Description
Human CA3 (Carbonic Anhydrase III, Muscle Specific) ELISA Kit
The Human CA3 (Carbonic Anhydrase III) Muscle Specific ELISA Kit is specifically designed for the accurate detection of CA3 levels in human samples such as serum, plasma, and cell culture supernatants. With its high sensitivity and specificity, this kit ensures reliable and reproducible results, making it an ideal tool for a wide range of research applications.CA3, a member of the carbonic anhydrase enzyme family, is predominantly expressed in skeletal muscle tissue and plays a crucial role in the regulation of acid-base balance.
Dysregulation of CA3 has been implicated in various muscle-related disorders, making it a valuable biomarker for studying muscle health and disease progression.By accurately measuring CA3 levels in human samples, researchers can gain insights into muscle metabolism, exercise physiology, and potential therapeutic targets for muscle disorders. The Human CA3 Muscle Specific ELISA Kit provides researchers with a reliable and efficient tool for studying the role of CA3 in muscle biology and disease.
| 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 CA3 in samples. No significant cross-reactivity or interference between Human CA3 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 CA3. 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 CA3 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 CA3, 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 CA3. The concentration of Human CA3 in samples can be calculated by comparing the OD of the samples to the standard curve.
| UniProt Protein Function: | CA3: Reversible hydration of carbon dioxide. Belongs to the alpha-carbonic anhydrase family. |
| UniProt Protein Details: | Protein type:Energy Metabolism - nitrogen; EC 4. 2. 1. 1; Lyase Chromosomal Location of Human Ortholog: 8q21. 2 Cellular Component: cytosol Molecular Function:carbonate dehydratase activity; zinc ion binding; nickel ion binding Biological Process: response to ethanol; bicarbonate transport; one-carbon compound metabolic process; response to oxidative stress |
| NCBI Summary: | Carbonic anhydrase III (CAIII) is a member of a multigene family (at least six separate genes are known) that encodes carbonic anhydrase isozymes. These carbonic anhydrases are a class of metalloenzymes that catalyze the reversible hydration of carbon dioxide and are differentially expressed in a number of cell types. The expression of the CA3 gene is strictly tissue specific and present at high levels in skeletal muscle and much lower levels in cardiac and smooth muscle. A proportion of carriers of Duchenne muscle dystrophy have a higher CA3 level than normal. The gene spans 10. 3 kb and contains seven exons and six introns. [provided by RefSeq, Oct 2008] |
| UniProt Code: | P07451 |
| NCBI GenInfo Identifier: | 134047703 |
| NCBI Gene ID: | 761 |
| NCBI Accession: | P07451. 3 |
| UniProt Secondary Accession: | P07451,O60842, B2R867, B3KUC8, |
| UniProt Related Accession: | P07451 |
| Molecular Weight: | 260 |
| NCBI Full Name: | Carbonic anhydrase 3 |
| NCBI Synonym Full Names: | carbonic anhydrase III, muscle specific |
| NCBI Official Symbol: | CA3 |
| NCBI Official Synonym Symbols: | Car3; CAIII |
| NCBI Protein Information: | carbonic anhydrase 3; CA-III; HEL-S-167mP; carbonate dehydratase III; epididymis secretory sperm binding protein Li 167mP |
| UniProt Protein Name: | Carbonic anhydrase 3 |
| UniProt Synonym Protein Names: | Carbonate dehydratase III; Carbonic anhydrase III; CA-III |
| Protein Family: | Carbonic anhydrase |
| UniProt Gene Name: | CA3 |
| UniProt Entry Name: | CAH3_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 | 2.394 2.418 | 2.406 | 2.344 |
| 100 | 1.563 1.567 | 1.565 | 1.503 |
| 50 | 0.897 0.883 | 0.89 | 0.828 |
| 25 | 0.419 0.453 | 0.436 | 0.374 |
| 12.5 | 0.213 0.201 | 0.207 | 0.145 |
| 6.25 | 0.153 0.151 | 0.152 | 0.09 |
| 3.13 | 0.101 0.117 | 0.109 | 0.047 |
| 0 | 0.054 0.07 | 0.062 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human CA3 were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human CA3 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) | 9.90 | 18.49 | 79.37 | 10.46 | 18.74 | 74.14 |
| Standard deviation | 0.65 | 1.11 | 4.24 | 0.73 | 1.07 | 4.00 |
| C V (%) | 6.57 | 6.00 | 5.34 | 6.98 | 5.71 | 5.40 |
Recovery
The recovery of Human CA3 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) | 94-106 | 100 |
| EDTA plasma (n=5) | 88-100 | 94 |
| Cell culture media (n=5) | 87-102 | 94 |
Linearity
Samples were spiked with high concentrations of Human CA3 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 (%) | 87-100 | 92-104 | 87-101 |
| Average (%) | 92 | 97 | 92 | |
| 1:4 | Range (%) | 87-101 | 85-98 | 82-94 |
| Average (%) | 93 | 91 | 88 | |
| 1:8 | Range (%) | 89-101 | 83-95 | 89-99 |
| Average (%) | 95 | 88 | 94 | |
| 1:16 | Range (%) | 88-102 | 86-98 | 86-99 |
| Average (%) | 95 | 92 | 92 |
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.
