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Description
Human CALP ELISA KitProduct Specification Usage Required experimental equipment: 1. Microplate reader (450nm) 2. High precision pipettes and pipette tips: 0. 5 10uL, 5 50uL, 20 200uL, 200 1000uL 3. 37C incubator 4. Distilled or deionized water Sample handling and requirements: The detection range of the kit is not equivalent to the concentration range of the analyte in the sample. Before the experiment, it is recommended to estimate the concentration of the analyte in
Product Specification
| Usage | Required experimental equipment: 1. Microplate reader (450nm) 2. High-precision pipettes and pipette tips: 0.5-10uL, 5-50uL, 20-200uL, 200-1000uL 3. 37°C incubator 4. Distilled or deionized water Sample handling and requirements: The detection range of the kit is not equivalent to the concentration range of the analyte in the sample. Before the experiment, it is recommended to estimate the concentration of the analyte in the sample based on relevant literature and conduct preliminary experiments to determine the actual concentration of the sample. If the concentration of the analyte in the sample is too high or too low, dilute or concentrate the sample appropriately. Stool storage: Stool samples should be collected in flat-bottomed test tubes and stored at 2-8°C for 6 days. Freezing stool samples will increase the calprotectin test value. If the sample needs to be stored for a long time, it is recommended to extract it first, then divide it into several tubes and store it at -20°C. It can be stored stably for at least 4 months. Fecal extraction: For each extraction, we recommend weighing 50-100 mg of fresh stool and adding 1 x 1 volume of extraction solution at a 1:19 (g:mL) ratio (for example, weigh 100 mg of stool and add 1.9 mL of 1 x 1 volume of extraction solution. The final sample concentration should be multiplied by the extraction dilution factor of 20). Cap the tube and mix thoroughly using a high-speed vortex mixer at maximum speed for 10-30 minutes. Transfer the mixed sample to a 2 mL microcentrifuge tube and centrifuge at 10,000 x g for 5 minutes. Aliquot the supernatant and store at -20°C for up to 4 months. If individual samples exceed or fall below the detection limit, we recommend adjusting the dilution factor and retesting. Serum: Collect whole blood in a serum separator tube at room temperature for 2 hours or at 4°C overnight, then centrifuge at 1000×g for 20 minutes. Remove the supernatant for analysis, or store at -20°C or -80°C. Avoid repeated freezing and thawing. Plasma: Collect the specimen using EDTA or heparin as an anticoagulant. Centrifuge at 1000×g for 15 minutes at 2-8°C within 30 minutes of collection. Remove the supernatant for analysis, or store at -20°C or -80°C. Avoid repeated freezing and thawing. Tissue homogenate: Rinse the tissue with pre-chilled PBS (0.01M, pH 7.4) to remove residual blood (lysed red blood cells in the homogenate may affect the measurement results). Weigh and mince the tissue. Mix the minced tissue with the appropriate volume of PBS (generally a 1:9 weight-to-volume ratio, e.g., 1g of tissue sample to 9mL of PBS. The specific volume can be adjusted according to experimental needs and recorded. It is recommended to add protease inhibitors to the PBS) in a glass homogenizer and grind thoroughly on ice. To further lyse tissue cells, the homogenate can be sonicated or repeatedly freeze-thawed. Finally, centrifuge the homogenate at 5000×g for 5-10 minutes and remove the supernatant for analysis. Cell culture supernatant: Centrifuge at 1000×g for 20 minutes. Remove the supernatant for analysis or store at -20°C or -80°C, but avoid repeated freeze-thaw cycles. Other biological fluids: Centrifuge at 1000×g for 20 minutes and remove the supernatant for analysis. Sample Dilution Scheme: Please estimate the sample concentration range in advance. If your test sample requires dilution, the following dilution scheme is recommended: 100-fold dilution: One-step dilution. Add 5 μL of sample to 495 μL of universal diluent for a 100-fold dilution. 1000-fold dilution: Two-step dilution. Add 5 μL of sample to 95 μL of universal diluent for a 20-fold dilution. Then, add 5 μL of the 20-fold diluted sample to 245 μL of universal diluent for a 50-fold dilution, for a total of 1000-fold dilution. 