High Sensitivity Direct Cyclic GMP - B
Contents
1. 70 C for later analysis Severely hemolyzed samples should not be used in this kit For samples containing low levels of cGMP the acetylated assay protocol must be used due to its enhanced sensitivity All standards and samples should be diluted in glass test tubes Cells Cell lysis buffers containing high concentrations of SDS or other detergents may not be compatible with this assay or may require extra dilution Please read Interferents section above for more information This kit is compatible with either adherent or non adherent cells The cells can be grown in any suitable sterile containers such as Petri dishes 12 48 or 96 well culture plates or flasks The cells must be isolated from the media prior to being lysed with the provided Sample Diluent The acidic Sample Diluent contains detergents to lyse the cells inactivate endogenous phosphodiesterases and stabilize the cGMP Some cell types are extremely hardy and the end user should optimize the lysis conditions utilizing freeze thaw cycles and ultrasonic treatments to fully lyse their cells For adherent cells the media should be aspirated from the cells and the cells washed with PBS The adherent cells should be treated directly with the Sample Diluent for 10 minutes at room temperature Cells can be scraped to dislodge them from the plate surface and cells should be inspected to ensure lysis Detergent has been added to the Sample Diluent to help lysis occur Centrifuge the sample2. Will Cause Assay To Fail 2 Pipet 75 uL Sample Diluent into the non specific binding NSB wells 3 Pipet 50 uL of Sample Diluent into wells to act as maximum binding wells BO or 0 pg mL 4 Pipet 50 uL of samples or standards into wells in the plate NOTE Sample Diluent will turn from orange to bright pink upon sample or standard addition to the Plate Primer in the wells 5 Add 25 uL of the diluted cGMP Conjugate to each well using a repeater or multichannel pipet 6 Add 25 uL of the cAMP Antibody to each well except the NSB wells using a repeater or multichannel pipet 7 Cover the plate with the plate sealer and shake the plate for 15 minutes at room temperature 7 B Bridge International Inc www b bridge com cGMP 150625 customersupport b bridge com 1 408 252 6200 8 Place the covered plate in a 4 C refrigerator for 16 hours 9 The next morning take the plate from the refrigerator and wash the plate 4 times with the wash buffer Tap the plate dry on absorbent towels 10 Add 100 uL of the mixed Chemiluminescent Substrate to each well using a repeater or multichannel pipet 11 Immediately read the luminescence generated from each well in a mutimode or chemiluminescent plate reader using a 0 1 second read time per well The chemiluminescent signal will decrease about 40 over 60 minutes CALCULATIONS All luminometers read Relative Light Units RLU These RLU readings will vary with make or model of
3. B Bridge International Inc kA High Sensitivity Direct Cyclic GMP Chemiluminescent Immunoassay Kit User Manual Catalog K3020 1 1 Plate Kit K3020 5 5 Plate Kit 1 www b bridge com cGMP 150625 B Bridge International Inc 1 408 252 6200 customersupport b bridge com TABLE OF CONTENTS Intended Use Background Assay Principle Kit Components Materials Required Precautions Interferents Sample Preparation Regular Format Assay Protocol Reagent Preparation Assay Protocol Calculations Typical Standard Curve Validation Data Acetylated Assay Protocol Reagent Preparation gt O O O O DW OO AaAaaeae herk A OOO O Acetylation O Assay Protocol k _ Calculations k Typical Standard Curve _ Validation Data 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 PAR
4. TICULAR 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 cGMP 150625 customersupport b bridge com 1 408 252 6200 INTENDED USE The B Bridge High Sensitivity Direct Cyclic GMP Chemiluminescent Immunoassay Kit is species independent and quantitatively measures cGMP in Cell Lysates Saliva Urine EDTA Plasma and Tissue Culture Media This kit can measure as little as 1 femtomol cGMP per sample Please read the complete kit insert before performing this assay BACKGROUND Guanosine 3 5 cyclic monophosphate cyclic GMP cGMP is a critical and multifunctional second messenger present at levels typically 10 100 fold lower than cAMP in most tissues Intracellular CGMP is formed by the action of the enzyme guanylate cyclase on GTP and degraded through phosphodiesterase hydrolysis Guanylate cyclases GC are either soluble or membrane bound Soluble GCs are nitric oxide responsive whereas membrane bound GCs respond to hormones such as acetylcholine insulin and oxytocin Other chemicals like serotonin and histamine also cause an increase in cGMP levels Cyclic GMP regulates cellular composition through cGMP dependent kinase cGMP dependent ion channels or transporters and through its hydrolytic degradation by phosphodiesterase ASSAY PRINCIPLE The B Bridge Direct High Sensitivity Cyclic GMP CGMP Chemilumi
