Description
Human CCNB (Cyclin-B) ELISA Kit
The Human CCNB (Cyclin B) ELISA Kit is a powerful tool for researchers looking to accurately measure Cyclin B levels in human samples. This kit provides high sensitivity and specificity, ensuring precise and consistent results for serum, plasma, and cell culture supernatants.Cyclin B is a key regulatory protein that controls cell cycle progression, specifically at the G2/M transition. Dysregulation of Cyclin B has been linked to various diseases, including cancer and reproductive disorders.
By using this ELISA kit, researchers can study the role of Cyclin B in these conditions and potentially identify new therapeutic targets.Overall, the Human CCNB ELISA Kit offers a reliable and efficient solution for studying Cyclin B dynamics and its implications in disease progression.
| Assay type: | Sandwich |
| Format: | 96T |
| Assay time: | 4.5h |
| Reactivity: | Human |
| Detection Method: | Colormetric |
| Detection Range: | 0.31-20 ng/mL |
| Sensitivity: | 0.19 ng/mL |
| Sample Volume Required Per Well: | 100µL |
| Sample Type: | Serum, plasma and other biological fluids |
| Specificity: | This kit recognizes Human CCNB in samples. No significant cross-reactivity or interference between Human CCNB 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 CCNB. 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 CCNB 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 CCNB, 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 CCNB. The concentration of Human CCNB in samples can be calculated by comparing the OD of the samples to the standard curve.
| UniProt Protein Function: | CCNB1: a member of the highly conserved cyclin family, whose members are characterized by a dramatic periodicity in protein abundance through the cell cycle. Cyclins function as regulators of CDK kinases. Complexes with cdc2 to form the maturation-promoting factor (MPF). Two alternatively spliced isoforms have been found, a constitutively expressed form and a cell cycle-regulated form that is expressed predominantly during G2/M phase. |
| UniProt Protein Details: | Protein type:Cell cycle regulation; Activator Chromosomal Location of Human Ortholog: 5q12 Cellular Component: nucleoplasm; spindle pole; centrosome; membrane; cytoplasm; nucleus; cytosol Molecular Function:protein binding; patched binding; histone kinase activity; protein kinase binding Biological Process: positive regulation of ubiquitin-protein ligase activity during mitotic cell cycle; positive regulation of histone phosphorylation; regulation of cell cycle; ventricular cardiac muscle cell development; positive regulation of attachment of spindle microtubules to kinetochore; mitotic nuclear envelope disassembly; positive regulation of mRNA 3'-end processing; positive regulation of mitotic cell cycle; positive regulation of fibroblast proliferation; negative regulation of protein amino acid phosphorylation; tissue regeneration; anaphase-promoting complex-dependent proteasomal ubiquitin-dependent protein catabolic process; protein complex assembly; G2/M transition of mitotic cell cycle; positive regulation of cardiac muscle cell proliferation; response to drug; in utero embryonic development; oocyte maturation; mitotic metaphase plate congression; response to DDT; gut development; regulation of ubiquitin-protein ligase activity during mitotic cell cycle; response to mechanical stimulus; cell division; spermatogenesis; regulation of cyclin-dependent protein kinase activity; mitotic cell cycle; mitotic spindle stabilization; G1/S transition of mitotic cell cycle |
| NCBI Summary: | The protein encoded by this gene is a regulatory protein involved in mitosis. The gene product complexes with p34(cdc2) to form the maturation-promoting factor (MPF). Two alternative transcripts have been found, a constitutively expressed transcript and a cell cycle-regulated transcript, that is expressed predominantly during G2/M phase. The different transcripts result from the use of alternate transcription initiation sites. [provided by RefSeq, Jul 2008] |
| UniProt Code: | P14635 |
| NCBI GenInfo Identifier: | 116176 |
| NCBI Gene ID: | 891 |
| NCBI Accession: | P14635. 1 |
| UniProt Secondary Accession: | P14635,Q5TZP9, A8K066, |
| UniProt Related Accession: | P14635 |
| Molecular Weight: | 44,102 Da |
| NCBI Full Name: | G2/mitotic-specific cyclin-B1 |
| NCBI Synonym Full Names: | cyclin B1 |
| NCBI Official Symbol: | CCNB1 |
| NCBI Official Synonym Symbols: | CCNB |
| NCBI Protein Information: | G2/mitotic-specific cyclin-B1; G2/mitotic-specific cyclin B1 |
| UniProt Protein Name: | G2/mitotic-specific cyclin-B1 |
| UniProt Gene Name: | CCNB1 |
| UniProt Entry Name: | CCNB1_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 |
| 20 | 2.457 2.499 | 2.478 | 2.393 |
| 10 | 1.678 1.68 | 1.679 | 1.594 |
| 5 | 0.927 0.903 | 0.915 | 0.83 |
| 2.5 | 0.437 0.447 | 0.442 | 0.357 |
| 1.25 | 0.256 0.25 | 0.253 | 0.168 |
| 0.63 | 0.189 0.181 | 0.185 | 0.1 |
| 0.31 | 0.132 0.14 | 0.136 | 0.051 |
| 0 | 0.075 0.095 | 0.085 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human CCNB were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human CCNB 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) | 0.94 | 2.89 | 8.63 | 0.99 | 2.83 | 8.10 |
| Standard deviation | 0.06 | 0.16 | 0.31 | 0.07 | 0.16 | 0.36 |
| C V (%) | 6.38 | 5.54 | 3.59 | 7.07 | 5.65 | 4.44 |
Recovery
The recovery of Human CCNB 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) | 92-103 | 98 |
| EDTA plasma (n=5) | 88-104 | 95 |
| Cell culture media (n=5) | 95-106 | 100 |
Linearity
Samples were spiked with high concentrations of Human CCNB 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 (%) | 88-102 | 95-108 | 92-106 |
| Average (%) | 93 | 101 | 98 | |
| 1:4 | Range (%) | 92-105 | 80-94 | 82-95 |
| Average (%) | 100 | 87 | 87 | |
| 1:8 | Range (%) | 90-106 | 79-90 | 83-96 |
| Average (%) | 97 | 85 | 88 | |
| 1:16 | Range (%) | 89-101 | 80-93 | 88-98 |
| Average (%) | 96 | 86 | 93 |
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.
