ab camp ELISA Kit

Transcription

1 ab camp ELISA Kit Instructions for Use For the quantitative determination of camp in saliva, serum, and culture supernatants. 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 4 4. Kit Contents 7 5. Storage and Handling 8 6. Additional Materials Required 8 7. Protocol 9 8. Calculation of Results Performance Characteristics Troubleshooting 26 2

4 1. Introduction ab is a competitive immunoassay for the quantitative determination of extracellular cyclic AMP in saliva, serum, and culture supernatants. The optional acetylated assay format provides an approximate 10 foldincrease 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 100 µl of standard diluent (Assay Buffer 2 or nonconditioned culture media) into the Non Specific Binding (NSB) and the Bo (0 pg/ml Standard) wells. Pipette 50 µl of Assay Buffer 2 to the NSB wells. Pipette 100 µl of Standards #1 through #5 to the bottom of the appropriate wells. Pipette 100 µl of the samples to the bottom of 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 Binding wells. Seal the plate and 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. 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. 5

7 Add 5 µl of the blue Conjugate to the TA wells. Add 200 µl of the pnpp Substrate solution to every well. Incubate at room temperature for 1 hour without shaking. Add 50 µl of Stop Solution to every well. Blank the plate reader against the Blank wells, read the 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. 6

8 4. Kit Contents Item Description Quantity Goat anti-rabbit IgG Microtiter Plate A clear plate of break apart strips coated with a goat anti rabbit polyclonal antibody 1x 96 wells camp ELISA Conjugate camp ELISA Antibody Wash Buffer Concentrate camp Standard pnpp Substrate Stop Solution Assay Buffer 2 Triethylamine Acetic Anhydride A blue solution of alkaline phosphatase conjugated with camp. A yellow solution of a rabbit polyclonal antibody to camp. Tris buffered saline containing detergents. A solution of 2,000 pmol/ml camp. A solution of p-nitrophenyl phosphate in buffer. Ready to use. A solution of trisodium phosphate in water. Keep tightly capped. Sodium acetate buffer containing proteins and sodium azide 5 ml 5 ml 27 ml 0.5 ml 20 ml 5 ml 27 ml Plate Sealer 1 2mL 1mL 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. Microplate reader capable of reading at 405 nm. 8

10 7. Protocol A. 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 until the kit expiration date, or for 3 months, whichever is earlier. 2. camp Standard, non-acetylated format Two diluent options are available for the preparation of the standard curve, NSB, and Bo wells. Use Assay Buffer 2 for the standard diluent when the sample is serum or saliva. For culture supernatants, use the same non conditioned media for the standard diluent. Allow the 2,000 pmol/ml standard stock to warm to room temperature. Label five 12mm x 75mm tubes #1 through #5. Pipette 990 μl of the appropriate sample diluent into tube #1. Pipette 750 μl of the appropriate sample diluent into tubes #2 through #5. Add 10 μl of the 2,000 pmol/ml standard stock into tube #1 and 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 thoroughly. Continue this for tubes #4 through #5. Diluted standards should be used within 60 minutes of preparation. 9

11 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. 4. camp Standard, acetylated format (optional) Two diluent options are available for the preparation of the standard curve, NSB, and Bo wells. Use Assay Buffer 2 for the standard diluent when the sample is serum or saliva. For culture supernatants, use the same non conditioned media for the standard diluent. Allow the 2,000 pmol/ml standard stock to warm to room temperature. Label five 12mm x 75mm tubes #1 through #5. Pipette 990 μl of the appropriate sample diluent into tube #1. Pipette 750 μl of the appropriate sample diluent into tubes #2 through #5. Add 10 μl of the 2,000 pmol/ml standard stock into tube #1 and 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 thoroughly. 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 0pg/mL Standard/Non Specific Binding tube. Pipette 1 ml of the appropriate standard diluent into 10

