User Manual - B-Bridge International, Inc.
Contents
1. 1414 customersupport b bridge com 1 408 252 62002. B Bridge International Inc kA Oxytocin Chemiluminescent Immunoassay Kit User Manual 1 Plate Kit Catalog K3048 C1 5 Plate Kit Catalog K3048 C5 1 B Bridge International Inc www b bridge com Oxytocin 111414 customersupport b bridge com 1 408 252 6200 TABLE OF CONTENTS Intended Use Background Assay Principle Kit Components Materials Required Precautions Reagent Preparation Sample Preparation Assay Protocol Calculations N NOD oO Ff A A WO W W Typical Standard Curve Example Notice to Purchaser This product is to be used for Research Purposes Only It is not to be used for Drug or Diagnostic Purposes nor is it intended for Human Use B Bridge products may not be resold modified for resale or used to manufacture commercial products without the express written consent of B Bridge International Inc EXCEPT AS OTHERWISE EXPRESSLY SET FORTH IN THIS USER MANUAL B BRIDGE DOES NOT MAKE ANY REPRESENTATION OR WARRANTIES OR CONDITIONS OF ANY KIND EITHER EXPRESSED OR IMPLIED WITH RESPECT TO THE PRODUCTS OR INFORMATION DISCLOSED HEREUNDER INCLUDING BUT NOT LIMITED TO THE IMPLIED WARRANTIES OF MERCHANTABILITY FIT FOR A PARTICULAR PURPOSE OR NONINFRINGEMENT OF THE INTELLECTUAL PROPERTY RIGHTS OF THIRD PARTIES B Bridge International Inc All Rights Reserved 2 B Bridge International Inc www b bridge com Oxytocin 111414 customersupport b bridge com 1 408 252 6200 INTENDED USE The B Bridge Oxytoci
3. The clarified milk sample i e the supernatant liquid can be stored at 20 C until needed Use all samples within 2 hour of preparation ASSAY PROTOCOL 1 The unused wells should be stored in the foil pouch with desiccant and stored at 4 C 2 Pipet 100 L of samples or standards into the appropriate number of wells in the plate 3 Pipet125 Lof 1X Assay Buffer into the non specific binding NSB wells 4 Pipet 100 Lof 1X Assay Buffer into wells to act as maximum binding wells BO or 0 pg mL 5 Add 25 L ofthe Oxytocin Conjugate to each well using a repeater or multichannel pipet 6 Add 25 L ofthe Oxytocin Antibody to each well except the NSB wells using a repeater or multichannel pipet 7 Shake the plate in a plate shaker at room temperature for 15 minutes to ensure adequate mixing of the reagents Cover the plate with the plate sealer and store at 4 C for 16 hours 6 B Bridge International Inc www b bridge com Oxytocin 111414 customersupport b bridge com 1 408 252 6200 8 Aspirate the plate and wash each well 4 times with 300 L 1X Wash Buffer Tap the plate dry on clean absorbent towels 9 Add 100 L of mixed chemiluminescent Substrate to each well using a repeater pipet 10 Incubate the plate at room temperature for 5 minutes without shaking 11 Read the luminescence generated from each well in a multimode or chemiluminescent plate reader using a 0 1 second read time per well The chemiluminescent signal will decr
4. ash Buffer 30 mL 125 mL Substrate Solution A 6 mL 28 mL Substrate Solution B 6 mL 28 mL Plate Sealer 1 each 5 each All components of this kit should be stored at 4 C until the expiration date of the kit MATERIALS REQUIRED BUT NOT SUPPLIED e Distilled or deionized water e Repeater pipet with disposable pipet tips capable of dispensing 25ul and 100ul e Amicroplate shaker e SpeedVac or other centrifugal evaporator to evaporate extracted samples e 96 well plate reader capable of reading glow chemiluminescence The number of RLUs obtained is dependant on the sensitivity and gain of the reader used If you are unsure of how to properly configure your reader contact your plate reader manufacturer or carry out the following protocol Dilute 5 ul of the Oxytocin conjugate into 45 ul of deionized water Pipet 5 ul of this dilution into an uncoated white well and add 100 ul of prepared CLIA substrate This well will give you an intensity 2 3x the maximum binding for the assay Adjust the gain or sensitivity so that your reader is giving close to the readers maximum signal To properly analyze the data software will be required for converting raw RLU readings from the plate reader and carrying out four parameter logistic curve 4PLC fitting Contact your plate reader manufacturer for details PRECAUTIONS As with all such products this kit should only be used by qualified personnel who have had laboratory safety instruction The complete inse
