Rat Uromodulin Glycoprotein, THP ELISA

Transcription

1 Rat Uromodulin Glycoprotein, THP ELISA Catalog Number M For the quantitative determination of Uromodulin (Tamm-Horsfall Glycoprotein, THP) in rat plasma, serum, urine, tissue and cell culture supernate samples. For research use only. This product insert must be read in its entirety before using this product.

2 summary and explanation The Rat Uromodulin (THP) ELISA Kit is designed to detect and quantify Uromodulin in rat serum, plasma, urine, cell culture supernatant and tissue samples. Uromodulin glycoprotein (also known as Tamm-Horsfall Glycoprotein, THP) is the most abundant protein found in the urine of mammals. Uromodulin is 616 amino acids in length with a molecular weight of 80 kda, and is synthesized in the thick ascending loop of Henle. While the specific function of Uromodulin is unknown, it appears to have a role in the regulation of salt and water excretion by the kidney. Soluble Uromodulin has been found to help protect against urinary tract infections of E.coli and P. mirabilis by inhibiting the binding of the bacteria to the epithelial cells of the urinary tract. In addition, it has been suggested that Uromodulin may help prevent renal stone formation. Defects in Uromodulin are associated with diseases such as Familial Juvenile Hyperuricemic Nephropathy (FJHN) and Medullary Cystic Kidney Disease (MCKD2). principle of the assay The assay employs a quantitative sandwich enzyme immunoassay technique which measures rat Uromodulin. It is based on the sandwich technique in which two monoclonal antibodies are directed against separate antigenic determinants on the Uromodulin molecule. During the assay Uromodulin in the sample reacts with anti-uromodulin antibodies bound to the microplate well and peroxidase-conjugated anti-uromodulin antibodies. A simple washing step removes unbound enzyme-labeled antibody. The bound conjugate is detected by a reaction with tetramethylbenzidin (TMB). This reaction is stopped by adding sulfuric acid to give a colorimetric endpoint that is read spectrophotometrically at 450 nm. The optical density (OD) recorded is proportional to the concentration of Uromodulin in the respective well. kit components Microplate - The plate contains 12 x 8-well strips coated with a antibody specific to THP. Ready to use. Standard (2X) Standard Diluent Biotinylated Antibody (100X) Biotinylated Antibody Diluent Wash Buffer Streptavidin-HRP (100X) Streptavidin-HRP Diluent Wash Buffer (25X) TMB Substrate Stop Solution Plate Sealers - 5 adhesive strips. IN30407.R00 page 2

3 Storage Unopened kit Opened/Reconstituted Reagents Store at 2-8º C. Do not use past the kit expiration date. Standard Diluent SA-HRP Diluent Biotinylated Ab Diluent Wash Buffer TMB Substrate Stop Solution Biotinylated Antibody SA-HRP Standard Microplate Wells Store at 2-8º C for up to 30 days. Store at -20º C for up to 30 days. Avoid repeated freeze-thaw cycles. Return unused wells to the foil pouch containing the desiccant and seal. Store at 2-8º C for up to 30 days. supplies required but not provided Pipettes or pipetting equipment with disposable polypropylene tips Glass measuring cylinders Distilled or deionized water Squirt bottle or automated microplate washer Microplate reader capable of measuring at 450 nm Shaker/Vortexer 37º C incubater precautions Stop Solution consists of diluted hydrochloric acid. Wear eye, hand, face, and clothing protection when using these materials. Avoid contact with skin and eyes. In case of contact wash immediately with water. All chemicals should be considered as being potentially hazardous. We therefore recommend that this product is handled only by those persons who have been trained in laboratory techniques and that it is used in accordance with the principles of good laboratory practice. For research use only. Not for internal or external use in humans or animals. This kit contains no material of human origin. For the handling of blood (serum), we recommend that precautions should be observed. Please refer to HHS Publication no. (CDC) or corresponding local/ national guidelines on laboratory safety procedures. IN30407.R00 products@mdbiosciences.com page 3

