Cyclic GMP (cGMP) ELISA Kit

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Antigen
Reactivity
Chemical
Alternatives
Kits with alternative reactivity to:
21
2
1
1
1
1
1
1
1
1
1
1
1
1
Method Type
Sandwich ELISA
Minimum Detection Limit
<1 fM cGMP/Sample
Application
ELISA
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Purpose The DetectX® Direct High Sensitivity Cyclic GMP (cGMP) Chemiluminescent Immunoassay kit isdesigned to quantitatively measure cGMP present in lysed cells, EDTA plasma, urine, saliva andtissue culture media samples.
Brand DetectX®
Sample Type Cell Lysate, Saliva, Urine, Plasma (EDTA), Tissue Culture Medium
Analytical Method Quantitative
Detection Method Chemiluminescent
Specificity Species Independent. Samples Types validated: Cell Lysates, Saliva, Urine, EDTA and Heparin Plasma, Tissue Culture Media
Cross-Reactivity (Details) (%) Cyclic GMP 100 % Cyclic AMP < 0.1 % GMP < 0.1 % AMP < 0.1 % ATP < 0.1 %
Sensitivity 0.034 pmol/mL
Characteristics The Direct Cyclic GMP (cGMP) Chemiluminescent Immunoassay kits will measure cGMP present in lysed cells, EDTA plasma, urine, saliva and culture media samples. 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. Acidified samples of cGMP are stable and endogenous phosphodiesterases are inactivated in the Sample Diluent. After an overnight incubation at 4°C, the plate is washed and the chemiluminescent substrate is added. The substrate generates light which is detected in a multi-label microtiter plate reader capable of reading luminescence. Cyclic GMP 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.
Components Coated White 96 Well Plates A white plastic microtiter plate(s) with break-apart strips coated with goat anti-mouse IgG. 1 Or 5 Each
Cyclic GMP Standard 70 μL Cyclic GMP at 640 pmol/mL in a special stabilizing solution.
DetectX® Cyclic GMP CLIA Antibody A mouse monoclonal antibody specific for cyclic GMP. 3 mL Or 13 mL
DetectX® Cyclic GMP CLIA Conjugate Stock A cyclic GMP-peroxidase conjugate concentrate in a special stabilizing solution. 150 μL Or 650 μL
Conjugate Diluent Contains special stabilizers and additives. 3 mL Or 13 mL
Sample Diluent Concentrate A 4X concentrate containing stabilizers and additives that must be diluted with deionized or distilled water. CAUSTIC 12 mL Or 60 mL
Plate Primer 25 mL A neutralizing solution containing special stabilizers and additives.
Acetic Anhydride 2mL Acetic Anhydride WARNING: Corrosive Lachrymator Triethylamine 4mL Triethylamine WARNING: Corrosive Lachrymator Wash Buffer Concentrate A 20X concentrate that must be diluted with deionized or distilled water. 30 mL Or 125 mL
Substrate Solution A 6 mL Or 28 mL
Substrate Solution B 6 mL Or 28 mL
Plate Sealer 1 Or 5 Each
Material not included Distilled or deionized water.
Repeater pipet and tips capable of dispensing 25 and 100 μL.
Glass test tubes.
Microplate shaker. 96 well microplate reader capable of reading glow chemiluminescence.
A list of some models of suitable readers can be found on our website at www.ArborAssays.com/resources/#general-info.
All luminometers read Relative Light Units (RLU).
These RLU readings will vary with make or model of plate reader.
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 μL of the Cyclic GMP CLIA Conjugate Concentrate into 1.495 mL of deionized water.
Pipet 5 μL of diluted conjugate into 45 μL of deionized water.
Pipet 5 μL of the mixture into a white well and add 100 μL of prepared CLIA substrate (see page 9 for details).
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.
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.
Alternative Name Cyclic GMP
Background Guanosine 3', 5'-cyclic monophosphate (cyclic GMP, cGMP) is a critical and multifunctional sec- ond messenger present at levels typically 10-100 fold lower than cAMP in most tissues. Intra- cellular cGMP is formed by the action of the enzyme guanylate cyclase on GTP and degraded through phosphodiesterase hydrolysis1-3. Guanylate cyclases (GC) are either soluble or membrane bound3,4. Soluble GCs are nitric oxide responsive, whereas membrane bound GCs respond to hor- mones such as acetylcholine, insulin and oxytocin. Other chemicals like serotonin and histamine also cause an increase in cGMP levels5,6. Cyclic GMP regulates cellular composition through cGMP- dependent kinase, cGMP-dependent ion channels or transporters, and through its hydrolytic deg- radation by phosphodiesterase1,7
Application Notes This assay has been validated for lysed cells, saliva, urine, EDTA plasma, tissue samples, and for tis- sue culture media samples.
Samples should be stored at -70 °C for long term storage. 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.
Cyclic GMP is identical across all species and we expect this kit may measure cGMP from sources other than human.
The end user should evaluate recoveries of cGMP in other samples being tested.
After dilution in the Sample Diluent (see page 9) there may be some precipitation of proteins and the supernatant from the centrifuged samples should be used.
After being diluted in Sample Diluent the samples can be assayed directly within 2 hours, or frozen at ≤ -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.
Comment