100,000-fold dilution: Three-step dilution. Add 5 μL of sample to 195 μL of universal diluent and dilute 40-fold. Then, add 5 μL of the 40-fold diluted sample to 245 μL of universal diluent and dilute 50-fold. Finally, add 5 μL of the 2,000-fold diluted sample to 245 μL of universal diluent and dilute 50-fold, for a total dilution of 100,000-fold. For each dilution step, add at least 3 μL of solution, and the dilution factor should not exceed 100-fold. Mix thoroughly at each dilution step to avoid foaming. Pre-test Preparation: 1. Remove the test kit from the refrigerator 10 minutes in advance and equilibrate to room temperature. 2. Prepare a gradient standard working solution: Add 1 mL of universal diluent to the lyophilized standard. Let stand for 15 minutes to completely dissolve, then gently mix (concentration 720 ng/mL). Then dilute to the following concentrations: 720 ng/mL, 360 ng/mL, 180 ng/mL, 90 ng/mL, 45 ng/mL, 22.5 ng/mL, 11.25 ng/mL, and 0 ng/mL. Serial dilution method: Take seven EP tubes and add 500 μL of universal diluent to each tube. Pipette 500 μL of the 720 ng/mL standard working solution into the first EP tube and mix thoroughly to make a 360 ng/mL standard working solution. Repeat this procedure for subsequent tubes. The last tube serves as a blank well; there is no need to pipette liquid from the penultimate tube. See the figure below for details. 3. Preparation of biotinylated detection antibody working solution: 15 minutes before use, centrifuge the concentrated biotinylated antibody at 1000×g for 1 minute. Dilute the 100× concentrated biotinylated antibody to a 1× working concentration with universal diluent (e.g., 10 μL concentrate + 990 μL universal diluent). Prepare and use immediately. 4. Preparation of the enzyme conjugate working solution: 15 minutes before use, centrifuge 100 μL of concentrated enzyme conjugate at 1000 × g for 1 minute. Dilute the 100 μL concentrated HRP conjugate with universal diluent to a 1 μL working concentration (e.g., 10 μL of concentrate + 990 μL of universal diluent). Prepare freshly prepared extract solution. 5. Preparation of the 1 μL extract solution: Dissolve 10 mL of the 20 μL extract solution in 190 mL of distilled water. (The concentrated extract solution may crystallize after removal from the refrigerator. This is normal. Allow to stand at room temperature and gently shake until the crystals are completely dissolved before reconstitution.) 6. Preparation of 1× Wash Buffer: Dissolve 10 mL of 20× Wash Buffer in 190 mL of distilled water (Concentrated Wash Buffer removed from the refrigerator may crystallize, which is normal. Allow to stand at room temperature until the crystals have completely dissolved before preparing the buffer). Procedure: 1. Remove the desired strips from the aluminum foil bag after equilibration at room temperature for 10 minutes. Seal the remaining strips in a ziplock bag and return them to 4°C. 2. Sample Loading: Add 100 μL of sample or standard of varying concentrations to the appropriate wells. Add 100 μL of Universal Diluent to the blank wells. Cover with a film sealer and incubate at 37°C for 60 minutes. (Recommendation: Dilute the sample to be tested at least 1-fold with Universal Diluent before loading into the plate. This minimizes matrix effects on the test results. When calculating the sample concentration, multiply by the dilution factor. It is recommended to run replicates for all samples and standards.) 3. Adding Biotinylated Antibody: Remove the ELISA plate, discard the liquid, and do not wash. Add 100 μL of biotinylated antibody working solution directly to each well. Cover with a sealing film and incubate at 37°C for 60 minutes. 4. Washing: Discard the liquid and add 300 μL of 1x Wash Buffer to each well. Let stand for 1 minute. Shake off the wash solution and pat dry on absorbent paper. Repeat this process three times (a microplate washer can also be used). 5. Adding Enzyme Conjugate Working Solution: Add 100 μL of enzyme conjugate working solution to each well. Cover with a sealing film and incubate at 37°C for 30 minutes. 6. Washing: Discard the liquid and wash the plate five times as in step 4. 7. Adding Substrate: Add 90 μL of substrate (TMB) to each well. Cover with a sealing film and incubate at 37°C in the dark for 15 minutes. 8. Add Stop Solution: Remove the ELISA plate and add 50 μL of Stop Solution directly to each well. Immediately measure the OD value of each well at a wavelength of 450 nm. Calculation of Experimental Results: Result Interpretation: 1. Calculate the average OD value of the standard and sample replicates and subtract the OD value of the blank well as a correction value. Plot a four-parameter logistic function standard curve on double-logarithmic graph paper, with concentration as the horizontal axis and OD value as the vertical axis. 2. If the sample OD value is above the upper limit of the standard curve, dilute the sample appropriately and re-measure. Multiply the sample concentration by the corresponding dilution factor. |
