BDNF (human) ELISA Kit rev 11/13
Contents
1. final wash empty or aspirate the wells and firmly tap the plate dry on a lint free paper towel to remove any remaining wash buffer Add 5 uL of the Progesterone Alkaline Phosphatase Conjugate to the TA wells Add 200 uL of the pNpp Substrate solution to every well Incubate at room temperature for 45 min without shaking Add 50 uL of Stop Solution to every well This stops the reaction and the plate should be read immediately Blank the plate reader against the Blank wells read the optical density at 405 nm preferably with correction between 570 and 590 nm If the plate reader is not able to be blanked against the Blank wells manually subtract the mean optical density of the Blank wells from all readings Calculation Several options are available for the calculation of the concentration of Progesterone in samples We recommend that the data be handled by an immunoassay software package utilizing a four parameter logistic curve fitting program Such software is often supplied by plate reader manufacturers Samples with concentrations outside of the standard curve range will need to be reanalyzed using a different dilution If this sort of data reduction software is not readily available the concentration of Progesterone can be calculated as 1 follows Calculate the average net OD bound for each standard and sample by subtracting the average NSB OD from the average OD bound Average Net OD Average OD NSB OD 2 Calculate the2. solution of trisodium phosphate This solution is caustic care should be taken in use e The activity of the alkaline phosphatase conjugate is dependent on the presence of Mg and Zn ions The activity of the conjugate is affected by concentrations of chelators gt 10 mM such as EDTA and EGTA e The performance of this kit has been tested using a variety of samples however it is possible that high levels of interfering substances may cause variation in assay results e The Progesterone Standard is supplied in ethanolic buffer at a pH optimized to maintain Progesterone integrity Care should be taken when handling this material because of the known and unknown effects of steroids on biological tissue Sample Handling This kit is compatible with Progesterone samples in a wide range of matrices Samples diluted sufficiently into Assay Buffer can be read directly from the standard curve Steroid Displacement Reagent should be added to serum plasma and other samples containing steroid binding proteins It will disassociate steroid from the binding protein allowing it to be detected by the assay Treat appropriate samples with Steroid Displacement Reagent in the following manner add one part of Steroid Displacement Reagent to 99 parts of sample Once the Steroid Displacement Reagent has been added to the neat samples briefly vortex the sample allow it to sit for approximately 5 min and then proceed with sample dilution The Steroid Displacement Reagent
3. 0 001 Danazol lt 0 001 Application Quantitative detection of Progesterone Specificity All species Sample Type Serum Saliva tissue culture media Kit Contents Components K7416 100 PartNo Goat anti Mouse IgG Plate 12 stripsx8 wells K7416 100 1 Progesterone Alkaline Phosphatase Conjugate 6 ml K7416 100 2 Progesterone Monoclonal Ab 6 ml K7416 100 3 Assay Buffer 30 ml K7416 100 4 Wash Buffer Concentrate 30 ml K7416 100 5 Progesterone Standard 100 ng ml in ethanol 0 5 ml K7416 100 6 ONpp Substrate 20 ml K7416 100 7 Stop Solution 5 ml K7416 100 8 Steroid Displacement Reagent 1 ml K7416 100 9 Plate Sealer 1 each K7416 100 10 User Supplied Reagents and Equipment e Microplate reader capable of measuring absorbance at 405 nm preferably with correction between 570 and 590 nm e Absorbent paper e Adjustable pipettes and pipette tips e Microplate shaker Storage Conditions and Reagent Preparation All components of the kit are stable at 4 C until the kit s expiration date e Wash Buffer Prepare Wash Buffer by diluting 5 mL of the concentrate supplied with 95 mL of deionized water This can be stored at room temperature until the kit expiration or for 3 months whichever is earlier Warning amp Precautions e Some kit components contain azide which may react with lead or copper plumbing When disposing of reagents always flush with large volumes of water to prevent azide build up e Stop Solution is a
4. Vi Vil VIII BioVision FOR RESEARCH USE ONLY Progesterone ELISA Kit 114 Catalog K7416 100 100 assays Store at 4 C Introduction BioVision s Progesterone ELISA kit is a competitive immunoassay for the quantitative determination of Progesterone in biological fluids The kit uses a polyclonal antibody to Progesterone to bind in a competitive manner Progesterone in a sample or Progesterone which has an alkaline phosphatase molecule covalently attached to it After a simultaneous incubation at room temperature the excess reagents are washed away and substrate is added After a short incubation time the enzyme reaction is stopped and the yellow color generated is read on a microplate reader at 405 nm The intensity of the bound yellow color is inversely proportional to the concentration of Progesterone in either standards or samples The measured optical density for the samples is used to calculate the concentration of Progesterone in the sample Sensitivity 8 57 pg ml range 15 62 500 pg ml The intra Assay reproducibility is CV lt 10 amp inter Assay is CV lt 12 Cross Reactivity Progesterone 100 5a Pregnane 3 20 dione 100 17 OH Progesterone 3 46 5 Pregnen 3B 01 20 one 1 43 Corticosterone 0 77 4 Androstene 3 17 dione 0 28 Deoxycorticosterone 0 056 DHEA 0 013 17B Estradiol lt 0 001 Estrone 0 001 Estriol lt 0 001 Testosterone lt 0 001 Hydrocortisone lt 0 001 5a Pregnane 3a 20a diol lt