Sample values: Four human plasma samples were tested in the assay.
Samples were diluted 10-20 fold and run in the assay.
Values ranged from 3.0 to 8.0 pmol/mL with an average for the samples of 5.48 pmol/mL.
Five normal human urine samples were diluted between 50 and 2,000 fold in Sample Diluent and values ranged in the neat samples from 44.2 to 564 pmol/mL with an average for the samples of 287.2 pmol/mL

Plate Pre-coated
Protocol 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.
A cGMP standard is provided to generate a standard curve for the assay and all samples should be read off the standard curve.
A white microtiter plate coated with an antibody to capture mouse IgG is provided and a neutralizing Plate Primer solution is added to all the used wells.
Standards or diluted samples, either with or without acetylation, are pipetted into the primed wells.
A cGMP- peroxidase conjugate is added to the standards and samples in the wells.
The binding reaction is initiated by the addition of a mouse monoclonal antibody to cGMP to each well.
After an over- night incubation at 4 °C, the plate is washed and the chemiluminescent substrate is added.
The substrate reacts with the bound cGMP-peroxidase conjugate to produce light The generated light is detected in a microtiter plate reader capable of reading luminescence.
The concentration of the cGMP in the sample is calculated, after making suitable correction for the dilution of the sample, using software available with most plate readers.
Reagent Preparation

