Description
Human CA2 (Carbonic Anhydrase II) ELISA Kit
The Human CA2 (Carbonic Anhydrase II) ELISA Kit is specifically designed for the precise quantification of CA2 levels in human samples including serum, plasma, and cell culture supernatants. This ELISA kit offers high sensitivity and specificity, ensuring accurate and reproducible results for a variety of research applications.CA2 is a key enzyme involved in maintaining acid-base balance and electrolyte transport in the body. It plays a critical role in various physiological processes such as bone resorption, calcification, and pH regulation.
Dysregulation of CA2 has been linked to conditions like osteoporosis, renal tubular acidosis, and glaucoma, highlighting its importance as a biomarker for studying these diseases and potential therapeutic interventions.Overall, the Human CA2 ELISA Kit is a valuable tool for researchers seeking to investigate the role of CA2 in health and disease, offering reliable and informative data for further study and clinical applications.
| Assay type: | Sandwich |
| Format: | 96T |
| Assay time: | 4.5h |
| Reactivity: | Human |
| Detection Method: | Colormetric |
| Detection Range: | 3.13-200 ng/mL |
| Sensitivity: | 1.88 ng/mL |
| Sample Volume Required Per Well: | 100µL |
| Sample Type: | Serum, plasma and other biological fluids |
| Specificity: | This kit recognizes Human CA2 in samples. No significant cross-reactivity or interference between Human CA2 and analogues was observed. |
This ELISA kit uses Sandwich-ELISA as the method. The micro ELISA plate provided in this kit has been pre-coated with an antibody specific to Human CA2. Standards or samples are added to the appropriate micro ELISA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Human CA2 and Avidin-Horseradish Peroxidase (HRP) conjugate are added to each micro plate well successively and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain Human CA2, biotinylated detection antibody and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by adding Stop Solution and the color turns yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The OD value is proportional to the concentration of Human CA2. The concentration of Human CA2 in samples can be calculated by comparing the OD of the samples to the standard curve.
| UniProt Protein Function: | CA2: Essential for bone resorption and osteoclast differentiation. Reversible hydration of carbon dioxide. Can hydrates cyanamide to urea. Involved in the regulation of fluid secretion into the anterior chamber of the eye. Interacts with SLC4A4. Interaction with SLC4A7 regulates SLC4A7 transporter activity. Activated by X-ray, histamine, L-adrenaline, L- and D-phenylalanine, L- and D-histidine, L-His-OMe and beta-Ala- His (carnosine). Competitively inhibited by saccharin, thioxolone, coumarins, 667-coumate, celecoxib (Celebrex), valdecoxib (Bextra), SC-125, SC-560, diclofenac, acetate, azide, bromide, sulfonamide derivatives such as acetazolamide (AZA), methazolamide (MZA), ethoxzolamide (EZA), dichlorophenamide (DCP), brinzolamide, dansylamide, thiabendazole-5-sulfonamide, trifluoromethane sulfonamide and N-hydroxysulfamide, fructose-based sugar sulfamate RWJ-37497, and Foscarnet (phosphonoformate trisodium salt). Repressed strongly by hydrogen sulfide(HS) and weakly by nitrate (NO(3)). Esterase activity weakly reduced by cyanamide. N- hydroxyurea interfers with zinc binding and inhibit activity. Belongs to the alpha-carbonic anhydrase family. |
| UniProt Protein Details: | Protein type:Lyase; EC 4. 2. 1. 1; Energy Metabolism - nitrogen Chromosomal Location of Human Ortholog: 8q22 Cellular Component: extracellular space; microvillus; apical part of cell; axon; basolateral plasma membrane; cytoplasm; plasma membrane; cytosol; myelin sheath Molecular Function:protein binding; carbonate dehydratase activity; zinc ion binding Biological Process: secretion; positive regulation of osteoclast differentiation; positive regulation of synaptic transmission, GABAergic; positive regulation of cellular pH reduction; one-carbon compound metabolic process; odontogenesis of dentine-containing teeth; response to zinc ion; bicarbonate transport; response to estrogen stimulus; regulation of intracellular pH; positive regulation of bone resorption; morphogenesis of an epithelium; kidney development; response to pH Disease: Osteopetrosis, Autosomal Recessive 3 |
| NCBI Summary: | The protein encoded by this gene is one of several isozymes of carbonic anhydrase, which catalyzes reversible hydration of carbon dioxide. Defects in this enzyme are associated with osteopetrosis and renal tubular acidosis. Two transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Jun 2014] |
| UniProt Code: | P00918 |
| NCBI GenInfo Identifier: | 115456 |
| NCBI Gene ID: | 760 |
| NCBI Accession: | P00918. 2 |
| UniProt Secondary Accession: | P00918,Q6FI12, Q96ET9, B2R7G8, |
| UniProt Related Accession: | P00918 |
| Molecular Weight: | 260 |
| NCBI Full Name: | Carbonic anhydrase 2 |
| NCBI Synonym Full Names: | carbonic anhydrase II |
| NCBI Official Symbol: | CA2 |
| NCBI Official Synonym Symbols: | CAC; CAII; Car2; CA-II; HEL-76 |
| NCBI Protein Information: | carbonic anhydrase 2; carbonic anhydrase B; carbonic anhydrase C; carbonic dehydratase; carbonate dehydratase II; epididymis luminal protein 76 |
| UniProt Protein Name: | Carbonic anhydrase 2 |
| UniProt Synonym Protein Names: | Carbonate dehydratase II; Carbonic anhydrase C; CAC; Carbonic anhydrase II; CA-II |
| Protein Family: | Beta-carotene hydroxylase |
| UniProt Gene Name: | CA2 |
| UniProt Entry Name: | CAH2_HUMAN |
As the OD values of the standard curve may vary according to the conditions of the actual assay performance (e. g. operator, pipetting technique, washing technique or temperature effects), the operator should establish a standard curve for each test. Typical standard curve and data is provided below for reference only.
