and precision across a greater range. In the example below, a 5-PL curve fit with 1/y 2 weighting was used to maximize the accuracy and precision of samples with low concentrations. However, the accuracy and precision of all models are limited at the lowest and highest ends of the detectable range due to the influence of individual laboratory conditions. As a result, caution should always be used when interpreting results where the analyte response becomes non-linear. 1 Extrapolating sample concentration values outside the range of the standard concentration values is not recommended. For any sample that is diluted, multiply the determined concentration by the dilution factor. Proinsulin (Total) Chemiluminescence ELISA Page 7 of March 16, 2017
8 TYPICAL STANDARD CURVE The following results are provided for demonstration purposes only and cannot be used in place of data obtained with the assay. A standard curve must be performed with each assay run and plate tested. In this example, a 5-parameter curve fit with 1/y 2 weighting is used for data analysis. Note: below is a table for converting pg/ml to pm. 9.4 pg/ml = 1 pm Standard Standard (pg/ml) Standard (pm) A B C D E F G 1, H 3, Zero 0 0 Standard Standard (pg/ml) Mean RLU RLU CV (%) S:N Mean Conc. (pg/ml) Conc. CV (%) % Recovery A 5 1, B 10 2, C 25 5, D 50 10, E , F , G 1, , ,276 1, H 3, , ,991 3, Zero Proinsulin (Total) Chemiluminescence ELISA Page 8 of March 16, 2017
9 EXPECTED VALUES The STELLUX Proinsulin (Total) Chemiluminescence ELISA is calibrated to the WHO Proinsulin 1st International Standard 09/296. It is recommended that each laboratory establish its own normal range for its individual patient population. PERFORMANCE CHARACTERISTICS (Refer to the kit specific Certificate of Analysis for the most relevant performance data.) Sensitivity Analytical sensitivity is determined by calculating the mean * standard deviation for 32 replicates of the Zero Standard. The sensitivity of the assay is pg/ml. Functional sensitivity is defined as the lowest concentration which demonstrates a calculated concentration CV 20% and an accuracy of %. The functional sensitivity of the assay is 2.5 pg/ml. The lower limit of quantitation (LLOQ) is defined as the lowest concentration which demonstrates a calculated concentration CV 20%, an accuracy of %, and an RLU value that is 5 times the background (Zero Standard). The LLOQ of the assay is 5 pg/ml. Proinsulin (Total) Chemiluminescence ELISA Page 9 of March 16, 2017
10 Precision: Within run (intra-assay) variation The within run precision is expressed as the percentage coefficient of variation (CV %). This was determined based on the mean and standard deviation of 16 replicates of a sample run in a single assay. The table below shows the results of 3 samples that span the range of the assay. Sample 1 Sample 2 Sample 3 Mean 30.6 pg/ml 191 pg/ml pg/ml CV (%) n Precision: Between run (inter-assay) variation The between run precision is expressed as the percentage coefficient of variation (CV %). This was determined based on the mean and standard deviation across 2 assays of 32 measurements of a single sample. The table below shows the results of 3 samples that span the range of the assay. Sample 1 Sample 2 Sample 3 Mean 30.5 pg/ml 199 pg/ml pg/ml CV (%) n Linearity The linearity of the assay was determined by preparing dilutions of samples with high proinsulin concentrations with the Zero Standard. The expected values were compared to the obtained values to determine a percent recovery. The mean recovery for serum samples within the range of the assay from 1:2 to 1:128 dilutions is 110% (range %). The mean recovery for heparin plasma samples within the range of the assay from 1:2 to 1:128 dilutions is 110% (range %). The mean recovery for EDTA plasma samples within the range of the assay from 1:2 to 1:128 dilutions is 104% (range %). The mean recovery of human islet tissue culture supernatants from 1:2 to 1:128 is 107% (range %). Proinsulin (Total) Chemiluminescence ELISA Page 10 of March 16, 2017
11 Spike and Recovery The spike and recovery of the assay was determined by adding various known amounts of proinsulin to samples. Serum, heparin plasma, EDTA plasma, and human islet tissue culture supernatants were evaluated. The measured concentration was compared to the expected concentration. The table below shows the recovery for the samples. Mean recovery (%) Min (%) Max (%) Serum low mid high Heparin plasma low mid high EDTA plasma low mid high Tissue culture supernatant low mid high Specificity The table below indicates the analyte and the percent cross-reactivity observed in the assay. Analyte % Cross-reactivity Human Intact Proinsulin 100 Human Des (31,32) Proinsulin 100 Human Des (64,65) Proinsulin 100 Human C-peptide < 0.1 Human Insulin < 0.6 Lispro < 0.2 Hook Effect No high dose hook effect was observed with analyte concentrations up to 300,000 pg/ml. REFERENCES 1. Finlay JWA, Dillard RF. Appropriate Calibration Curve Fitting in Ligand Binding Assays. AAPS Journal. 2007; 9(2): E260-E267. Proinsulin (Total) Chemiluminescence ELISA Page 11 of March 16, 2017
12 SHORT ASSAY PROTOCOL Add 50 µl standards, controls, and samples Add 50 µl Working Strength Protease Inhibitor Incubate for 1 hour at RT, shake at rpm Wash 6 times Add 100 µl Working Strength Detector Antibody Incubate for 1 hour at RT, shake at rpm Wash 6 times Add 100 µl Working Strength SA-HRP Incubate for 30 min at RT, shake at rpm Wash 6 times Add 100 µl Working Strength Chemi Substrate Incubate for 1 minute at RT Read plate Total Time = 2 hours 31 minutes SUGGESTED PLATE LAYOUT Below is a suggested plate layout for running standards, controls, and up to 36 samples in duplicate A Std A Std A Zero Zero B Std B Std B Ctrl 1 Ctrl C Std C Std C Ctrl 2 Ctrl D Std D Std D Ctrl 3 Ctrl E Std E Std E F Std F Std F G Std G Std G H Std H Std H Std= Standard Ctrl = Control Numbered wells = Samples Proinsulin (Total) Chemiluminescence ELISA Page 12 of March 16, 2017
13 APPENDIX Instrument settings: Please contact the microplate reader manufacturer s technical services department for additional assistance. These instrument settings are to be used as a guideline. Biotek Synergy2 Detection Method: Luminescence Read Type: Endpoint Integration: 0:01.00 (MM:SS.ss) (1 second) Emission: Hole Optics Position: Top Sensitivity: 150 Top Probe Vertical Offset: 1.00mm Molecular Devices Spectramax L Read Mode: Luminescence Integration Time: 1000 milliseconds Sensitivity: PMT; MaxRange, Target Wave: 470 nm Automix: Classic 30 mm/s Injection and Delay: Off Injection wells: None Dark Adapt: Off AutoRead: Off Molecular Devices Spectramax M5 Read Mode: Luminescence (LUM) Read Type: Endpoint Wavelength: All Plate Type: 96 well standard opaque Read Area: Variable based on experiment PMT and Optics: Integration Time 1000 ms Shake: Off More Settings: Calibrate (on); Carriage Speed (Normal); Read Order (Column); Setting Time (off) Tecan Infinite 200 Plate: Corning 96 Flat Bottom Black Polystyrol Mode: Luminescence Attenuation: NONE Integration Time: 1000 ms Settle Time: 0 ms Proinsulin (Total) Chemiluminescence ELISA Page 13 of March 16, 2017
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