Direct camp ELISA Kit

Transcription

1 ab Direct camp ELISA Kit Instructions for Use For the quantitative determination of cyclic AMP in cells, tissue, and culture supernatants treated with HCl. This product is for research use only and is not intended for diagnostic use.

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3 Table of Contents 1. Introduction 3 2. Principle of the Assay 4 3. Assay Summary 5 4. Kit Contents 7 5. Storage and Handling 8 6. Additional Materials Required 8 7. Protocol 9 8. Calculation of Results Performance Characteristics Trouble shooting 26 2

4 1. Introduction ab is a competitive immunoassay for the quantitative determination of camp in cells, tissue, and culture supernatants treated with HCl. The optional acetylated assay format provides an approximate 10 fold increase in sensitivity and is ideal for samples with extremely low levels of camp. If expected levels of camp are unknown, the investigator may evaluate a few samples in the non acetylated format in order to determine if higher sensitivity is required. Adenosine 3, 5 cyclic monophosphate (cyclic AMP; camp) is one of the most important second messengers involved as a modulator of physiological processes. camp is also involved in regulating neuronal, glandular, cardiovascular, immune and other functions. A number of hormones are known to activate camp through the action of the enzyme adenylate cyclase which converts ATP to camp. These hormones include a variety of anterior pituitary peptide hormones such as corticotropin (ACTH), glucagon, calcitonin, thyroid stimulating hormone (TSH), and luteinizing hormone (LH). Because camp has been shown to be involved in the cardiovascular and nervous systems, immune mechanisms, cell growth and differentiation, and general metabolism, there remains considerable interest in the measurement of intracellular camp in tissues and cell cultures. The investigation of camp may help to provide a clearer understanding of the physiology and pathology of many disease states. 3

5 2. Principle of the Assay Standards and samples are added to wells coated with a GxR IgG antibody. A blue solution of camp conjugated to alkaline phosphatase is then added, followed by a yellow solution of rabbit polyclonal antibody to camp. During a simultaneous incubation at room temperature the antibody binds, in a competitive manner, the camp in the sample or conjugate. The plate is washed, leaving only bound camp. pnpp substrate solution is added. The substrate generates a yellow color when catalyzed by the alkaline phosphatase on the camp conjugate. Stop solution is added. The yellow color is read at 405nm. The amount of signal is indirectly proportional to the amount of camp in the sample. 4

6 3. Assay Summary Refer to the Assay Layout Sheet to determine the number of wells to be used Pipette 50 μl of Neutralizing Reagent into each well except the Total Activity (TA) and Blank wells. Pipette 100 μl of the 0.1M HCL into the NSB (Non Specific Standard) and Bo (0 pmol/ml Standard) wells. Pipette 50 μl of the 0.1M HCL into the NSB wells. Pipette 100 μl of Standards #1 through #5 into the appropriate wells. Pipette 100 μl of the Samples into the appropriate wells Pipette 50 μl of blue Conjugate into each well, except the Total Activity (TA) and Blank wells. Pipette 50 μl of yellow Antibody into each well, except the Blank, TA and Non Specific Standard wells. Incubate the plate at room temperature on a plate shaker for 2 hours at ~500 rpm. Empty the contents of the wells and wash by adding 400 μl of wash solution to every well. Repeat the wash 2 more times for a total of 3 washes. 5

7 After the final wash, empty or aspirate the wells, and firmly tap the plate on a lint free paper towel to remove any remaining wash buffer. Add 5 μl of the blue Conjugate to the TA wells. Add 200 μl of the Substrate solution to every well. Incubate at room temperature for 1 hour without shaking. Add 50 μl 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. 6

8 4. Kit Contents Item Description Quantity Goat anti-rabbit IgG Microtiter Plate camp Conjugate camp ELISA Antibody Neutralizing Reagent Wash Buffer Concentrate camp Standard A plate using breakapart strips coated with goat anti-rabbit polyclonal antibody A blue solution of camp conjugated to alkaline phosphatase A yellow solution of a rabbit polyclonal antibody to camp Tris buffered saline containing detergents A solution of 2,000 pg/ml camp. pnpp Substrate A solution of p- nitrophenyl phosphate in buffer. Stop Solution A solution of trisodium phosphate in water. Keep tightly capped 0.1 M HCl 0.1 M hydrochlorid acid in water. Triethylamine Acetic Anhydride 1x 96 wells 5 ml 5 ml 5 ml 27 ml 0.5 ml 20 ml 5 ml 27 ml Plate Sealer 1 2 ml 1 ml 7