5. U 10 000 10 o o 0 01 0 1 1 Cyclic GMP pmol mL VALIDATION DATA ACETYLATED FORMAT Sensitivity and Limit of Detection Acetylated Sensitivity was calculated by comparing the RLU s for twenty wells run for each of the acetylated BO and standard 6 The sensitivity was determined at two 2 standard deviations from the BO along the standard curve Sensitivity was determined as 0 023 pmol mL This is equivalent to 1 15 fmol well The Limit of Detection for the assay was determined in a similar manner by comparing the RLU s for twenty runs for each of acetylated zero standard and a low concentration acetylated human sample Limit of Detection was determined as 0 019 pmol mL This is equivalent to 0 95 fmol well 11 B Bridge International Inc www b bridge com cGMP 150625 customersupport b bridge com 1 408 252 6200
6. Working Stock 1 2 3 4 5 6 Sample Diluent 150 ul 585 ul 300 ul 300 ul 300 ul 300 ul 300 ul cGMP Standard Stock Solution 10 ul Working Stock 15 ul Standard 1 300 ul Standard 2 300 ul Standard 3 300 ul Standard 4 300 ul Standard 5 300 ul Final Concentration pM ml 40 1 0 5 0 25 0 125 0 0625 0 03125 STANDARD AND SAMPLE ACETYLATION Pipet 300 uL of Sample Diluent into a glass tube to act as the Zero standard NSB tube 2 Add 15 uL of Acetylation Reagent to this tube and vortex immediately Proceed to assay within 30 minutes 3 Pipet 200 uL of each standard and sample to be tested into glass tubes 4 Add 10 uL of the Acetylation Reagent into each tube and vortex immediately Proceed to assay within 30 minutes NOTE Samples and Sample Diluent will turn from orange to pale yellow upon acetylation ACETYLATED ASSSAY PROTOCOL 1 Add 50 uL of Plate Primer into all wells used Failure To Add Plate Primer To ALL Wells First Will Cause Assay To Fail 2 Pipet 75 uL acetylated Sample Diluent into the non specific binding NSB wells 3 Pipet 50 uL of acetylated Sample Diluent into wells to act as maximum binding wells BO or 0 pg mL 4 Pipet 50 uL of acetylated samples or standards into wells in the plate 5 Add 25 uL of the diluted cGMP Conjugate to each well using a repeater or multichannel pipet 6 Add 25 uL of the cGMP Antibody to each well except the NSB wells using a repeater or multi
7. channel pipet 7 Cover the plate with the plate sealer and shake the plate for 15 minutes at room temperature 8 Place the covered plate in a 4 C refrigerator for 16 hours 9 The next morning take the plate from the refrigerator and wash the plate 4 times with the wash buffer Tap the plate dry on absorbent towels B Bridge International Inc customersupport b bridge com 10 www b bridge com cGMP 150625 1 408 252 6200 10 Add 100 uL of the mixed Chemiluminescent Substrate to each well using a repeater or a multichannel pipet 11 Immediately read the luminescence generated from each well in a mutimode or chemiluminescent plate reader using a 0 1 second read time per well The chemiluminescent signal will decrease about 40 over 60 minutes 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 should be multiplied by the dilution factor to obtain neat sample values ACETYLATED FORMAT TYPICAL STANDARD CURVE EXAMPLE 90 70 000 N RN x ee e E me B BO g 8 g TT E sala A E ee a ee ee AE a a E ed ed ae ee S 8 8 Net RL