4 Sample COLLECTION & STORAGE Plasma - Collect plasma using tubes containing EDTA or sodium citrate as an anticoagulant. Centrifuge samples at RPM for 15 minutes and assay. (EDTA or Heparin can also be used as an anticoagulant) Serum - Allow the blood sample to clot for 2 hours or overnight at room temperature (22-25 C) or overnight at 4 C. After clot formation, centrifuge samples at RPM for 15 minutes. When sediments occurred during storage, centrifugation should be performed again. Urine - Collect in sterile tubes. Centrifuge samples at RPM for 15 minutes. Collect the supernatants carefully. When sediments occurred during storage, centrifugation should be performed again. Cell Culture Supernatants - Collect in sterile tubes. Centrifuge samples at RPM for 15 minutes. Collect the supernatants carefully. When examining cell extracts, use PBS (ph ) to dilute cell suspension to approximately 1 million/ml. Centrifuge at RPM for 15 minutes. Collect the supernatants carefully. Avoid repeated freeze-thaw cycles. When sediments occurred during storage, centrifugation should be performed again. Tissue - Excise sample and weigh up. Add a certain amount of PBS (ph 7.4). Freeze with liquid nitrogen immediately for later use. Thaw the sample and keep it at 2-8 C. Add PBS (ph 7.4) and then homogenize the sample thoroughly either by hand, with a homogenizer or through repeated freeze-thaw cycles. Centrifuge at RPM for 15 minutes. Collect the supernatants carefully. Aliquot and keep one for examination and freeze the others for later use. After collecting the sample, extraction should be immediately carried out in accordance with related documents. After extraction, experiment should be conducted immediately as well. Otherwise, keep the sample at -20 C. Avoid repeated freeze-thaw cycles. Samples containing NaN3 must not be tested as it inhibits the activity of Horse Radish Peroxidase (HRP). reagent preparation Bring all reagents to room temperature (22-25 C) before use. Reagents can easily be trapped in the caps of small microfuge tubes. It is recommended to briefly centrifuge these reagents before use. Wash Buffer - If crystals have formed in the concentrate, warm to room temperature (22-25 C) and mix gently until the crystals have completely dissolved. Dilute 30 ml of the Wash Buffer Concentrate into 720 ml of deionized water to prepare 750 ml of Wash Buffer. Store for up to 30 days at 2-8 C. Standards - Prepare standard 15 minutes before use. Label 5 standard tubes as shown below. Reconstitute the Standard with 1 ml of Sample Diluent. Let stand for 10 minutes until fully dissolved. Serially dilute the original standard solution (200 ng/ml) two-fold with equal volume of standard diluent to produce 100 ng/ml, 50 ng/ml, 25 ng/ml, 12.5 ng/ml, 6.25 ng/ml and 3.13 ng/ml. Any remaining standard should be frozen at -20 C and used within 30 days. 500 μl Serial Dilutions using 500 μl 200 ng/ml THP Standard 100 ng/ml 50 ng/ml 25 ng/ml 12.5 ng/ml 6.25 ng/ml 3.13 ng/ml IN30407.R00 page 4

5 Biotinylated Antibody - Calculate the required amount before performing the assay (100 µl/well). Centrifuge the stock before use. Dilute the concentrated Biotinylated Antibody 1:100 using the Biotinylated Antibody Diluent. Streptavidin-HRP - Calculate the required amount before performing the assay (100 µl/well). Centrifuge the stock before use. Dilute the concentrated Streptavidin-HRP Conjugate 1:100 using the Streptavidin-HRP Diluent. assay protocol Read the entire protocol before beginning the assay. It is recommended that all standards and samples be assayed in duplicate. Reagents can easily be trapped in the caps of small microfuge tubes. It is recommended to briefly centrifuge these reagents before use. Note: Reagents and samples may require specific handling temperatures and need preparation prior to the assay. See the Reagent and Sample Preparation sections before proceeding. Note: Bring all reagents and samples to room temperature (22-25 C) before use Prepare all reagents and samples as described in the previous sections. Remove any excess microplate strips from the plate frame and return them to the foil pouch containing the desiccant pack. Pipette 100 µl of Standard or sample in duplicate into the wells using a clean pipette tip for each standard or sample. Cover with the plate sealer provided and incubate for 90 minutes at 37 C. Blot the plate on paper towels to remove residual fluid from the plate. Add 100 µl of Biotinylated Antibody into each well. Cover with the plate sealer provided and incubate for 1 hour at 37 C. Aspirate and wash the wells 5 times with 200 µl per well of Wash Buffer. Take care that all wells are filled and emptied at each wash. Blot the plate on paper towels to remove residual fluid from the plate. Add 100 µl of SA-HRP Conjugate to each well. Cover with the plate sealer provided and incubate for 30 minutes at 37 C. Aspirate and wash the wells 5 times with 200 µl per well of Wash Buffer. Take care that all wells are filled and emptied at each wash. Blot the plate on paper towels to remove residual fluid from the plate. Add 90 µl each of TMB Substrate to each well. Cover with the plate sealer provided and incubate for 15 minutes at 37 C. Gently tap the side of the plate to ensure thorough mixing. Note: Substrate is sensitive to light and therefore should not be exposed to light for too long. Stop the reaction by adding 50 µl of Stop Solution to each well. Read the plate at 450 nm. If wavelength correction is available, subtract readings at 570 nm from those at 450 nm to correct optical imperfections. If it is not available, read plate at 450 nm only. Note: some unstable black particles may be generated at high concentration points after stopping the reaction at 10 minutes, which may reduce the absorbance readings. IN30407.R00 products@mdbiosciences.com page 5