9 5. Storage and Handling All components of this kit, except the Conjugate and Standard, are stable at 4 C. The Conjugate and Standard must be stored at -20 C. 6. Additional Materials Required Deionized or distilled water. Precision pipettes for volumes between 5 μl and 1,000 μl. Repeater pipettes for dispensing 50 μl and 200 μl. Disposable beaker for diluting buffer concentrates. Graduated cylinders. A microplate shaker. Lint free paper toweling for blotting Triton X-100 (optional for sample preparation) Liquid nitrogen, mortar & pestle, and concentrated HCl (optional - for tissue samples) Microplate reader capable of reading at 405 nm 8

10 7. Protocol A. Sample Handling Treatment of cells and tissue with HCl will stop endogenous phosphodiesterase activity and allow for the direct measurement of these samples in the assay without evaporation or further processing. Recommended treatment protocols follow. Samples containing rabbit IgG will interfere with the assay. Culture supernatants may be run directly in the assay after treatment with concentrated HCl (not the 0.1M HCl supplied). Add 10 μl concentrated HCl to each 1 ml of culture media to be assayed. Centrifuge at 600 x g at room temperature. Please note that some samples may contain high levels of camp and additional dilution may be required. Samples with low levels of camp may be assayed in the acetylated format or the samples may be concentrated. Protocol for Cell Lysates The concentration of cells used must be optimized for the specific cell line and treatment conditions. Cells may be grown in typical containers such as Petri dishes, culture plates (e.g., 48 well, 12 well, or 96 well), culture flasks, etc. Some cells are particularly hardy (e.g., bacteria) and may require the addition of 0.1 to 1% Triton X 100 to the 0.1M HCl for enhanced lysis. If Triton X 100 is added to 9

11 samples it should also be added to the standard dilution as a modest increase in optical density may occur. 1. Pellet suspension cells and aspirate the media. Treat cells with 0.1M HCl. A general starting concentration of 1 x 106 cells per ml of 0.1M HCl is recommended. Remove the media from adherent cells and add enough 0.1M HCl to cover the bottom of the plate. Avoid over diluting the sample with an excessive volume of HCl. Please note that the culture media may be saved and assayed separately, if desired. 2. Incubate the cells in 0.1M HCl for 10 minutes at room temperature. 3. Inspect the cells under a microscope to ensure uniform lysis. Continue incubating for an additional 10 minutes, if necessary. 4. Centrifuge 600 x g to pellet the cellular debris. 5. The supernatant may be assayed immediately or stored frozen for later analysis. Note: Standards must be diluted in 0.1 M HCl and Neutralizing Reagent used. 10

12 Protocol for Tissue Samples 1. After collection, tissue samples should be flash frozen in liquid nitrogen. If analysis cannot be carried out immediately, store tissue at 80 C. 2. Grind frozen tissue to a fine powder under liquid nitrogen in a stainless steel mortar. 3. When liquid nitrogen has evaporated, weigh the frozen tissue and homogenize in 10 volumes of 0.1M HCl (e.g., 0.1 g of tissue should be homogenized in 1 ml of 0.1M HCl). 4. Centrifuge 600 x g to pellet the debris (~10 minutes). 5. The supernatant may be further diluted in the 0.1M HCl provided and run directly in the assay or stored frozen for later analysis. Note: Standards must be diluted in 0.1 M HCl and Neutralizing Reagent used. 11