8. e and Sample Diluent Immediately vortex each treated standard sample or Sample Diluent after addition of the Acetylation Reagent and use within 30 minutes of preparation Note Upon Acetylation all of the standards and samples diluted in the orange Sample Diluent will change to a pale yellow colour ACETYLATED ASSAY REAGENT PREPARATION We recommend that all standards and samples be run in duplicate to allow the end user to accurately determine cAMP concentrations Ensure that all samples have reached room temperature and have been diluted as appropriate prior to running them in the kit Wash Buffer Dilute Wash Buffer Concentrate 1 20 by adding one part of the concentrate to nineteen parts of deionized water Once diluted this is stable at room temperature for 3 months Cyclic GMP Conjugate Concentrate The supplied Cyclic GMP Conjugate Concentrate should be diluted 1 20 with the Conjugate Diluent ie 125uL Conjugate Concentrate with 2 375 mL of Conjugate Diluent for a total solution volume of 2 5 mL Once diluted the Cyclic GMP conjugate is stable for one month when stored at 4 C Chemiluminescent Substrate Mix one part of the Substrate Solution A with one part of Substrate Solution B in a brown bottle Once mixed the substrate is stable for one month when stored at 4 C Acetylation Reagent Working in a fume hood mix one part of Acetic Anhydride with 2 parts of Triethylamine in a glass test tube Use the following table to help determ
9. ine the amount of Acetylation Reagent to make Use the Acetylation Reagent within 60 minutes of preparation Number of Samples to be Tested Reagent 20 40 100 Acetic Anhydride Volume uL 200 400 1 000 Triethylamine Volume uL 400 800 2 000 Acetylation Reagent Volume mL 06 12 3 Standard Preparation All standards and samples should be diluted in glass test tubes 1 Label one glass test tube as Working Stock and six tubes as 1 through 6 2 Pipet 150 uL of Sample Diluent into the Working Stock tube and 585 uL of Sample Diluent into tube 1 3 Pipet 300 uL of Sample Diluent into tubes 2 to 6 9 B Bridge International Inc www b bridge com cGMP 150625 customersupport b bridge com 1 408 252 6200 4 The Cyclic GMP stock solution contains an organic solvent Prerinse the pipet tip several times to ensure accurate delivery Carefully add 10 uL of the cGMP stock solution to the Working Stock tube and vortex completely 5 Take 15 uL of the cGMP solution from the Working Stock tube and add it to tube 1 and vortex completely 6 Take 300 uL of the cGMP solution in tube 1 and add it to tube 2 and vortex completely 7 Repeat the serial dilutions for tubes 3 through 6 The concentration of Cyclic GMP in tubes 1 through 6 will be 1 0 5 0 25 0 125 0 0625 and 0 0313 pmol mL Standard Standard Standard Standard Standard Standard Reagent
10. nescent Immunoassay kit is designed to quantitatively measure cGMP present in Cell Lysates Saliva Urine EDTA Plasma and Tissue Culture Media For samples where the levels of cGMP are expected to be relatively high the regular format for the assay can be used For samples with expected low levels of cGMP an optional acetylation protocol can be used The kit is unique in that all samples and standards are diluted into an acidic Sample Diluent which contains special additives and stabilizers for CGMP measurement This allows plasma urine and saliva samples to be read in an identical manner to lysed cells Acidified samples of cGMP are stable and endogenous phosphodiesterases are inactivated in the Sample Diluent 1 Plate primer added to microtiter plate wells followed by sample or standard 2 The reaction is initiated with the addition of the c MP peroxidase conjugate and chemiluminescent substrate 3 Incubate overnight and use plate reader to ascertain luminescence 4 Calculate cGMP concentration from standard curve 3 B Bridge International Inc www b bridge com cGMP 150625 customersupport b bridge com 1 408 252 6200 KIT COMPONENTS Component Cat K3020 1 K3020 5 Coated white 96 well plate 1 plate 5 plates Cyclic GMP Standard 640 pmol mL 70 ul 70 ul Cyclic GMP CLIA Antibody 3 ml 13 ml Cyclic AMP Conjugate CLIA Concentrate 150 ul 650 ul Conjugate Diluent 3 ml 13 ml Sample Diluent Concentrate 12 ml 60 ml WARNING Caus
11. nless steel mortar under liquid nitrogen until it is a fine powder Allow the liquid nitrogen to evaporate and weigh the powdered tissue Add 1 mL of ice cold 5 TCA weight volume for every 100 mg of tissue and grind in a glass Teflon mortar Incubate in the TCA for 10 minutes on ice and then centrifuge at 2600 x g at 4 C for 15 minutes Collect the supernatant For every 1 mL of TCA supernatant add 3 mL of water saturated diethyl ether and shake in a glass vial Allow the ether to separate as the top layer remove it and discard the ether Dry the aqueous layer by lyophilization or using a vacuum centrifuge Reconstitute by adding 1 mL of Sample Diluent for every mL of 5 TCA used to extract and run in the assay immediately or store at lt 70 C Diethyl ether is extremely flammable and should be used in a hood Tissue Culture Media For measuring cGMP in tissue culture media TCM samples should be read off a standard curve generated in TCM Samples may need to be diluted further in TCM We have validated the assay using RPMI 1640 Plasma Samples Plasma samples should be diluted 2 1 10 with the supplied Sample Diluent and acetylated prior to running in the Acetylated Format assay Urine Samples Urine samples should be diluted 1 20 with the supplied Sample Diluent prior running in the assay Due to the high concentration of cGMP in urine samples may need to be diluted further Saliva Samples Saliva samples should be diluted 2 1 4 with