| Concentration (ng/mL) | O.D | Average | Corrected |
| 200 | 2.161 2.207 | 2.184 | 2.129 |
| 100 | 1.448 1.49 | 1.469 | 1.414 |
| 50 | 0.839 0.833 | 0.836 | 0.781 |
| 25 | 0.42 0.424 | 0.422 | 0.367 |
| 12.5 | 0.247 0.225 | 0.236 | 0.181 |
| 6.25 | 0.159 0.133 | 0.146 | 0.091 |
| 3.13 | 0.093 0.113 | 0.103 | 0.048 |
| 0 | 0.052 0.058 | 0.055 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human CA2 were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human CA2 were tested on 3 different plates, 20 replicates in each plate.
| Intra-assay Precision | Inter-assay Precision | |||||
| Sample | 1 | 2 | 3 | 1 | 2 | 3 |
| n | 20 | 20 | 20 | 20 | 20 | 20 |
| Mean (ng/mL) | 9.12 | 27.51 | 77.27 | 8.25 | 26.56 | 79.73 |
| Standard deviation | 0.55 | 1.40 | 3.21 | 0.54 | 1.34 | 2.70 |
| C V (%) | 6.03 | 5.09 | 4.15 | 6.55 | 5.05 | 3.39 |
Recovery
The recovery of Human CA2 spiked at three different levels in samples throughout the range of the assay was evaluated in various matrices.
| Sample Type | Range (%) | Average Recovery (%) |
| Serum (n=5) | 86-101 | 93 |
| EDTA plasma (n=5) | 90-107 | 98 |
| Cell culture media (n=5) | 85-98 | 92 |
Linearity
Samples were spiked with high concentrations of Human CA2 and diluted with Reference Standard & Sample Diluent to produce samples with values within the range of the assay.
| Serum (n=5) | EDTA plasma (n=5) | Cell culture media (n=5) | ||
| 1:2 | Range (%) | 86-97 | 98-111 | 92-106 |
| Average (%) | 91 | 104 | 97 | |
| 1:4 | Range (%) | 89-106 | 86-99 | 85-97 |
| Average (%) | 97 | 92 | 90 | |
| 1:8 | Range (%) | 86-100 | 83-96 | 87-98 |
| Average (%) | 93 | 89 | 93 | |
| 1:16 | Range (%) | 91-108 | 81-93 | 85-98 |
| Average (%) | 99 | 88 | 91 |
An unopened kit can be stored at 4°C for 1 month. If the kit is not used within 1 month, store the items separately according to the following conditions once the kit is received.
| Item | Specifications | Storage |
| Micro ELISA Plate(Dismountable) | 8 wells ×12 strips | -20°C, 6 months |
| Reference Standard | 2 vials | |
| Concentrated Biotinylated Detection Ab (100×) | 1 vial, 120 µL | |
| Concentrated HRP Conjugate (100×) | 1 vial, 120 µL | -20°C(shading light), 6 months |
| Reference Standard & Sample Diluent | 1 vial, 20 mL | 4°C, 6 months |
| Biotinylated Detection Ab Diluent | 1 vial, 14 mL | |
| HRP Conjugate Diluent | 1 vial, 14 mL | |
| Concentrated Wash Buffer (25×) | 1 vial, 30 mL | |
| Substrate Reagent | 1 vial, 10 mL | 4°C(shading light) |
| Stop Solution | 1 vial, 10 mL | 4°C |
| Plate Sealer | 5 pieces | |
| Product Description | 1 copy | |
| Certificate of Analysis | 1 copy |
- Set standard, test sample and control (zero) wells on the pre-coated plate and record theirpositions. It is recommended to measure each standard and sample in duplicate. Note: addall solutions to the bottom of the plate wells while avoiding contact with the well walls. Ensuresolutions do not foam when adding to the wells.
- Aliquot 100 µL of standard solutions into the standard wells.
- Add 100 µL of Sample / Standard dilution buffer into the control (zero) well.
- Add 100 µL of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids) into test sample wells.
- Cover the plate with the sealer provided in the kit and incubate for 90 min at 37 °C.
- Aspirate the liquid from each well, do not wash. Immediately add 100 µL of BiotinylatedDetection Ab working solution to each well. Cover the plate with a plate seal and gently mix. Incubate for 1 hour at 37 °C.
- Aspirate or decant the solution from the plate and add 350 µL of wash buffer to each welland incubate for 1-2 minutes at room temperature. Aspirate the solution from each well andclap the plate on absorbent filter paper to dry. Repeat this process 3 times. Note: a microplatewasher can be used in this step and other wash steps.
- Add 100 µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37 °C.
- Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
- Add 90 µL of Substrate Reagent to each well. Cover with a new plate seal and incubate forapproximately 15 min at 37 °C. Protect the plate from light. Note: the reaction time can beshortened or extended according to the actual color change, but not by more than 30min.
- Add 50 µL of Stop Solution to each well. Note: Adding the stop solution should be done inthe same order as the substrate solution.
- Determine the optical density (OD value) of each well immediately with a microplate readerset at 450 nm.
