ab133058 HO-1 Human ELISA development set Instructions for Use For the quantitative measurement of HO-1 concentrations in cell lysates and microsomes. This product is for research use only and is not intended for diagnostic use.
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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 13 9. Performance Characteristics 15 10. Troubleshooting 18 2
1. Introduction Heme oxygenase (Hsp32) is the rate-limiting enzyme that breaks down heme to iron, carbon monoxide and biliverdin, which is then metabolized to bilirubin by biliverdin reductase. In mammalians heme oxygenase exists as two primary isoforms, the inducible isoform HO-1 and the constitutively expressed HO-2, both catalyzing the same reaction. HO-1 is expressed in erythrocyte and hemoglobin metabolizing tissues of the spleen, liver and bone marrow, with localization to the membranes of the ER, mitochondria and caveolae. HO-1 expression is induced in response to an array of oxidative, stress-inducing factors, including heat shock, heme accumulation, hypoxia, UV radiation, nitric oxide, cytokines and heavy metals. 3
2. Principle of the Assay ab133058 contains the basic components for the development of a HO-1 Human Immunometric Enzyme-Linked Immunosorbent Assay (ELISA). Each kit contains sufficient reagents for five 96-well plates. This assay detects natural HO-1 in cell lysates, serum, EDTA plasma, and microsomes of mouse origin. 4
3. Assay Summary Pipette 100 μl of Assay Buffer into the control (0 ng/ml standard) wells. Pipette 100 μl of standards and samples into the appropriate wells. Seal the plate. Incubate for 1 hour at room temperature. Seal the plate. Incubate for 1 hour at room temperature. Empty the wells and wash with 400µl Wash buffer, repeat 3 or more times. 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. Pipette 100 μl of Detection Antibody into each well, except the Blank, wells. Seal the plate. Incubate for 1 hour at room temperature. Wash as described above. Add 100µl of the diluted conjugate to each well except the blank. Seal the plate. Incubate for 30 minutes at room temperature. Wash as described above. Pipette 100µl of TMB solution into each well. Seal the plate. Incubate for 30 minutes at room temperature. Pipette 100µl 1N HCL into each well. 5
Blank the plate reader against the substrate, read the optical density at 450 nm. If the plate reader is not capable of adjusting for the blank, manually subtract the mean OD of the substrate blank from all readings. 6
4. Kit Contents Item Description Quantity HO-1 (human) Capture Antibody HO-1 (human) Standard HO-1 (human) Detection Antibody SA-HRP One vial containing 219 μg lyophilized HO-1 (human) monoclonal antibody One vial containing 156.25 ng lyophilized recombinant HO-1 (human) protein One vial containing 18.75 μg lyophilized HO-1 (human) polyclonal antibody. One vial containing 12.5 μg lyophilized Streptavidin conjugated to horseradish peroxidase 1 vial 1 vial 1 vial 1 vial 7
5. Storage and Handling Store all components at 4 C. 6. Additional Materials Required RIPA cell lysis buffer 96 well high-binding polystyrene microtiter plates Precision pipettes Microplate reader capable of reading at 450 nm Phosphate buffered saline Tween 20 Bovine serum albumin 3,3, 5,5 tetramethylbenzidine (TMB) solution 1N hydrochloric acid Sucrose 8
7. Protocol A. Buffer Formulations 1. Coating buffer : 10mM sodium phosphate, 15mM NaCl, ph 7.4 2. Blocking buffer : 10mM sodium phosphate, 15mM NaCl, 1.0% BSA, 1.0% sucrose, ph 7.4 3. Assay buffer : 100mM sodium phosphate, 150mM NaCl, 1.0% BSA, 0.1% Tween-20, ph 7.4 4. Wash buffer: 10mM sodium phosphate, 15mM NaCl, 0.1% Tween-20, ph 7.4 B. Plate Coating 1. Reconstitute HO-1 (human) Capture Antibody with 250µl deionized water for a 250x stock. Use immediately, or make aliquots and freeze at -20 C for up to 3 months. For prolonged storage, aliquot and freeze at -80 C. Avoid repeated freeze/thaw cycles. 2. Dilute the stock 1/250 in Coating buffer. Immediately dispense into 96 well microtiter plates using 100µl of the diluted capture antibody per well, Seal the plate and incubate overnight at room temperature. 3. Aspirate each well to remove coating solution. Immediately add 200µl Blocking buffer per well. Seal the plate and incubate for at least 1 hour. 9
4. Aspirate each well to remove blocking solution. Plates may be used immediately or dried and stored with dessicant at 4 C. C. Procedural Notes 1. If buffers other than those recommended are used in the assay, the end user must determine the appropriate dilution and assay validation. 2. Pipette the reagents to the sides of the wells to avoid possible contamination. 3. Pre-rinse the pipette tip with the reagent, use fresh pipette tips for each sample, standard and reagent. 4. Insufficient washing or residual wash buffer in the wells may cause variation in assay results. 5. Bring all reagents to room temperature for at least 30 minutes prior opening. 6. All standards, controls and samples should be assayed in duplicate. 10