5. ease about 40 over 60 minutes 12 Use the plate reader s built in 4PLC software capabilities to calculate Oxytocin concentrations for each sample CALCULATIONS All luminometers read Relative Light Units RLU These RLU readings will vary with make or model of plate reader Average the duplicate RLU readings for each standard and sample Create a standard curve by reducing the data using the 4PLC fitting routine on the plate reader after subtracting the mean RLU s for the NSB The sample concentrations obtained calculated from the B BO curve and should be multiplied by the dilution factor to obtain neat sample values Conversion factor 1 ng mL of Oxytocin is equivalent to 0 993 nM TYPICAL STANDARD CURVE EXAMPLE ONLY 100 rrt 140 000 90 120 000 80 70 100 000 60 80 000 5 o m 50 a amp 60 000 40 30 40 000 20 20 000 10 1 10 100 1 000 10 000 Oxytocin Conc pg mL Always run your own standard curve for calculations of results 7 B Bridge International Inc www b bridge com Oxytocin 111414 customersupport b bridge com 1 408 252 6200 TYPICAL DATA EXAMPLE ONLY e Standard 1 20735 14135 Standard 2 29 455 22855 25365 708160 14750 108 150 76 06 Cazes w215 100 Standard 7 123370 19320 s392 Standard 6 114 750 Always run your own standard curve for calculations of results 8 B Bridge International Inc www b bridge com Oxytocin 11
6. ith one part Substrate Solution B in a brown bottle Once diluted this is stable for one month when stored at 4 C SAMPLE PREPARATION This assay has been validated for serum EDTA and heparin plasma milk and tissue culture samples Samples containing visible particulate should be centrifuged prior to use Oxytocin is identical across all species and we expect this kit may measure Oxytocin from sources other than human Because of the cross reactivity to mesotocin this kit should also be able to measure mesotocin from birds fish and amphibians The end user should evaluate recoveries of Oxytocin in other samples being tested Serum and Plasma Samples Serum and plasma samples should be extracted with the provided Extraction Solution or with a solid phase C18 column extraction protocol prior to running in the kit Protocol Using Extraction Solution e Mix 1 part sample with 1 5 parts of Extraction Solution Vortex and then nutate at room temperature for 90 minutes e Centrifuge for 20 minutes at 4 C at 1660 x g SpeedVac supernatant to dryness at 37 C Reconstitute sample with 250 uL of Assay Buffer Milk Samples Milk samples should be clarified by centrifuging at 10 000 x g for 15 minutes Pierce the top fatty layer and collect the supernatant liquid Repeat the centrifugation and collection two more times The collected supernatant liquid must then be diluted 1 10 with the provided Assay Buffer before using in the assay
7. n Chemiluminescent Immunoassay kit is designed to quantitatively measure Oxytocin present in serum EDTA and heparin plasma clarified milk and tissue culture media samples This assay is species independent BACKGROUND Oxytocin is a neurohypophysial peptide which is produced in the paraventricular nuclei of the hypothalamus and stored in the posterior pituitary The molecule consists of nine amino acids linked with a 1 6 disulfide bond and a semi flexible carboxyamidated tail A hormone once thought to be limited to female smooth muscle reproductive physiology and neurotransmitter recent studies have begun to investigate Oxytocin s role in various behaviors including orgasm social recognition pair bonding anxiety and maternal behaviors and is important in male reproductive physiology Oxytocin and the related neurohypophysial peptide Arg8 Vasopressin maintain renal water and sodium balance Oxytocin OH Tyr 0O HN I o cys N Tu ys HN S Pro GyS lle u O HN O N Gin AANO ae HaN No OL nor ly o FF NH NH Highly conserved across species boundaries Oxytocin like neurohypophysial peptides are substituted primarily at residues 4 and or 8 In the Oxytocin like peptide mesotocin a common peptide found in some fishes reptiles birds amphibians marsupials and non mammalian tetrapods the leucine at residue 8 is substituted for isoleucine Acting in classical endocrine fashion Oxytocin elicits regulatory effects b