13 B. Reagent Preparation 1. Wash Buffer Prepare the Wash Buffer by diluting 5 ml of the supplied concentrate with 95 ml of deionized water. This can be stored at room temperature for 3 months. 2. camp standard non-acetylated format Allow the 2,000 pmol/ml standard solution to warm to room temperature. Label five 12 x 75 mm glass tubes #1 through #5. Pipette 900 μl of the 0.1M HCl into tube #1. Pipette 750 μl of standard diluent into tubes #2 through #5. Add 100 μl of the 2,000 pmol/ml standard to tube #1. Vortex thoroughly. Add 250 μl of tube #1 to tube #2 and vortex thoroughly. Add 250 μl of tube #2 to tube #3 and vortex. Continue this for tubes #4 through #5. Diluted standards should be used within 60 minutes of preparation 3. Acetylation Reagent (optional) Prepare the Acetylating Reagent by adding 0.5 ml of Acetic Anhydride to 1 ml of Triethylamine. Note that this volume is sufficient to add to 30 ml of diluted standards and samples. Use the prepared reagent within 60 minutes of preparation. Discard any unused portion of the Acetylating Reagent. 12

14 4. camp Standard, acetylated format (optional) Allow the 2,000 pmol/ml standard solution to warm to room temperature. Label five 12 x 75 mm glass tubes #1 through #5. Pipette 990 μl of the 0.1M HCl into tube #1. Pipette 750 μl of standard diluent into tubes #2 through #5. Add 10 μl of the 2,000 pmol/ml standard to tube #1. Vortex thoroughly. Add 250 μl of tube #1 to tube #2 and vortex thoroughly. Add 250 μl of tube #2 to tube #3 and vortex. Continue this for tubes #4 through #5. Acetylate all standards and samples by adding 10 μl of the Acetylating Reagent for each 200 μl of the standard or sample. Add the Acetylating Reagent directly to the diluted standard or sample and vortex immediately after the addition of the Acetylating Reagent. Label one 12mm x 75mm tube as the 0 pg/ml Standard/Non Specific Standard tube. Pipette 1 ml of the 0.1M HCl into this tube. Add 50 μl of the Acetylating Reagent to the 0 pg/ml Standard/Non Specific Standard tube and use in Steps 2 and 3 of the Assay Procedure. The acetylated standards and samples should be used within 30 minutes of preparation. 13

15 C. Assay Procedure Refer to the Assay Layout Sheet to determine the number of wells to be used. Remove the wells not needed for the assay and return them, with the desiccant, to the mylar bag and seal. Store unused wells at 4 C.Note: If the acetylated format of the assay is to be run, all standards, samples, and the diluent for the NSB and Bo wells must be acetylated as per the instructions in the Reagent Preparation section. Acetylated standards and samples must be used within 30minutes Refer to the Assay Layout Sheet to determine the number of wells to be used and put any remaining wells with the desiccant back into the pouch and seal the ziploc. Store unused wells at 4 C. 1. Pipette 50 µl of Neutralizing Reagent into each well except the Total Activity(TA) and Blank wells. 2. Pipette 100 µl of the 0.1M HCl into the NSB (non specific binding) and Bo (0pmol/mL standard) wells. 3. Add 50 µl of 0.1M HCl to the NSB wells. 4. Pipette 100 µl of Standards #1 through #5 to the bottom of the appropriate wells. 5. Pipette 100 µl of the samples to the bottom of the appropriate wells. 6. Pipette 50 µl of the blue conjugate into each well except the TA and Blank wells. 7. Pipette 50 µl of the yellow antibody into each well except the Blank, TA, and NSB wells. 14

16 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. 8. Seal the plate. Incubate for 2 hours on a plate shaker (~500 rpm) at room temperature. 9. Empty the contents of the wells and wash by adding 400 µl of wash buffer to every well. Repeat 2 more times for a total of 3 washes. After the final wash, empty or aspirate the wells and firmly tap the plate on a lint free paper towel to remove any remaining wash buffer. 10. Pipette 5 µl of the blue conjugate to the TA wells. 11. Add 200 µl of the substrate solution into each well. 12. Incubate for 1 hour at room temperature without shaking. 13. Pipette 50 µl stop solution into each well. 14. After blanking the plate reader against the substrate blank, read optical density at 405 nm. If plate reader is not capable of adjusting for the blank, manually subtract the mean OD of the substrate blank from all readings 15

17 Assay Layout sheet: 16

18 8. Calculation of Results Several options are available for the calculation of the concentration of camp in the samples. We recommend that the data be handled by an immunoassay software package utilizing a 4 parameter logistic curve fitting program. Samples with concentrations outside of the standard curve range will need to be re-analyzed using a different dilution. To normalize for protein content, divide the resulting picomole per ml determinations (pmol/ml) by the total protein concentration (mg/ml) in each sample. This is expressed as pmol camp per mg of total protein. 17

