Mouse Sclerostin ELISA

Transcription

1 Mouse Sclerostin ELISA For the quantitative determination of sclerostin in mouse serum and plasma. For Research use Only. Not For Use In Diagnostic Procedures. Catalog Number: Size: 41-SCLMS-E01 96 Wells Version: 4-ALPCO July 21, G Keewaydin Drive, Salem, NH P: (800) F: (603)

2 INTENDED USE The ALPCO Mouse Sclerostin ELISA is designed for the quantitative determination of sclerostin in mouse serum and plasma. PRINCIPLE OF THE ASSAY The ALPCO Mouse Sclerostin ELISA is a sandwich type immunoassay. The 96-well microplate is coated with a polyclonal antibody specific for mouse sclerostin. The standards, control, and samples are added to the microplate wells. After incubation, the wells are washed with Wash Buffer and blotted dry. The polyclonal detection antibody conjugate is then added, and the microplate is incubated a second time on a microplate shaker at rpm, washed and blotted dry. TMB Substrate is added, and after a short incubation on a microplate shaker at rpm, Stop Solution is added, and the optical density (OD) is measured by a spectrophotometer at 450 nm. The intensity of the color generated is directly proportional to the amount of mouse sclerostin in the sample. MATERIALS SUPPLIED Component Quantity Preparation Mouse Sclerostin Microplate (coated with polyclonal anti-sclerostin antibody) 12 x 8 well strips Ready to use Calibrator* 1 ml Lyophilized Sclerostin Control* 0.5 ml Lyophilized Assay Diluent 60 ml Ready to use Biotin Conjugate ml 100X Streptavidin (SA)-HRP Conjugate ml 100X Conjugate Buffer 25 ml Ready to use Wash Buffer 50 ml 20X TMB Substrate 12 ml Ready to use Stop Solution 12 ml Ready to use Plate Sealers 4 Ready to use *Please refer to the Certificate of Analysis enclosed with each kit for the lot specific concentrations. 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 or deionized water for reagent preparation Microplate washer or wash bottle Microplate shaker capable of rpm Microplate reader with 450 nm filter Vortex for sample preparation Centrifuge Heparin tubes (for collection of plasma samples) Mouse Sclerostin ELISA Page 2 of 8

3 PRECAUTIONS 1. All samples and 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 BSE negative. However, all materials should be kept from ruminating animals. 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. SAMPLE HANDLING AND PREPARATION Serum and plasma (heparin) samples are appropriate for use in this assay. It is recommended that all samples are run in duplicates. Dilute samples 1:10 with Assay Diluent and gently vortex. For duplicate samples, dilute 25 ul of sample in 225 ul of Assay Diluent. If the sample concentration is greater than that of the highest standard, use Assay Diluent to dilute the sample further and repeat analysis. In order to reduce potential time-associated sample drift, it is recommended to thoroughly vortex each sample before use and perform each pipetting action without pausing. REAGENT PREPARATION AND STORAGE CONDITIONS The kit should be stored at 2-8ºC. The kit is stable until the expiry date on the box label. All reagents must be equilibrated to room temperature prior to preparation and subsequent use in the assay. Calibrator is provided in a lyophilized form. Reconstitute with 1 ml deionized or distilled water and vortex to mix. Let stand for 10 minutes prior to use. This is the Reconstituted Stock Calibrator. Prepare dilution series using the Reconstituted Stock Calibrator and Assay Diluent as in the following example. Actual Calibrator and Standard concentrations are provided on the lot specific Certificate of Analysis that is provided with each kit. Standard Volume of Calibrator/Standard Volume of Assay Diluent Final Concentration (pg/ml) 6 20 l Reconstituted Stock Calibrator 980 l l Standard l l Standard l l Standard l l Standard l l Standard l l 0 Mouse Sclerostin ELISA Page 3 of 8

4 If multiple assays are to be performed, the Reconstituted Stock Calibrator should be stored in aliquots at -20ºC. Thawed, Reconstituted Stock Calibrator is not stable and should be discarded after use. Only thaw enough Reconstituted Stock Calibrator for desired run. Biotin Conjugate is a 100X concentrate. Dilute Conjugate with 10 parts Conjugate Buffer. For example, to prepare enough Working Strength Conjugate for one complete microplate, dilute 0.1 ml of Conjugate 100X with 9.9 ml of Conjugate Buffer (1:100, 1 part to 99 parts). Working Strength Conjugate is stable for 4 weeks at 2-8ºC. SA-HRP Conjugate is a 100X concentrate. Dilute SA-HRP Conjugate with 10 parts Conjugate Buffer. For example, to prepare enough Working Strength Conjugate for one complete microplate, dilute 0.1 ml of Conjugate 100X with 9.9 ml of Conjugate Buffer (1:100, 1 part to 99 parts). Working Strength Conjugate is stable for 4 weeks at 2-8 C. Wash Buffer is a 20X concentrate. Dilute Wash Buffer with 20 parts deionized water. For example, to prepare Working Strength Wash Buffer, dilute 20 ml of Wash Buffer 20X with 380 ml of deionized water (1:20, 1 part to 19 parts). Working Strength Wash Buffer is stable for 4 weeks at room temperature (18-25 C). Control is provided in a lyophilized form. Reconstitute with 0.5 ml deionized water and vortex to mix. Let stand for 10 minutes prior to use. If multiple assays are to be performed, the control should be stored in aliquots at -20ºC. Thawed, reconstituted control is not stable and should be discarded. Only thaw enough reconstituted control for desired run. Control needs to be diluted 1:10 as for the samples. QUALITY CONTROL It is recommended that the Control provided with the ALPCO Mouse Sclerostin ELISA be included in every assay. The concentration range of the control is provided on the Certificate of Analysis provided with each kit. ASSAY PROCEDURE All reagents and microplate strips should be equilibrated to room temperature (18-25 C) 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. Designate enough microplate strips for duplicate determinations of the Standards, control, and samples. The remaining microplate strips should be stored at 2-8 C in the tightly sealed foil pouch containing the desiccant. 2. Pipette 100 µl of each Standard (0-6), diluted control, and diluted sample (see Sample Handling and Preparation and Reagent Preparation) into their respective wells. See Reagent Preparation for Calibrator and Control reconstitution instructions. Mouse Sclerostin ELISA Page 4 of 8

