Heme Oxygenase (Decycling) 1 (HMOX1) ELISA Kit

Antigen: Heme Oxygenase (Decycling) 1 (HMOX1)

Synonyme: HO1, HO-1, Hmox, Hemox, Hsp32, D8Wsu38e, Ho1, Heox, Ho-1, HEOXG, hsp32, HMOX1, MGC132176, zgc:65984, ARABIDOPSIS THALIANA HEME OXYGENASE 1, ATHO1, F18A8.4, F18A8_4, GENOMES UNCOUPLED 2, GUN2, HEME OXYGENASE, HEME OXYGENASE 1, HEME OXYGENASE 6, HY6, PLASTID HEME OXYGENASE, REVERSAL OF THE DET PHENOTYPE 4, TED4, Hmox1, DKFZp469G141

Reaktivität: Human

Applikation: ELISA

Zertifikate: ISO 9001:2008

Produktnummer: ABIN366536

Produktbeschreibung

Produktmerkmale: This immunoassay kit allows for the in vitro quantitative determination of human HO-1 concentrations in cell culture supernates, serum, plasma and other biological fluids.

Weitere Bezeichnung: heme oxygenase 1 (HO-1)

Proben: Serum, Plasma

Beschreibung: Heme oxygenase, an essential enzyme in heme catabolism, cleaves heme to form biliverdin, which is subsequently converted to bilirubin by biliverdin reductase, and carbon monoxide, a putative neurotransmitter. Heme oxygenase activity is induced by its substrate heme and by various nonheme substances. Heme oxygenase occurs as 2 isozymes, an inducible heme oxygenase-1 and a constitutive heme oxygenase-2. HMOX1 and HMOX2 belong to the heme oxygenase family. The HMOX gene is located on the long (q) arm of chromosome 22 at position 13.1, from base pair 34,101,636 to base pair 34,114,748. Heme Oxygenase-1 (HO-1) also known as Hsp32, is the inducible isoform of heme oxygenase that catalyzes the NADPH, O2 and cytochrome P450 reductase dependent oxidation of heme to carbon monoxide, ferrous iron and biliverdin which is rapidly reduced to bilirubin. These products of the HO reaction have important physiological effects: carbon monoxide is a potent vasodilator and has been implicated to be a physiological regulator of cGMP and vascular tone, biliverdin and its product bilirubin are potent antioxidants, “free” iron increases oxidative stress and regulates the expression of many mRNAs by affecting the conformation of iron regulatory protein (IRP)-1 and its binding to iron regulatory elements (IREs) in the 5’- or 3’- UTRs of the mRNAs. To date, three identified heme oxygenase isoforms are part of the HO system that catalyze heme into biliverdin and carbon monoxide. These are inducible HO-1 or Hsp32, constitutive HO-2 that is abundant in the brain and testis, and HO-3 which is related to HO-2 but is the product of a different gene. HO-1 is a vital component of neuronal defense mechanisms and oxidative stress has been postulated to be the underlying basis for neuronal cell death in neurodegenerative diseases such as Alzheimer’s disease (AD) and Parkinson’s disease. The expression of HO-1 is normally very low in the brain but increases markedly after heat shock, ischemia or glutathione depletion. Spatial distribution of HO-1 expression in AD brain is essentially identical to that of the pathogenic conformational changes of tau protein that is the major component of the intraneuronal lesion of AD, neurofibrillary tangles.

Spezifität: This assay has high sensitivity and excellent specificity for detection of Human heme oxygenase 1 (HO-1). No significant cross-reactivity or interference between Human heme oxygenase 1 (HO-1) and analogues was observed.
Note: Limited by current skills and knowledge, it is impossible for us to complete the cross-reactivity detection between Human heme oxygenase 1 (HO-1) and all the analogues, therefore, cross reaction may still exist.

Sensitivität: The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest protein concentration that could be differentiated from zero. It was determined the mean O.D value of 20 replicates of the zero standard added by their three standard deviations.

Kommentare:

Precision:
1. Intra-assay Precision (Precision within an assay): CV%<8%. Three samples of known concentration were tested twenty times on one plate to assess.
2. Inter-assay Precision (Precision between assays): CV%<10%. Three samples of known concentration were tested in twenty assays to assess.