19 Typical Results- Non-acetylated assay format The results shown below are for illustration only and should not be used to calculate results. Sample Average Net OD Percent Bound camp (pmol/ml) Blank OD (0.088) TA Non Specific Standard 0 pg/ml Stadard % % 0 S % 200 S % 50 S % 12.5 S % S % Unknown % Unknown %

20 Typical Standard Curve- Non-acetylated assay format A typical standard curve is shown below. This curve must not be used to calculate camp concentrations; each user must run a standard curve for each assay. 19

21 Typical Results- acetylated assay format Sample Average Net OD Percent Bound camp (pmol/ml) Blank OD (0.097) TA Non Specific Standard 0 pg/ml Standard % % 0 S % 20 S % 5 S % 1.25 S % S % Unknown % 4.90 Unknown %

22 Typical Standard Curve- acetylated assay format A typical standard curve is shown below. This curve must not be used to calculate camp concentrations; each user must run a standard curve for each assay. 21

23 9. Performance Characteristics Sensitivity The sensitivity of the assay, defined as the concentration of camp measured at 2 standard deviations from the mean of 16 zeros along the standard curve, was determined to be 0.39 pmol/ml in the non acetylated assay format and pmol/ml in the acetylated assay format. Linearity A buffer sample containing camp was serially diluted 1:2 in the kit assay buffer and measured in the assay. The results are shown in the table below. Non-acetylated Dilution Expected Observed Recovery (%) (pmol/ml) (pmol/ml) Neat : % 1: % 1: % 22

24 Acetylated Dilution Expected Observed Recovery (%) (pmol/ml) (pmol/ml) Neat : % 1: % 1: % Precision Intra assay precision was determined by assaying 20 replicates of three buffer controls containing camp in a single assay. Non-acetylated Format Acetylated Format pmol/ml %CV pmol/ml %CV Inter assay precision was determined by measuring buffer controls of varying camp concentrations in multiple assays over several days. 23

25 Non-acetylated Format Acetylated Format pmol/ml %CV pmol/ml %CV Cross Reactivities The cross reactivities for a number of related compounds were determined by diluting the cross reactants in the kit assay buffer at a concentration of ten times the high standard. These samples were then measured in the assay. Compound Cross Reactivity camp 100% AMP 0.33% ATP 0.12% cgmp <0.001% GMP <0.001% GTP <0.001% cump <0.001% CTP <0.001% 24

26 Sample Recoveries camp standard was spiked into treated culture media, diluted with 0.1M HCl, and measured in the kit. The results were as follows: Non-acetylated Format Acetylated Format Sample Recovery Recommend ed Dilution Recovery Recommende d Dilution Tissue Culture Media 94.8% 1:4 95.2% 1:4 0.1 M HCl should not be used to dilute culture supernatants (without pre treatment with concentrated HCl), serum, or saliva samples. 25

27 10. Trouble shooting For technical questions please do not hesitate to contact us by or phone (select contact us on for the phone number for your region). Weak Color Development How long was the substrate incubation? What were the conditions of the substrate incubation? Were reagents brought to room temperature prior to use? It is possible that Stop Solution was added to the plate without allowing the full substrate incubation. If a plate is left to incubate on a cold lab bench or under a drafty area during ambient incubations, signal values (e.g. optical density) may be lower than expected. It is important to ensure that all reagents are brought to room temperature prior to use, or as mentioned in the product specific instruction manual. Usually leaving the kit out on the bench top at ambient temperature for about half an hour prior to setting up the assay will be sufficient, when the reagents can be stored at 4 C. Frozen volumes take a little more time to come to room temperature. Do not thaw frozen reagents in a water bath. If a different standard/sample diluent is used (such as culture media) this must also be warmed. 26

