Product Specification Sheet
Contents
1. the standard solution X The Porcine MHB concentration of the samples can be interpolated from the standard curve Summary Prepare reagents samples and standards Lt Add 100ul prepared samples or standards and incubate the plate at 37 C for 90min wash plate twice with 300 ul TBS Add 100ul biotinylated antibodies and incubate the plate at 37 C for 60min wash plate 3 times with TBS tt Add 100ul ABC working solution and incubate the plate at 37 C for 30 min wash plate five times with TBS tt Add 90ul TMB color developing agent and incubate at 37 C Add 100ul stop solution tt Read the O D absorbance at 450nm within 30 min Calculate the Porcine MHB concentration in the samples by plotting graph between Concentrations and corresponding absorbencies of Standards2. ELISA kit User Manual Porcine Methemoglobin MHB ELISA Kit Catalog No BMA 22427 96T For Research Use Only Not for In Vitro Diagnostic Use Intended use The ELISA Kit is to be used for quantitative determination of Porcine Methemoglobin MHB in serum Plasma and other biological fluids Kit is intended for research use only not for diagnostics 1 Principles of Method The Porcine MHB ELISA Kit is an Vitro enzyme linked immunosorbent assay for the quantitative measurement of Porcine MHB in serum plasma tissue lysates cell culture supernatants and urine The Porcine MHB polyclonal antibodies are pre coated onto 96 well plate Standard and samples are aspirated into the wells and Porcine MHB present in a sample is bound to the wells by the immobilized antibody The biotinylated detection antibodies are added to the wells and then followed by washing with PBS or TBS buffer After washing away unbound biotinylated antibody Avidin Biotin Peroxidase Complex is added to the wells The wells are washed again a TMB substrate solution is added to the wells and the color changes after adding acidic stop solution The intensity of the color is proportional to the amount of Porcine MHB bound in samples and measured at 450nm 10nm 2 Reagents and Materials Provided Composition Quantity Composition Quantity Pre coated Micro titer plate 96 wells 1 Sample Diluent Buffer 2X 15ml Standards 2 ABC Diluent Buffer 1 X 12ml D
3. cell culture supernatants to each empty well Seal the plate with the cover and incubate at 37 C for 90 min Remove the cover discard plate content and blot the plate onto paper towels or other absorbent material Do NOT let the wells completely dry at any time washing twice with 300ul of wash buffer Add 100ul of biotinylated anti Porcine MHB antibody working solution into each well and incubate the plate at 37 C for 60 min Wash the plate three times with 0 01M TBS and each time let washing buffer stay in the wells for 1 min Add 100ul of prepared ABC working solution into each well and incubate the plate at 37 C for 30 min Wash plate 5 times with 0 01M TBS and each time let washing buffer stay in the wells for 1 2 min Add 100ul of prepared TMB color developing agent into each well and incubate plate at 37 C for 10 15 minutes away from light observe the color at all times when shades of blue can be seen in the wells with the three four most concentrated Porcine MHB standard solutions the other wells show no obvious color Add 100ul of prepared stop solution into each well to stop the reaction The color changes into yellow immediately Read the O D absorbance at 450nm in a micro plate reader within 30 min after adding the stop solution or even 620 630nm Secondary wavelength 41 The standard curve can be plotted as the relative O D 450 of each standard solution Y vs the respective concentration of
4. etection Antibody 1X120ul Antibody Diluent Buffer 1X 12ml Avidin Biotin Peroxidase Complex ABC 1 100 1X 120 ul Stop solution 1X 10ml TMB color developing reagent A 1X 10ml TBS Diluent Wash Buffer 25X 1 X 20ml TMB color developing reagent B 1X 1 5 ml Instruction manual 1 Note Standard Frozen dried TMB color developing reagent A Avoid light 3 Materials Required But Not Provided G Micro titer plate reader in standard size 2 Polypropylene tubes for diluting and for aliquoting standard 3 Distilled or de ionized water 4 Calibrated adjustable precision pipettes preferably with disposable plastic tips 4 Performance Characteristic Normal Range 20ug ml 0 312ug mIl Sensitivity lt 0 06uUg ml Specificity The ELISA Kit shows no cross reactivity with any of the cytokines 5 Reference curve Porcine MHB 229 2 ee E ii l 0 5 0 0 8 16 24 Concentration yug ml 6 Storage and stability All the components in the kit can be stored up to 1 year at 20 C and 4 weeks at 2 8 C Please avoid repeated freeze thaw cycles and do not mix reagents from different kits unless they have the same lot numbers 7 Reagents Preparation Plate washing Discard the solution in the plate without touching the side walls Blot the plate onto paper towels or other absorbent material Soak each well with at least 0 35 ml PBS or TBS buffer for 1 2 minutes then discard the rinse solution Repeat this
5. g solution The solution should be prepared no more than 2 hours prior to the experiment Note please make centrifuging for the vial before preparing a The total volume should be 100ul or 0 1ml well x the number of wells Allowing 0 1 0 2ml more than total volume b Biotinylated anti Porcine MHB antibody should be diluted in 1 100 1 99 with the antibody diluent buffer and mixed thoroughly C Preparation of Avidin Biotin Peroxidase Complex ABC working solution The solution should be prepared no more than 1 hour prior to the experiment Note please make centrifuging for the vial before preparing a The total volume should be 100uI or 0 1ml well x the number of wells Allowing 0 1 0 2ml more than total volume b Avidin Biotin Peroxidase Complex ABC should be diluted in 1 100 1 99 with the ABC dilution buffer and mixed thoroughly D Preparation of TMB working solution transfer 9 volumes of TMB color developing Reagent A in one volume of TMB color developing Reagent B for 30 minutes in 37 C before using to make TMB substrate mixing thoroughly 8 Assay Procedure The user should decide sample dilution fold by crude estimation of Porcine MHB amount in samples o 100ul per well of the grades Porcine MHB standard solutions into the pre coated 96 well plate Add 100ul of the sample diluent buffer into the control wells Add 100ul of each properly diluted sample of sera plasma body fluids tissue lysates or
6. process for several times Sample Preparation and Storage Stored samples to be assayed within 24 hours at 2 8 C For long term storage aliquot and freeze samples at 20 C Avoid repeated freeze thaw cycles Cell culture supernatant tissue lysates or body fluids Remove particulates by centrifugation analyze immediately or aliquot and store at 20 C Serum Allow the serum to clot in a serum separator tube about 2 hours or 4 C at room temperature Centrifuge at approximately 1000 X g for 10 min Analyze the serum immediately or aliquot and store frozen at 20 C Plasma Collect plasma using heparin EDTA citrate as an anticoagulant Centrifuge for 15 minutes at 1000 x g within 30 min of collection Analyze immediately or aliquot and store frozen at 20 C Sample Dilution Guideline User needs to estimate the concentration of the target protein in the samples and select proper dilution factor so that the diluted target protein concentration falls near the middle of the linear regime in the standard curve Reagent Preparation and Storage A Preparation of the standard Standard solution should be prepared no more than 2 hours prior to the experiment Add 1 ml sample diluent buffer into one tube dissolve the standard thoroughly and make times dilution 500uL S500nl S500OuL S500uL 20 10 5 2 5 1 25 0625 0312 pg ml ug ml ug ml ug ml g m pg m pg ml B Preparation of biotinylated anti Porcine MHB antibody workin
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