12 this tube. Add 50 μl of the Acetylating Reagent to the 0pg/mL Standard/Non Specific Binding tube and use in Steps 2 and 3 of the Assay Procedure. The acetylated standards should be used within 30 minutes of preparation. B. Sample Handling Samples containing rabbit IgG will interfere with the assay. EDTA plasma may precipitate during acetylation. Biological fluids, such as serum and saliva, should be diluted in Assay Buffer 2 and run directly in the assay. A minimum 1:10 dilution is required for serum and a 1:4 dilution for saliva (see Sample Recoveries section). These are the minimum dilutions required to remove matrix interference of these samples. Culture supernatants may be run directly in the assay provided the same non-conditioned media is used as the standard diluent. 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. 11

13 C. Procedural Notes 1. Do not mix components from different kit lots. 2. Allow all reagents to warm to room temperature for at least 30 minutes before opening. 3. Standards can be made up in either glass or plastic tubes. 4. Pre-rinse the pipette tip with the reagent, use fresh pipette tips for each sample, standard and reagent. 5. Pipette standards and samples to the bottom of the wells. 6. Add the reagents to the side of the well to avoid contamination. 7. This kit uses break-apart microtiter strips, which allow the user to measure as many samples as desired. Unused wells must be kept desiccated at 4 C in the sealed bag provided. The wells should be used in the frame provided. 8. Care must be taken to minimize contamination by endogenous alkaline phosphatase. Contaminating alkaline phosphatase activity, especially in the substrate solution, may lead to high blanks. Care should be taken not to touch pipette tips and other items that are used in the assay with bare hands. 9. Prior to addition of substrate, ensure that there is no residual wash buffer in the wells. Any remaining wash buffer may cause variation in assay results. 12

14 D. 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. 1. Pipette 100 µl of the appropriate standard diluent (Assay Buffer 2 or non conditioned culture media) into the NSB (non specific binding) and Bo (0pmol/mL standard) wells. 2. Add 50 µl of Assay Buffer 2 to the NSB wells. 3. Pipette 100 µl of Standards #1 through #5 to the bottom of the appropriate wells. 4. Pipette 100 µl of the samples to the bottom of the appropriate wells. 5. Pipette 50 µl of the blue conjugate into each well except the TA and Blank wells. 6. Pipette 50 µl of the yellow antibody 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. 13

15 7. Seal the plate. Incubate for 2 hours on a plate shaker (~500 rpm) at room temperature. 8. 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. 9. Pipette 5µL of the blue conjugate to the TA wells. 10. Add 200 µl of the substrate solution into each well. 11. Incubate for 1 hour at room temperature without shaking. 12. Pipette 50 µl stop solution into each well. 13. 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. 14

16 Assay Layout sheet 15

17 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 (4PL) 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. 16

18 Typical Results- Non-acetylated assay format The results shown below are for illustration only and should not be used to calculate results. Sample Mean OD (- Blank) Percent Bound camp (pmol/ml) Blank OD (0.086) TA Non specific Binding 0 pg/ml Standard % % 0 S % 200 S % 50 S % 12.5 S % S % Unknown % Unknown %

19 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. 18

20 Typical Results- acetylated assay format Sample Mean OD (- Blank) Percent Bound camp (pmol/ml) Blank OD (0.093) TA Non specific Binding 0 pg/ml standard % % 0 S % 20 S % 5 S % 1.25 S % S % Unknown % 9.20 Unknown %

21 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. 20

22 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 0.30 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: % 1: % 21

23 Acetylated Dilution Expected Observed Recovery (%) (pmol/ml) (pmol/ml) Neat : % 1: % 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

24 Inter assay precision was determined by measuring buffer controls of varying camp concentrations in multiple assays over several days. Non-acetylated Format Acetylated Format pmol/ml %CV pmol/ml %CV

25 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 the following matrices diluted with Assay Buffer 2 and measured in the kit. The results were as follows: Non-acetylated Format Acetylated Format Sample Recovery Recommended Dilution Recovery Recommended Dilution Tissue Culture Media 96.2% None 101.2% None Human Serum 101.5% 1: % 1:64 Human Saliva 103.2% 1:4 94.9% 1:4 25

27 10. Troubleshooting 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 pipetted 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 pipetting (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 pipetting 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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