5. ay Buffer or Tissue Culture Media into tube 1 Pipet 500 uL of standard diluent into tubes 2 through 6 Remove 10 uL of diluent from tube 1 Add 10 uL of the 100 ng mL standard to tube 1 Vortex thoroughly Add 500 uL of tube 1 to tube 2 and vortex thoroughly Add 500 uL of tube 2 to tube 3 and vortex Continue this for tubes 4 through 6 The concentration of Progesterone in tubes 1 through 6 will be 500 250 125 62 5 31 25 and 15 62 pg mL respectively Pipet 100 uL of standard diluent Assay Buffer or Tissue Culture Media into the NSB non specific binding and the Bo binding for 0 pg mL Standard wells Pipet 100 uL of Standards 1 6 and samples into the appropriate wells Pipet 50 uL of Assay Buffer into the NSB wells Pipet 50 uL of Progesterone Alkaline Phosphatase Conjugate into each well except the Total Activity TA and Blank wells Pipet 50 uL of Progesterone Antibody Solution into each well except the Blank TA and NSB wells Note Every well used should be Green in color except the NSB wells which should be Blue The Blank and TA wells are empty at this point and have no color Tap the plate gently to mix Incubate at room temperature on a plate shaker for 2 hrs at 500 rom The plate may be covered with the plate sealer provided if so desired At the end of the first incubation empty the contents of the wells and wash by adding 400 uL of wash solution to every well Repeat the wash 2 more times for a total of 3 Washes After the
6. binding of each pair of standard wells as a percentage of the maximum binding wells Bo Percent Bound Net OD x 100 Net Bo OD Graph the data points and the best fit line through the points The concentration of Progesterone in the unknowns can be determined by interpolation 10 Optical Density B Bo e B Bo TTT 0 1 o Optical Density Progesterone Conc pg mL Progesterone Standard Curve This standard curve is for demonstration only A standard curve must be run with each assay RELATED PRODUCTS Progesterone human ELISA Kit 4714 Testosterone human ELISA Kit K7417 Testosterone mouse rat ELISA Kit K7418 Androgen Receptor Antibody 6710 Androgen Receptor Antibody Clone 549CT16 1 4 6711 FOR RESEARCH USE ONLY Not to be used on humans 155 S Milpitas Blvd Milpitas CA 95035 USA T 408 493 1800 F 408 493 1801 www biovision com tech biovision com
7. needs to be added prior to any subsequent dilutions of the sample Samples in the majority of tissue culture media can also be read in the assay after being diluted provided the standards have been diluted into the tissue culture media instead of Assay Buffer There will be a small change in the binding associated with running the standards and samples in media Users should only use standard curves generated in media or buffer to calculate concentrations of Progesterone in the appropriate matrix Notes a Tissue culture media may need to be diluted in the Assay Buffer in order to avoid matrix interference If samples require further dilution in Assay Buffer the standard curve must be prepared in a like manner e g 1 10 dilution of tissue culture media in Assay Buffer b Recommended dilution for tissue culture media 1 10 human serum and saliva 1 10 Please note however the end user must verify that the recommended dilutions are appropriate for their samples c Samples containing mouse IgG may interfere with the assay 155 S Milpitas Blvd Milpitas CA 95035 USA T 408 493 1800 F 408 493 1801 www biovision com tech biovision com xI xII Biovision FOR RESEARCH USE ONLY d Some samples may have very low levels of Progesterone present and extraction may be necessary for accurate measurement Extraction procedure 1 Prepare 1 ng mL Progesterone Standard solution for determination of extraction efficiency in sample matri
8. x This solution can be made by diluting 5 uL of the supplied Standard with 500 uL of ethanol ACS grade or equivalent 2 Add sufficient Progesterone to a sample matrix for determination of extraction efficiency For a typical experiment based on a one mL sample starting volume add 25 50 pg mL of the 1 ng mL Progesterone solution 3 In a fume hood add 1 mL of Diethyl Ether ACS Grade for every mL of sample Put Stopper and shake sample 4 Allow layers to separate Carefully pipet off the top ether layer and place in a clean test tube 5 Repeat steps 2 and 3 twice more combining the ether layers 6 Evaporate the ether to dryness under nitrogen Dissolve the extracted Progesterone with at least 250 uL of Assay Buffer Vortex well then allow to sit for 5 min at room temperature Repeat vortex step twice 7 Run the reconstituted samples in the assay immediately If analysis is to be delayed store evaporated samples desiccated at or below 20 C Assay Protocol Bring all reagents to room temperature for at least 30 min prior to opening All standards and samples should be run in duplicate 1 2 m i Place the desired no of coated strips into the holder Keep any unused wells with the desiccant back into the pouch seal Ziploc and store at 4 C Progesterone Standard Allow the 100 ng mL Progesterone Standard solution to warm to room temperature Label six 12 x 75 mm glass tubes 1 through 6 Pipet 2 mL of standard diluent Ass
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