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| Sensitivity | 6.1 ng/mL | ||||||||||||||||||||||||||||||||||||
| Theory | This kit uses a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA). Samples, standards, biotin-labeled detection antibodies, and HRP conjugates are sequentially added to microwells pre-coated with human calprotectin (CALP) capture antibodies. After incubation and washing, the sample is developed with the substrate TMB. TMB converts to blue under the catalysis of HRP and to yellow under the action of acid. The intensity of the color is positively correlated with the amount of human calprotectin (CALP) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | ||||||||||||||||||||||||||||||||||||
| Source | Human | ||||||||||||||||||||||||||||||||||||
| Synonym | Human Calprotectin ELISA Kit | ||||||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | ||||||||||||||||||||||||||||||||||||
| Composition |
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| Background | Calprotectin (CALP) is a calcium-binding protein (approximately 36 kDa) produced by neutrophils, constituting 5% of total cellular protein. It exhibits tissue- or cell-specific properties and serves as a marker of acute inflammatory cell activation. It is composed of a calcium-binding protein heterotrimer covalently linked by two 14 kDa heavy chains and one 8 kDa light chain. It exhibits heat resistance and enhanced hydrolysis resistance. It was first isolated from neutrophils by Fagerhol et al. in 1980 and named L1 protein. Dorin and Freemont et al. further confirmed that this protein shares structural features with the S-100 protein and subsequently named this protective, multifunctional, calcium-binding protein calprotectin. | ||||||||||||||||||||||||||||||||||||
| General Notes | 1. Strictly adhere to the specified incubation time and temperature to ensure accurate results. All reagents must be at room temperature (20-25°C) before use. Refrigerate reagents immediately after use. 2. Improper plate washing may result in inaccurate results. Ensure that all liquid in the wells is aspirated thoroughly before adding substrate. Do not allow the wells to dry out during incubation. 3. Remove any residual liquid and fingerprints from the bottom of the plate, as this will affect the OD value. 4. The substrate developer solution should be colorless or very light in color. Do not use substrate solution that has turned blue. 5. Avoid cross-contamination of reagents and specimens to prevent erroneous results. 6. Avoid direct exposure to strong light during storage and incubation. 7. Do not expose any reagents to bleaching solvents or the strong fumes emitted by bleaching solvents. Any bleaching agent will destroy the biological activity of the reagents in the kit. 8. Do not use expired products, and do not mix components with different product numbers and batches. 9. Recombinant proteins from sources other than the kit may not be compatible with the antibodies in this kit and will not be recognized. 10. If there is a possibility of disease transmission, all samples should be managed properly and samples and testing devices should be handled according to prescribed procedures. |
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| Storage Temp. | If the unopened kit is stored at 4°C, the shelf life is 6 months. | ||||||||||||||||||||||||||||||||||||
| Test Range | 11.25-720 ng/mL | ||||||||||||||||||||||||||||||||||||
| Applications | Serum, plasma, tissue homogenates, cell culture supernatants, stool samples, and other biological fluids |
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4.3 ★★★★★
Based on 25 reviews
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Product Reviews
★★★★★ 5
An indispensible Hebrew Language resource.