6 Summary Prepare reagents and samples as previously described. ] ] ] ] ] ] Pipette 100 µl Standard or sample in duplicate into the wells. Incubate for 90 min. at 37 C. Remove residual fluid from each well. Do not wash. Add 100 µl of Biotinylated Antibody to each well. Incubate for 1 hr. at 37 C. Aspirate and wash 5 times. Add 100 µl of HRP Conjugate to each well. Incubate for 30 min. at 37 C. Aspirate and wash 5 times. ] Add 90 µl of TMB Substrate to each well. Incubate 15 min. at 37 C. ] Add 50 µl of Stop Solution to each well. Read at 450 nm. IN30407.R00 page 6

7 calculation of results Calculate the mean value of the duplicate or triplicate readings for each standard and sample. To generate a standard curve, plot the graph using the standard concentrations on the x-axis and the corresponding mean 450 nm absorbance on the y-axis. The best-fit line can be determined by regression analysis of the linear portion of the curve. Determine the unknown sample concentration from the Standard Curve and multiply the dilution factor. The curve is provided for illustration only. A standard curve should be generated each time the assay is performed. PERFORMANCE CHARACTERISTICS Assay Range 3.13 ng/ml ng/ml Sensitivity Sensitivity is defined as the minimal detectable dose determined by adding two standard deviations of the mean optical density value for replicates of the zero standard and calculating the corresponding concentration. The sensitivity of the Rat Uromodulin ELISA is typically ~ 1.88 ng/ml. Reproducibility Intra-assay Precision (Precision within an assay) - The intra-assay precision was measured by assaying control samples multiple times on one plate. The intra-assay precision coefficient of variation (CV) is <10%. Inter-assay Precision (Precision between assays) - The inter-assay precision was assessed by repeated measurements of control samples in successive assays with multiple users. The inter-assay precision coefficient of variation (CV) is <10%. Specificity The ELISA kit recognizes natural and recombinant rat Uromodulin. No significant cross-reactivity or interference between rat Uromodulin and analogues was observed. NOTE: Limited by existing techniques, cross reaction may still exist, as it is impossible for us to complete the cross-reactivity detection between rat Uromodulin and all analogues. IN30407.R00 products@mdbiosciences.com page 7

8 TROUBLESHOOTING Problem Low Absorbance High Absorbance/high zero standard value Flat curve/poor reproduciblity Recommendation Check reagents for proper storage. Control expiration date. Check preparation of reagents. Control incubation times and temperature. Check reader wavelength. Check preparation of reagents. Control incubation times and temperature. Equilibrate ELISA reagents to room temperature. Ensure that every well of the ELISA plate is completely filled and emptied at every wash step. Check that plates are blotted on tissue paper after washing. Check reagents for proper storage. Control expiration date. Check preparation of working standards. Check incubation times and temperatures. Use separate reservoirs for pipetting different solutions with multichanned pipettes. Always use new pipette tips. Check pipette calibration. Ensure efficient washing procedure. IN30407.R00 page 8

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12 North America MD Bioproducts Divison of MD Biosciences, Inc University Ave. W. Ste. 100 St. Paul, MN USMDBIO Tel: Fax: International/Headquarters MD Bioproducts Division of MD Biosciences GmbH Gewerbestrasse Egg b Zurich Switzerland Tel: Fax: products@mdbiosciences.com