Synonyme: HO1, HO-1, Hmox, Hemox, Hsp32, D8Wsu38e, Ho1, Heox, Ho-1, HEOXG, hsp32, HMOX1, MGC132176, zgc:65984, ARABIDOPSIS THALIANA HEME OXYGENASE 1, ATHO1, F18A8.4, F18A8_4, GENOMES UNCOUPLED 2, GUN2, HEME OXYGENASE, HEME OXYGENASE 1, HEME OXYGENASE 6, HY6, PLASTID HEME OXYGENASE, REVERSAL OF THE DET PHENOTYPE 4, TED4, Hmox1, DKFZp469G141

Anwendungen

Prinzip: This assay employs the quantitative sandwich enzyme immunoassay technique. Antibody specific for heme oxygenase 1 (HO-1) has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any heme oxygenase 1 (HO-1) present is bound by the immobilized antibody. After removing any unbound substances, a biotin-conjugated antibody specific for heme oxygenase 1 (HO-1) is added to the wells. After washing, avidin conjugated Horseradish Peroxidase (HRP) is added to the wells. Following a wash to remove any unbound avidin-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of heme oxygenase 1 (HO-1) bound in the initial step. The color development is stopped and the intensity of the color is measured.

Aufbereitung der Reagenzien: 1. Biotin-antibody (1x). Centrifuge the vial before opening. Biotin-antibody requires a 100-fold dilution. A suggested 100-fold dilution is 10 μl of Biotin-antibody + 990 μl of Biotin-antibody Diluent.

2. HRP-avidin (1x). Centrifuge the vial before opening. HRP-avidin requires a 100-fold dilution. A suggested 100-fold dilution is 10 μl of HRP-avidin + 990 μl of HRP-avidin Diluent.

3. Wash Buffer(1x). If crystals have formed in the concentrate, warm up to room temperature and mix gently until the crystals have completely dissolved. Dilute 20 ml of Wash Buffer Concentrate (25 x) into deionized or distilled water to prepare 500 ml of Wash Buffer (1 x).

4. Standard. Centrifuge the standard vial at 6000-10000rpm for 30s. Reconstitute the Standard with 1.0 ml of Sample Diluent. Do not substitute other diluents. This reconstitution produces a stock solution of the highest concentration. Mix the standard to ensure complete reconstitution and allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making dilutions.
Pipette 250 μl of Sample Diluent into each tube. Use the stock solution to produce a 2-fold dilution series (below). Mix each tube thoroughly before the next transfer. The undiluted Standard serves as the high standard. Sample Diluent serves as the zero standard (0 pg/ml).


Note:

Kindly use graduated containers to prepare the reagent. Please don't prepare the reagent directly in the Diluent vials provided in the kit.

Bring all reagents to room temperature (18-25°C) before use for 30min.

Prepare fresh standard for each assay. Use within 4 hours and discard after use.

Making serial dilution in the wells directly is not permitted.

Please carefully reconstitute Standards according to the instruction, and avoid foaming and mix gently until the crystals have completely dissolved. To minimize imprecision caused by pipetting, use small volumes and ensure that pipettors are calibrated. It is recommended to suck more than 10μl for once pipetting.

Distilled water is recommended to be used to make the preparation for reagents or samples. Contaminated water or container for reagent preparation will influence the detection result.

Probennahme: Serum: Use a serum separator tube (SST) and allow samples to clot for two hours at room temperature or overnight at 4° C before centrifugation for 15 minutes at 1000 x g. Remove serum and assay immediately or aliquot and store samples at -20° C or -80° C. Avoid repeated freeze-thaw cycles.
Plasma: Collect plasma using EDTA, or heparin as an anticoagulant. Centrifuge for 15 minutes at 1000 x g at 2-8° C within 30 minutes of collection. Assay immediately or aliquot and store samples at -20° C or -80° C. Avoid repeated freeze-thaw cycles.

Testdurchführung: Bring all reagents and samples to room temperature before use. Centrifuge the sample again after thawing before the assay. It is recommended that all samples and standards be assayed in duplicate.

1. Prepare all reagents, working standards, and samples as directed in the previous sections.

2. Refer to the Assay Layout Sheet to determine the number of wells to be used and put any remaining wells and the desiccant back into the pouch and seal the ziploc, store unused wells at 4° C.

3. Add 100μl of standard and sample per well. Cover with the adhesive strip provided. Incubate for 2 hours at 37° C. A plate layout is provided to record standards and samples assayed.

4. Remove the liquid of each well, don’t wash.

5. Add 100μl of Biotin-antibody (1x) to each well. Cover with a new adhesive strip. Incubate for 1 hour at 37° C. (Biotin-antibody (1x) may appear cloudy. Warm up to room temperature and mix gently until solution appears uniform.)

6. Aspirate each well and wash, repeating the process two times for a total of three washes. Wash by filling each well with Wash Buffer (200μl) using a squirt bottle, multi-channel pipette, manifold dispenser, or autowasher, and let it stand for 2 minutes, complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining wash Buffer by aspirating ordecanting. Invert the plate and blot it against clean paper towels.

7. Add 100μl of HRP-avidin (1x) to each well. Cover the microtiter plate with a new adhesive strip. Incubate for 1 hour at 37° C.