Format: Hardcover
This book is an amazing resource for anyone who has completed preliminary Hebrew such as with Pratico and Van Pelt in their Basics of Biblical Hebrew. This book goes above and beyond, it gives the diagnostic origin patterns for adjectives, patterns in noun formation as well as other components of speech, contrasts each of the seven major verb forms and the minor forms as well, and gives an excellent evolution of the utilization of the Hebrew language and its update in characters.
It explains in its initial history a lot of unique statements in the Torah, and illustrations throughout give the reader an excellent idea about the Hebrew Scriptures. I bought this to aid in designing a language studies course at seminary and it has stood me in great help so far, going beyond anything I would have expected to get out of an intermediate level grammar and is an amazing, invaluable, and priceless resource, both to the professor, and the student. I recommend all people taking Hebrew purchase this book and add it to their collection and read it through cover to cover as well as using it as a topical reference guide.
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Reviewed in the United States on October 30, 2016
★★★★★ 5
can potentially be overwhelming
Format: Hardcover
This is a good resource for introduction to syntax. However, the vast amount of examples has benefits as well as disadvantages. The benefit is that it is comprehensive in information which makes it good for a reference. The disadvantage is that the many subcategories can cause one to be discouraged as the quantity of information may be overwhelming. Nonetheless that is the nature of learning a language.
It is still a good resource if you can maintain the brain power to understand the technical language if you have a novice background to linguistics. The volume could be more pedagogical however that would cater to the self learner and perhaps that was not the intent of the author. This volume seems to be designed for the classroom setting. However it is still decent for the self learner.
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Reviewed in the United States on June 30, 2014
★★★★★ 5
Favorite Hebrew Reference Book
Format: Hardcover
This is the Hebrew grammar that I use the most often. While I also use Williams and Arnold and Choi, this grammar is more comprehensive, and its size certainly reflects this. It probably betrays my inclination towards the exactness of Greek to like this grammar so much. As my Hebrew professor often tells us, all these grammar categories are contrived to some extent, so at some point it isn't helpful anymore to go further in depth on the uses of the so-called "cases."
All in all, I usually find it easy to locate the grammatical point in which I am interested, though sometimes I have to resort to looking up every instance a Hebrew particle occurs throughout the book. For this looking up of various nuances of a particle, I sometimes find Williams more user friendly. Nevertheless, Waltke and O'Connor's volume has been my resource of choice.
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Reviewed in the United States on November 6, 2012
★★★★★ 4
deep
Format: Hardcover
If you are a serious student of Hebrew this is the only way to go. The book explains how the language is built from the ground up. If you are like me you have found learning Hebrew challenging (that's a joke). I have tried learning vocabulary, rote memorization, even coming up with silly English sentences to help me remember words (The goddess "Hera" is quick) (sad, I know). This tool is essential approaching the language from the building block side of things and I am now able to look at the prayer book and recognize forms and tenses I never understood before. It is for the serious student who REALLY wants to learn the language.
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Reviewed in the United States on February 27, 2009
★★★★★ 5
Can't do Without
Format: Hardcover
I have been a fan of Gesenius for a long time. While I knew there was updated information to consider, I thought his text was enough. But although his text is certainly still necessary, and necessarily covers issues that the book reviewed here does not, it is clear that one must turn to Waltke & O'Connor to reach a greater level of accuracy. Writing so long after Gesenius, they have presented new and accurate information based on the latest research, not only of Ancient Hebrew, but also based on cognate Semitic languages.
I have found the sections on Hebrew verbs the most enlightening and correcting, e.g., that the Pi'el stem is NOT intensive. Such intenseness must be detected from a combination of the lexical verb meaning, the stem, and the text/co-text. This book also presents other up-to-date information on the forming of nouns from verbs, etc.
As others have noted, the layout and presentation is great. It really can't be presented any easier, I don't think.
One must read Gesenius, for he presents very important information across the board, including much on phonology and linguistics not covered by Waltke & O'Connor. But to stick with Gesenius will leave one in error on some points. Waltke & O'Connor, simply put, make the corrections and make studying Biblical Hebrew exciting, and even much easier to learn.
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Reviewed in the United States on January 13, 2004
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