5 3. Cover microplate with a plate sealer and incubate for 4.5 hours at room temperature, shaking at rpm on a microplate shaker. *Alternatively, this step can be carried out overnight at room temperature, without shaking. Contact ALPCO for more details. 4. Decant the contents of the wells and wash the microplate 4 times with 350 µl of Working Strength Wash Buffer per well (see Reagent Preparation) using a microplate washer. Alternatively, fill the wells with Working Strength Wash Buffer using a wash bottle (do not use a multichannel pipette). Discard the liquid, invert, and firmly tap the microplate on absorbent paper towels between 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. 5. Pipette 100 µl of Working Strength Conjugate (see Reagent Preparation) into each well. 6. Cover microplate with a plate sealer and incubate for 20 min at room temperature shaking at rpm on a microplate shaker. 7. Decant the contents of the wells and wash the microplate 4 times with 350 µl of Working Strength Wash Buffer per well (see Reagent Preparation) using a microplate washer. Alternatively, fill the wells with Working Strength Wash Buffer using a wash bottle (do not use a multichannel pipette). Discard the liquid, invert, and firmly tap the microplate on absorbent paper towels between 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. 8. Pipette 100 µl SA-HRP cover microplate with a plate sealer and incubate for 20 min at room temperature, shaking at rpm on a microplate shaker 9. Decant the contents of the wells and wash the microplate 4 times with 350 µl of Working Strength Wash Buffer per well (see Reagent Preparation) using a microplate washer. Alternatively, fill the wells with Working Strength Wash Buffer using a wash bottle (do not use a multichannel pipette). Discard the liquid, invert, and firmly tap the microplate on absorbent paper towels between 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. 10. Pipette 100 µl of TMB Substrate into each well. 11. Cover microplate with a plate sealer and incubate for 10 min at room temperature, shaking at rpm on a microplate shaker. 12. Pipette 100 µl of Stop Solution into each well and gently shake the microplate to mix the contents. Remove any bubbles before proceeding with the next step. 13. Place the microplate in a microplate reader capable of reading the absorbance at 450 nm. The microplate should be analyzed within 30 min following the addition of the Stop Solution. Mouse Sclerostin ELISA Page 5 of 8

6 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. It is recommended to use a 4 parameter logistic (lin/log) curve fit for this assay, with subtraction of the Zero Standard s OD. If samples were diluted, the appropriate dilution factor should be used to calculate the actual sample values. TYPICAL STANDARD CURVE The following results are provided for demonstration purposes only and cannot be used instead of data obtained with the assay. A standard curve must be performed with each assay run and plate tested. Standards (pg/ml) Absorbance (450 nm) EXPECTED VALUES A survey of mouse sclerostin levels across a sampling of mouse strains showed normal mouse sclerostin ranges to be ng/ml. Note that mouse sclerostin values may vary among different mouse strains and treatments. Therefore, it is recommended that each laboratory establish its own normal range for its sample populations. Conversion for Mouse Sclerostin: 1 pg/ml = 0.44 pmol/l (MW: 22.5 kd) PERFORMANCE CHARACTERISTICS Sensitivity The analytical sensitivity (Limit of Detection) was determined by calculating the mean ± 3 standard deviations for 24 replicates of the Assay Diluent. The sensitivity of the assay is 17.4 pg/ml. Lower Limit of Detection (LLOQ) The LLOQ was determined to be 37 pg/ml. Precision: Within run (intra-assay) variation Mouse Sclerostin ELISA Page 6 of 8

7 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 for 3 samples that span the range of the assay. Sample 1 Sample 2 Sample 3 Mean pg/ml 1226 pg/ml 537 pg/ml Std. Dev pg/ml 36 pg/ml 12 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 8 assays of duplicate measurements of a single sample. The table below shows the results of 4 samples that span the range of the assay. Sample 1 Sample 2 Sample 3 Sample 4 Mean 1693 pg/ml 1413 pg/ml 1209 pg/ml pg/ml Std. Dev. 188pg/ml 186 pg/ml 178 pg/ml 11.9 pg/ml CV % n Linearity The linearity of the assay was determined by preparing dilutions of 3 samples with high mouse sclerostin concentrations with the Assay Diluent. The expected values were compared to the obtained values to determine a percent recovery. The range of recovery was %, and the average recovery was 113%. Spike and Recovery The spike and recovery of the assay was determined by adding various known amounts of mouse sclerostin to a sample. This spiked sample was evaluated in the assay and the measured concentration was compared to the expected concentration (endogenous + spiked). Across 3 samples, the range of recovery was % with an average of 103 %. Limitations The effect of various potentially interfering substances was determined in the ALPCO Mouse Sclerostin ELISA. The table below indicates the highest concentration tested with no interference in the assay. Interfering Substance Heparin EDTA Hemoglobin Concentration Tested 30 mg/ml 50.6 mg/ml 1.0 mg/ml Mouse Sclerostin ELISA Page 7 of 8

8 SHORT ASSAY PROTOCOL Mouse Sclerostin ELISA Page 8 of 8