12. 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 should be multiplied by the dilution factor to obtain neat sample values REGULAR FORMAT TYPICAL STANDARD CURVE EXAMPLE B BO Net RLU Pi GMP Conc Pa VALIDATION DATA REGULAR FORMAT Sensitivity and Limit of Detection Sensitivity was calculated by comparing the RLU s for twenty wells run for each of the BO and standard 7 The sensitivity was determined at two 2 standard deviations from the BO along the standard curve Sensitivity was determined as 0 034 pmol mL 8 B Bridge International Inc www b bridge com cGMP 150625 customersupport b bridge com 1 408 252 6200 The Limit of Detection for the assay was determined in a similar manner by comparing the RLU s for twenty runs for each of the zero standard and a low concentration human urine sample Limit of Detection was determined as 0 047 pmol mL ACETYLATED ASSAY Use this format for any sample with low cGMP concentrations Prior to running the acetylated assay all standards samples and the Sample Diluent used for the BO and NSB wells must be acetylated Acetylation is carried out by adding 10 uL of the Acetylation Reagent as prepared below for each 200 uL of the standard sampl
13. s at 2600 x g at 4 C for 15 minutes and assay the supernatant directly If required the TCM can be assayed for cGMP as outlined below For non adherent cells pellet and wash the cells with PBS by centrifuging the samples at 2600 x g at 4 C for 15 minutes as described above Treat the aspirated washed pellet directly with the Sample Diluent for 10 minutes at room temperature Cells should be inspected to ensure lysis Detergent has been added to the Sample Diluent to help lysis occur Centrifuge the samples at 2600 x g at 4 C for 15 minutes and assay the supernatant directly If required the TCM can be assayed for cGMP as outlined below 5 B Bridge International Inc www b bridge com cGMP 150625 customersupport b bridge com 1 408 252 6200 Tissue Samples Tissues samples should be frozen in liquid nitrogen and stored at 80 C if analysis is not to be carried out immediately Grind the frozen tissue in a stainless steel mortar under liquid nitrogen until it is a fine powder Allow the liquid nitrogen to evaporate and weigh the powdered tissue Add 1 mL of Sample Diluent for every 100 mg of tissue Incubate in the Sample Diluent for 10 minutes on ice and then centrifuge at 2600 x g at 4 C for 15 minutes Collect the supernatant and run in the assay immediately or store frozen at lt 70 C For samples that require concentration and delipidation a trichloroacetic acid TCA ether protocol can be used Grind the frozen tissue in a stai
14. the supplied Sample Diluent prior running in the assay Use all samples within 2 hours of dilution in Sample Diluent REGULAR FORMAT ASSAY REGULAR FORMAT REAGENT PREPARATION Allow the kit reagents to come to room temperature for 30 60 minutes We recommend that all standards and samples be run in duplicate to allow the end user to accurately determine cAMP concentrations Ensure that all samples have reached room temperature and have been diluted as appropriate prior to running them in the kit Wash Buffer Dilute Wash Buffer Concentrate 1 20 by adding one part of the concentrate to nineteen parts of deionized water Once diluted this is stable at room temperature for 3 months Cyclic GMP Conjugate Concentrate The supplied Cyclic GMP Conjugate Concentrate should be diluted 1 20 with the Conjugate Diluent ie 125uL Conjugate Concentrate with 2 375 mL of Conjugate Diluent for a total solution volume of 2 5 mL Once diluted the Cyclic GMP conjugate is stable for one month when stored at 4 C Chemiluminescent Substrate 6 B Bridge International Inc www b bridge com cGMP 150625 customersupport b bridge com 1 408 252 6200 Mix one part of the Substrate Solution A with one part of Substrate Solution B in a brown bottle Once mixed the substrate is stable for one month when stored at 4 C Standard Preparation All standards and samples should be diluted in glass test tubes 1 Label one glass test tube as Working Stock and seven