Allow the kit reagents to thaw and 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 cGMP 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.
Sample Diluent Prepare the Sample Diluent by diluting the Sample Diluent Concentrate 1:4, adding one part of the concentrate to three parts of deionized water.
Once diluted this is stable at 4 °C for 3 months.
Cyclic GMP Conjugate The supplied Cyclic GMP CLIA Conjugate Concentrate should be diluted 1:20 with the Conjugate Diluent as indicated in the table below.
Once diluted the Cyclic GMP conjugate is stable for one month when stored at 4 °C. 1 Plate 2 Plates 3 Plates 4 Plates 5 Plates Conjugate Concentrate 125 μL 250 μL 375 μL 500 μL 625 μL Conjugate Diluent 2.375 mL 4.75 mL 7.125 mL 9.5 mL 11.875 mL Final Mixture 2.5 mL 5 mL 7.5 mL 10 mL 12.5 mL 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. ® www.ArborAssays.com 9 reagent preparatiOn - regular fOrmat All standards and samples should be diluted in glass test tubes.
Standard Preparation - regular fOrmat Label one glass test tube as Stock 2 and seven tubes as #1 through #7.
Pipet 150 μL of Sample Diluent into the Stock 2 tube and 296 μL of Sample Diluent into tube #1.
Pipet 150 μL of Sample Diluent into tubes #2 to #7.
The Cyclic GMP stock solution contains an organic solvent.
Prerinse the pipet tip several times to ensure accurate delivery.
Carefully add 10 μL of the cGMP stock solution to the Stock 2 tube and vortex completely.
Take 24 μL of the cGMP solution in the Stock 2 tube and add it to tube #1 and vortex completely.
Take 150 μL of the cGMP solution in tube #1 and add it to tube #2 and vortex completely.
Repeat the serial dilutions for tubes #3 through #7.
The concentration of Cyclic GMP in tubes 1 through 7 will be 3, 1.5, 0.75, 0.375, 0.188, 0.094, and 0.047 pmol/mL.
Non-Acetylated Stock 2 Std 1 Std 2 Std 3 Std 4 Std 5 Std 6 Std 7 Sample Diluent (μL) 150 296 150 150 150 150 150 150 Addition Cyclic Stock Std 1 Std 2 Std 3 Std 4 Std 5 Std 6 GMP Std. 2 Vol of Addition (μL) 10 24 150 150 150 150 150 150 Final Conc (pM/ mL) 40 3 1.5 0.75 0.375 0.1875 0.0938 0.0469 Use Standards within 1 hour of preparation. ® www.ArborAssays.com 10 aSSay prOtOcOl - regular fOrmat 1.
Use the plate layout sheet on the back page to aid in proper sample and standard identification.
Determine the number of wells to be used and return unused wells to the foil pouch with desiccant.
Seal the ziploc plate bag and store at 4°C. 2.
Add 50 μL of Plate Primer into all wells used. failure tO add plate primer tO all WellS firSt Will cauSe aSSay tO fail. 3.
Pipet 75 μL Sample Diluent into the non-specific binding (NSB) wells. 4.
Pipet 50 μL of Sample Diluent into wells to act as maximum binding wells (B0 or 0 pmol/ mL). 5.
Pipet 50 μL 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. 6.
Add 25 μL of the diluted DetectX® cGMP CLIA Conjugate to each well using a repeater pipet. 7.
Add 25 μL of the DetectX® cGMP CLIA Antibody to each well, except the NSB wells, using a repeater pipet. 8.
Cover the plate with the plate sealer and shake the plate for 15 minutes at room temperature. 9.
Place the covered plate in a 4 °C refrigerator for 16 hours. 10.
The following day remove the Chemiluminescent Substrate from the refrigerator and allow to come to room temperature for at least 30 minutes.
Addition of cold Substrate will cause depressed signal. 11.
Take the plate from the refrigerator and wash each well 4 times with 300 μL wash buffer.
Tap the plate dry on clean absorbent towels. 12.
Add 100 μL of the mixed Chemiluminescent Substrate to each well, using a repeater pipet. 13.
Incubate the plate at room temperature for 5 minutes without shaking. 14.
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. 15.
Use the plate reader's built-in 4PLC software capabilities to calculate cGMP concentration for each sample.

Sample Preparation

Cells Cell lysis buffers containing high concentrations of SDS or other detergents may not be compat- ible with this assay or may require extra dilution. Please read Interferents section on page 22 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. We used ~ 107 Jurkat cells per mL of Sample Diluent. Cell number needs to be determined by the end user since it will be dependant on cell type and treatment conditions. Care must be taken not to over dilute the samples. 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 samples at ≥600 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 ≥600 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 ≥600 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.