8. rt should be read and understood before attempting to use the product The antibody coated plate needs to be stored desiccated The silica gel pack included in the foil ziploc bag will keep the plate dry The silica gel pack will turn from blue to pink if the ziploc has not been closed properly This kit utilizes a peroxidase based readout system Buffers including other manufacturers Wash Buffers containing sodium azide will inhibit color production from the enzyme Make sure all buffers used for samples are azide free Ensure that any plate washing system is rinsed well with deionized water prior to 4 B Bridge International Inc www b bridge com Oxytocin 111414 customersupport b bridge com 1 408 252 6200 using the supplied Wash Buffer In all cases please consult your institution s safety procedures for working with hazardous chemicals REAGENT PREPARATION Allow the kit reagents to thaw and come to room temperature for 30 minutes We recommend that all standards and samples be run in duplicate to allow the end user to accurately determine Oxytocin concentrations Ensure that all samples have reached room temperature and have been diluted as appropriate prior to running them in the kit Assay Buffer Dilute 5X Assay Buffer 1 5 by adding one part Assay Buffer to four parts of deionized water Once diluted this is stable at 4 for 3 months Wash Buffer Dilute 20X Wash Buffer 1 20 by adding one part of Wash Buffer to nineteen pa
9. rts of deionized water Once diluted this is stable at 4 C for 3 months Standard Preparation 1 Label test tubes 1 through 8 2 Pipet 450 L of 1X Assay Buffer into tube 1 and 300 L of Assay Buffer into tubes 2 8 3 Carefully add 50 L of the Oxytocin stock solution to tube 1 and vortex completely Note The Oxytocin stock solution contains an organic solvent Prerinse the pipet tip several times to ensure accurate delivery 4 Take 200 L of the solution in tube 1 and add it to tube 2 and vortex completely 5 Repeat the serial dilutions for tubes 3 through 8 The concentration of Oxytocin in tubes 1 through 8 will be 5 000 2 000 800 320 128 51 2 20 48 and 8 192 pg mL respectively Use all Standards within 2 hours of preparation Standard Standard Standard Standard Standard Standard Standard Standard 4 Reagent 1 2 3 5 6 7 8 1X Assay Buffer 450 ul 300 ul 300 ul 300 ul 300 ul 300 ul 300 ul 300 ul Oxytocin Stock 50 ul Standard 1 200 ul Standard 2 200 ul Standard 3 200 ul Standard 4 200 ul Standard 5 200 ul Standard 6 200 ul Standard 7 200 ul Final Oxytocin Concentration 5 000 2 000 800 320 128 51 2 20 48 8 192 pg mL 5 B Bridge International Inc www b bridge com Oxytocin 111414 customersupport b bridge com 1 408 252 6200 Chemiluminescent Substrate Mix one part Substrate Solution A w
10. y binding specific cell surface receptors which in turn initiate a secondary intracelluar response cascade via a phosphoinositide signaling pathway ASSAY PRINCIPLE The B Bridge Oxytocin Chemiluminescent Immunoassay kit is designed to quantitatively measure Oxytocin present in serum plasma clarified milk and tissue culture media samples This assay is species independent Please read the complete kit insert before performing this assay 1 Sample or standards are added to the well in a microtiter plate coated with an antibody to capture rabbit IgG 2 Oxytocin peroxidase conjugate is added to each well containing either standards or sample 3 The binding reaction is initiated by the addition of a polyclonal antibody to Oxytocin 4 Incubate overnight at 4 C wash plate and add substrate to each well 5 Substrate reacts with the bound Oxytocin peroxidase conjugate to produce light The intensity of the chemiluminescent signal is detected by a plate reader capable of measuring luminescence 6 Calculate Oxytocin concentration from standard curve 3 B Bridge International Inc www b bridge com Oxytocin 111414 customersupport b bridge com 1 408 252 6200 KIT COMPONENTS Component Cat K3048 C1 K3048 C5 Coated White 96 Well Plates 1 plate 5 plates Oxytocin Standard 50 000 pg mL in solution 125 uL 625 ul Oxytocin Antibody 3 mL 13 mL Oxytocin Conjugate 3 mL 13 mL 5X Assay Buffer 28 mL 55 mL Extraction Solution 50 mL 250 mL 20X W
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