15. tic Plate Primer 25 ml 25 ml Acetic Anhydride 2 ml 2 ml WARNING Corrosive Lachrymator Triethylamine 4ml 4ml WARNING Corrosive Lachrymator 20X Wash Buffer Concentrate 30 ml 125 ml Substrate Solution A 6 ml 28 ml Substrate Solution B 6 ml 28 ml Plate Sealer 1 each 5 each Store all components at 4 C MATERIALS REQUIRED BUT NOT SUPPLIED Deionized or distilled water Microplate Shaker Glass test tubes 96 well plate reader capable of reading glow chemiluminescence Software for converting raw relative light unit RLU readings from the plate reader and carrying out four parameter logistic curve 4PLC fitting Note Dilute 5 uL of the Cyclic GMP CLIA Conjugate Concentrate into 495 uL of deionized water Pipet 5 uL of diluted conjugate into a white well and add 100 uL of prepared CLIA substrate This well will give you an intensity slightly above the maximum binding for the assay Adjust the gain or sensitivity so that your reader is giving close to the maximum signal 4 www b bridge com cGMP 150625 1 408 252 6200 B Bridge International Inc customersupport b bridge com PRECAUTIONS This kit should only be used by qualified personnel who have had laboratory safety instruction The complete User Manual should be read and understood before using this product Sodium azide in buffers will irreversibly inhibit the activity of peroxidase enzymes Samples in any buffer containing azide will need to be diluted s
16. tubes as 1 through 7 2 Pipet 150 uL of Sample Diluent into the Working Stock tube and 296 uL of Sample Diluent into tube 1 Pipet 150 uL of Sample Diluent into tubes 2 to 7 3 4 The Cyclic GMP stock solution contains an organic solvent Prerinse the pipet tip several times to ensure accurate delivery Carefully add 10 uL of the cGMP stock solution to the Stock 2 tube and vortex completely 5 Take 24 uL of the cGMP solution in the Working Stock tube and add it to tube 1 and vortex completely Take 150 uL of the cGMP solution in tube 1 and add it to tube 2 and vortex completely ND through 7 will be 3 1 5 0 75 0 375 0 188 0 094 and 0 047pmol mL Use Standards within 1 hour of preparation Repeat the serial dilutions for tubes 3 through 7 The concentration of Cyclic GMP in tubes 1 Working Standard Standard Standard Standard Standard Standard Reagent Stock 1 2 3 4 5 6 Standard Y Sample Diluent 150ul 296l 150l 150l 150l 150p 1504 150 ul cGMP Standard Stock Solution 10 ul Working Stock 24 ul Standard 1 150 ul Standard 2 150 ul Standard 3 150 ul Standard 4 150 ul Standard 5 150 ul Standard 6 150 ul Final Concentration pM ml 40 3 1 5 0 75 0 375 0 1875 0 0938 0 0469 REGULAR FORMAT ASSAY PROTOCOL 1 Add 50 uL of Plate Primer into all wells used Failure To Add Plate Primer To ALL Wells First
17. ubstantially typically gt 1 100 The supplied Sample Diluent and Sample Diluent Concentrate are acidic Take appropriate precautions when handling these reagents The kit uses acetic anhydride and triethylamine as acetylation reagents Triethylamine and acetic anhydride are lachrymators Caution corrosive flammable and harmful vapor Use in hood with proper ventilation and wear appropriate protective safety wear In all cases please consult your institution s safety procedures for working with hazardous chemicals INTERFERENTS A variety of detergents were tested as possible interfering substances in the assay CHAPS and Tween 20 at 0 1 increased measured cAMP by 8 9 and decreased measured cAMP by 0 9 respectively Triton X 100 at 2 increased measured cAMP by 1 8 and CTAC at 0 05 increased measured cAMP by 6 3 Samples containing SDS above 0 01 should not be used in the assay SAMPLE PREPARATION Samples can be used within 2 hours or preparation or stored long term at 70 C 24 Hour urine samples may need to have 1 mL concentrated hydrochloric acid added for every 100 mL volume to act as a preservative Samples containing visible particulate should be centrifuged prior to using After dilution in the Sample Diluent see page 9 there may be some precipitation of proteins and the supernatant from the centrifuged samples used After being diluted in Sample Diluent the samples can be assayed directly within 2 hours or frozen at lt
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