28 What were the conditions of the incubations? How was the plate shaken during incubations (if required)? If the incubation times and temperatures are not observed, this can lead to lower than expected signal values (e.g. optical density). Pay attention that in air-conditioned rooms the temperature does not drop below 21 C. If customers do not have a plate shaker, they will often use an orbital flask shaker or some other piece of equipment. This is not a problem as long as the liquid is vigorously displaced about 3/4 of the way up the sides of the wells without coming out. It is very important that the plate is secured into place. If the plate is not shaken and it is required in the procedure, a longer incubation may be necessary to bring the reagents to equilibrium. How long after the addition of Stop Solution was the plate read? The plate needs to be read at the correct wavelength as soon as possible after the addition of the Stop Solution. We generally recommend that the plate be read within 10 minutes. 27

29 High Background How was the plate washed? It is important that the plate is washed thoroughly. If plate washing is troublesome, a squirt bottle can be filled with diluted Wash Buffer and all of the wells completely filled from this. The plate contents can be dumped into the sink and shaken to remove excess buffer. This should be repeated for the number of times recommended in the instruction manual. It is important to remember that adding too little Wash Buffer can result in high background, while adding too much is not a problem. The contents of the wells should be aspirated and the plate tapped dry on lint-free paper towels. What were the incubation times and temperatures? If the plate was incubated for too long or at a higher than recommended temperature, high background could result. Drift Were reagents brought to room temperature prior to use? If the reagents are not at a constant temperature prior to their addition into the wells, the results from one side of the plate to the other can differ depending on the temperatures at addition. 28

30 Was the set-up of the assay interrupted? If the assay is interrupted at any point during the addition of reagents, it is possible that differing results will be seen before the interruption versus after. The wells that had reagents added before the interruption will have been incubating for longer than those after. Poor Precision Were the wells washed properly? All wells receive the same treatment during the wash step. If some are washed less than others, this can translate to poor precision. It is important that the plate is washed thoroughly. If plate washing is troublesome, a squirt bottle can be filled with diluted Wash Buffer and all of the wells completely filled from this. The plate contents can be dumped into the sink and shaken to remove excess buffer. This should be repeated for the number of times recommended in the instruction manual. It is important to remember that adding too little Wash Buffer can result in high background, while adding too much is not a problem. The contents of the wells should be aspirated and the plate tapped dry on lint-free paper towels. 29

31 Were the wells aspirated sufficiently after the wash steps? How were reagents pipetteted into wells? It is very important that as little Wash Buffer as possible remains in the wells after aspiration. Residual buffer can cause dilution of subsequent reagents. After the last wash step, it is a good idea to hit the plate several times over a piece of paper toweling to remove excess buffer. In order to eliminate precision error, customers need to remember to prerinse all pipette tips used in the assay. We usually recommend that the customer draw up the liquid into the tip and aspirate it three times prior to addition into the well. Regular pipette calibration and maintenance is also essential to ensure that the tips fit properly and that the correct volumes are dispensed. Be sure reagents are not splashed between wells or outside of the wells during pipetteing (especially if using repeater pipettes). Poor Standard Curve What was used as the standard diluent? Diluents other than the supplied assay buffer may contain interfering substances that can affect the standard curve. 30

32 How was the precision of the standard curve? Were the Blank and NSB values subtracted out? How were the standard dilutions prepared? If the %CV values for the standard curve signal values (e.g. optical density) are consistently above 5%, it may be a good idea to pay particular attention to pipetteting technique. If the standard curve signal values were acceptable but the sample precision was not, the problem relates to the sample. Also, see recommendations under "Poor Precision". If the net signal values (e.g. optical density) are not used, the signal values will appear higher than those presented in the sample data in the instruction manual. It is important that test tubes of an appropriate size and material are used. Standard dilutions must be properly mixed (e.g. vortexed) while preparing the serial dilutions. It is also crucial that the standard dilutions be prepared and used within the time specified in the product specific instruction manual. Never store unused standard dilutions for a later use. 31

33 Edge Effects Where was the plate incubated? Often times the conditions for ambient incubations can be less than ideal. If there is a draft in the area or the plate is incubated on a cold lab bench, this can lead to uneven color development. If multiple plates were run, were they stacked on top of each other during incubation? If a non-ambient incubation was required, was the plate properly sealed? Multiple plates should only be incubated in a single layer. This will assure that no area of the plate is at a different temperature than any other. Making sure that the plate sealer is tightly covering all of the wells will help to discourage uneven evaporation of the well contents, or condensation for colder incubation conditions. 32

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