8. Repeat the aspiration/wash process for five times as in step 6.

9. Add 90μl of TMB Substrate to each well. Incubate for 15-30 minutes at 37° C. Protect from light.

10. Add 50μl of Stop Solution to each well, gently tap the plate to ensure thorough mixing.

11. Determine the optical density of each well within 5 minutes, using a microplate reader set to 450 nm. If wavelength correction is available, set to 540 nm or 570 nm. Subtract readings at 540 nm or 570 nm from the readings at 450 nm. This subtraction will correct for optical imperfections in the plate. Readings made directly at 450 nm without correction may be higher and less accurate.

Samples may require dilution. Please refer to Sample Preparation section.


Note:

1. The final experimental results will be closely related to validity of the products, operation skills of the end users and the experimental environments.

2. Samples or reagents addition: Please use the freshly prepared Standard. Please carefully add samples to wells and mix gently to avoid foaming. Do not touch the well wall as possible. For each step in the procedure, total dispensing time for addition of reagents or samples to the assay plate should not exceed 10 minutes. This will ensure equal elapsed time for each pipetting step, without interruption. Duplication of all standards and specimens, although not required, is recommended. To avoid cross-contamination, change pipette tips between additions of each standard level, between sample additions, and between reagent additions. Also, use separate reservoirs for each reagent.

3. Incubation: To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary. Do not allow wells to sit uncovered for extended periods between incubation steps. Once reagents have been added to the well strips, DO NOT let the strips DRY at any time during the assay. Incubation time and temperature must be observed.

4. Washing: The wash procedure is critical. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Solution by aspirating or decanting and remove any drop of water and fingerprint on the bottom of the plate. Insufficient washing will result in poor precision and falsely elevated absorbance reading. When using an automated plate washer, adding a 30 second soak period following the addition of wash buffer, and/or rotating the plate 180 degrees between wash steps may improve assay precision.

5. Controlling of reaction time: Observe the change of color after adding TMB Substrate (e.g. observation once every 10 minutes), TMB Substrate should change from colorless or light blue to gradations of blue. If the color is too deep, add Stop Solution in advance to avoid excessively strong reaction which will result in inaccurate absorbance reading.

6. TMB Substrate is easily contaminated. TMB Substrate should remain colorless or light blue until added to the plate. Please protect it from light.

7. Stop Solution should be added to the plate in the same order as the TMB Substrate. The color developed in the wells will turn from blue to yellow upon addition of the Stop Solution. Wells that are green in color indicate that the Stop Solution has not mixed thoroughly with the TMB Substrate.

Ergebnisberechnung: Average the duplicate readings for each standard and sample and subtract the average zero standard optical density. Create a standard curve by reducing the data using computer software capable of generating a four parameter logistic (4-PL) curve-fit. As an alternative, construct a standard curve by plotting the mean absorbance for each standard on the x-axis against the concentration on the y-axis and draw a best fit curve through the points on the graph. The data may be linearized by plotting the log of the Human heme oxygenase 1 (HO-1) concentrations versus the log of the O.D. and the best fit line can be determined by regression analysis. This procedure will produce an adequate but less precise fit of the data. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.

Applikationshinweise: Detection Wavelength: 450 nm

Bestandteile: 1. Assay plate (12 x 8 coated Microwells). Quantity: 1(96 wells)
2. Standard (Freeze dried). Quantity: 2
3. Biotin-antibody (100 x concentrate) Quantity: 1 x 120 µl
3.HRP-avidin (100 x concentrate). Quantity: 1 x 120 µl
4. Biotin-antibody Diluent. Quantity: 1 x 10 ml
5. HRP-avidin Diluent. Quantity: 1 x 10 ml
6. Sample Diluent. Quantity: 1 x 20 ml
7. Wash Buffer (25 x concentrate). Quantity: 1 x 20 ml
8. TMB Substrate. Quantity: 1 x 10 ml
9. Stop Solution. Quantity: 1 x 10 ml
10. Adhesive Strip (For 96 wells). Quantity: 4
11. Instruction manual

Benötigtes Material: 1. Microplate reader capable of measuring absorbance at 450 nm, with the correction wavelength set at 540 nm or 570 nm.
2. An incubator which can provide stable incubation conditions up to 37° C +/- 0.5° C.
3. Squirt bottle, manifold dispenser, or automated microplate washer.
4. Absorbent paper for blotting the microtiter plate.
5. 100 mL and 500 mL graduated cylinders.
6. Deionized or distilled water.
7. Pipettes and pipette tips.
8. Test tubes for dilution.

Lagerung: Unopened: Store at 2 - 8° C. Do not use past kit expiration date.
Opened: May be stored for up to 1 month at 2-8° C. Try to keep coated assay plate in a sealed aluminum foil bag, and avoid the damp.

Forschungsgebiet: Enzyme, Metabolismus

Beschränkungen: Nur für Forschungszwecke einsetzbar