Calculation of Results

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.
Cre- ate 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/B0 curve, should be multiplied by the dilution factor to obtain neat sample values.
Or use the online tool from http://www.myassays.com/arbor-assays-cyclic-gmp-direct-chemiluminescent-kit-non-acetyl.assay to calculate the data. *The MyAssays logo is a registered trademark of MyAssays Ltd. typical data - regular fOrmat Sample Mean RLU Net RLU % B/B0 Cyclic GMP Conc. (pmol/mL) NSB 12,945 0 - - Standard 1 22,530 9,585 13.52 3 Standard 2 29,605 16,660 23.51 1.5 Standard 3 37,480 24,535 34.62 0.75 Standard 4 48,805 35,860 50.60 0.375 Standard 5 60,930 47,985 67.70 0.1875 Standard 6 71,465 58,520 82.57 0.0938 Standard 7 76,600 63,655 89.81 0.0469 B0 83,820 70,875 100 0 Sample 1 26,655 13,710 19.34 1.86 Sample 2 40,695 27,750 39.15 0.62 Always run your own standard curve for calculation of results. ® www.ArborAssays.com 12 Typical Standard Curve - Regular Format 100 70,000 90 80 60,000 70 50,000 60 40,000 50 %B/B0 Net RLU 40 30,000 30 20,000 20 10,000 10 0 0 0.01 0.1 1 10 cGMP Conc. (pmol/mL) Always run your own standard curve for calculation of results.
Do not use this data.
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 B0 and standard #7.
The sensitivity was determined at two (2) standard deviations from the B0 along the standard curve.
Sensitivity was determined as 0.034 pmol/mL.
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 ® www.ArborAssays.com 13 %B/B0 Net RLU acetylated prOtOcOl - OVerVieW 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 B0 and NSB wells must be acetylated.
Acetylation is carried out by adding 10 μL of the Acetyla- tion Reagent (as prepared below) for each 200 μL of the standard, sample and Sample Diluent.
Immediately vortex each treated standard, sample or Sample Diluent after addition of the Acetyla- tion 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. reagent preparatiOn - acetylated fOrmat 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 determine the amount of Acetylation Reagent to make.
Reagents Number of Samples to be Tested 20 40 100 200 Acetic Anhydride Volume (μL) 200 400 1,000 2,000 Triethylamine Volume (μL) 400 800 2,000 4,000 Acetylation Reagent Vol (mL) 0.6 1.2 3 6 Use the Acetylation Reagent within 60 minutes of preparation. ® www.ArborAssays.com 14 Standard Preparation - Acetylated Format All standards and samples should be diluted in glass test tubes.
Label one glass test tube as Stock 2 and six tubes as #1 through #6.
Pipet 150 μL of Sample Diluent into the Stock 2 tube and 585 μL of Sample Diluent into tube #1.
Pipet 300 μL of Sample Diluent into tubes #2 to #6.
The Cyclic GMP stock solution contains an organic solvent.
Prerinse the pipet tip several times to ensure accurate delivery.
Carefully add 10 μL of the cGMP stock solution to the Stock 2 tube and vortex completely.
Take 15 μL of the cGMP solution in the Stock 2 tube and add it to tube #1 and vortex completely.
Take 300 μL of the cGMP solution in tube #1 and add it to tube #2 and vortex completely.
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.
Acetylated Stock 2 Std 1 Std 2 Std 3 Std 4 Std 5 Std 6 Sample Diluent (μL) 150 585 300 300 300 300 300 Addition Cyclic Stock 2 Std 1 Std 2 Std 3 Std 4 Std 5 GMP Std.
Vol of Addition (μL) 10 15 300 300 300 300 300 Final Conc (pM/mL) 40 1 0.5 0.25 0.125 0.0625 0.03125 Standard and Sample Acetylation Pipet 300 μL of Sample Diluent into a glass tube to act as the Zero standard/NSB tube.
Add 15 μL of Acetylation Reagent to this tube and vortex immediately.
Proceed to assay within 30 minutes.
Pipet 200 μL of each standard or sample to be tested into fresh glass tubes.
Add 10 μL 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.
Use Acetylated Standards and Samples within 30 minutes of preparation.

Restrictions For Research Use only
Precaution of Use • As with all such products, this kit should only be used by qualified personnel who have had labo- ratory safety instruction.
The complete insert 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 using the supplied Wash Buffer as prepared on Page 8. • The Sample Diluent Concentrate is acidic.
Take appropriate precautions when handling the reagent.
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.
Storage 4 °C
Storage Comment All components of this kit should be stored at 4°C until the expiration date of the kit.
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Product cited in: Miwa, Koseki, Oshima, Hattori, Kase, Kondo, Fukui, Tomita, Ohda, Watari: "Impairment of gastric accommodation induced by water-avoidance stress is mediated by 5-HT2B receptors." in: Neurogastroenterology and motility : the official journal of the European Gastrointestinal Motility Society, Vol. 28, Issue 5, pp. 765-78, 2016 (PubMed).

Cortese-Krott, Kuhnle, Dyson, Fernandez, Grman, DuMond, Barrow, McLeod, Nakagawa, Ondrias, Nagy, King, Saavedra, Keefer, Singer, Kelm, Butler, Feelisch: "Key bioactive reaction products of the NO/H2S interaction are S/N-hybrid species, polysulfides, and nitroxyl." in: Proceedings of the National Academy of Sciences of the United States of America, Vol. 112, Issue 34, pp. E4651-60, 2015 (PubMed).