D. Reagent Preparation 1. Recombinant HO-1 (human) Standard Reconstitute vial contents with 250µl deionized water for a 50x stock. Aliquot and store at -20 C. For prolonged storage aliquot and freeze at -80 C. Avoid repeated freeze/thaw cycles. The recommended standard curve range is 12.5ng/ml to 0.195ng/ml, using 2-fold serial dilutions in Assay Buffer. Do not store diluted standard. 2. HO-1 (human) Detection Antibody Reconstitute vial contents with 250µl deionized water for a 250x stock. Store at 4 C. For prolonged storage aliquot and freeze at - 20 C. Avoid repeated freeze/thaw cycles. For best results, reconstitute the Detection Antibody at the time of plate coating and wait at least one day before freezing. Dilute the stock 1/250 in Assay Buffer for a working solution. Do not store diluted antibody. 3. SA-HRP Reconstitute vial contents with 250µl deionized water for a 600x stock. Store at 4 C. For prolonged storage aliquot and freeze at - 20 C. Avoid repeated freeze/thaw cycles. 11
Dilute the stock 1/600 in Assay Buffer for a working solution. Do not store diluted antibody. E. Assay Procedure Bring all reagents to room temperature for at least 30 minutes prior to opening. All standards and samples should be run in duplicate. 1. Pipette 100 μl of Assay Buffer into the control (0 ng/ml standard) wells. 2. Pipette 100 μl of standards and samples prepared in Assay Buffer to the bottom of the appropriate wells. 3. Seal the plate. Incubate for 1 hour at room temperature. 4. Empty the contents of the wells and wash by adding 400µl of Wash Buffer to every well. Repeat 3 or more times for a total of 4 washed. 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. Pipette 100µl of the diluted detection antibody into each well except the blank. 6. Seal the plate. Incubate for 1 hour at room temperature. 7. Wash as above. 8. Add 100µl of the diluted conjugate to each well except the blank. 9. Seal the plate. Incubate for 30 minutes at room temperature. 10. Wash as above. 11. Pipette 100µl of TMB solution into each well. 12
12. Seal the plate. Incubate for 30 minutes at room temperature. 13. Pipette 100µl 1M HCL into each well. 14. After blanking the plate reader against the substrate, read optical density at 450 nm. If the plate reader is not capable of adjusting for the blank, manually subtract the mean OD of the substrate blank from all readings. 13
8. Calculation of Results Several options are available for the calculation of the relative levels of HO-1 in samples. We recommend that the data be handled by an immunoassay software package utilizing a 4 parameter logistic curve-fitting program. Typical Standard Curve A typical standard curve is shown below. This curve must not be used to calculate HO-1 concentrations; each user must run a standard curve for each assay. 14
9. Performance Characteristics Sensitivity The sensitivity or limit of detection of this assay is 0.049 ng/ml. It was determined by interpolation at 2 standard deviations above the mean signal at background, using data from 8 standard curves. Dilutional Linearity To determine possible interference from the sample matrix, the indicated sample types were serially diluted into the assay buffer. The concentrations of human HO-1 were measured in the assay and the results analyzed to determine the range over which a linear response was obtained. These data may be used as a guideline to determine minimal recommended dilution (MRD) for similar samples. 15
Dilution Factor HeLa CL Liver MS Neat 104%* 29%** 1:2 97% 57% 1:4 109% 74% 1:8 100% 78% 1:16 --- 88% 1:32 --- 88% 1:64 --- 96% 1:128 --- 100% CL: Cell lysate, MS: Microsome *Cell lysate was diluted 1/80 in Assay Buffer for levels to be within the dynamic range of the assay. **Microsomes were diluted 1/10 in Assay Buffer for levels to be within the dynamic range of the assay. 16
Parallelism Dose-response curves from cell lysates diluted into assay buffer (using the MRD) were compared to the recombinant human HO-1 standard curve. Parallelism indicates antibody-binding characteristics of the native and standard proteins are similar, allowing accurate determination of analyte. Specificity This assay detects natural HO-1 in cell lysates and microsomes of Human origin. There is no cross reactivity with human HO-2 or HO-3. 17
10. Troubleshooting For technical questions please do not hesitate to contact us by email (technical@abcam.com) or phone (select contact us on www.abcam.com 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. 18
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. 19
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. 20
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. 21
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. 22
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. 23
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. 24
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