Human Cell Biology ELISA Kits 3
Human alphaFP (Alpha-Fetoprotein) CLIA Kit (HUES00068)
- SKU:
- HUES00068
- Product Type:
- ELISA Kit
- ELISA Type:
- CLIA Kit
- Size:
- 96 Assays
- Sensitivity:
- 0.19ng/mL
- Range:
- 0.31-20ng/mL
- ELISA Type:
- Sandwich
- Synonyms:
- AFP, FETA, HPAFP, Alpha-fetoglobulin, oralpha fetal protein
- Reactivity:
- Human
- Sample Type:
- Serum, plasma and other biological fluids
- Research Area:
- Cell Biology
Description
| Assay type: | Sandwich |
| Format: | 96T |
| Assay time: | 4.5h |
| Reactivity: | Human |
| Detection method: | Chemiluminescence |
| Detection range: | 0.31-20 ng/mL |
| Sensitivity: | 0.19 ng/mL |
| Sample volume: | 100µL |
| Sample type: | Serum, plasma and other biological fluids |
| Repeatability: | CV < 15% |
| Specificity: | This kit recognizes Human alphaFP in samples. No significant cross-reactivity or interference between Human alphaFP and analogues was observed. |
This kit uses Sandwich-CLIA as the method. The micro CLIA plate provided in this kit has been pre-coated with an antibody specific to Human alphaFP. Standards or samples are added to the appropriate micro CLIA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Human alphaFP 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 alphaFP, biotinylated detection antibody and Avidin-HRP conjugate will appear fluorescence. The Relative light unit (RLU) value is measured spectrophotometrically by the Chemiluminescence immunoassay analyzer. The RLU value is positively associated with the concentration of Human alphaFP. The concentration of Human alphaFP in the samples can be calculated by comparing the RLU of the samples to the standard curve.
| UniProt Protein Function: | AFP: Binds copper, nickel, and fatty acids as well as, and bilirubin less well than, serum albumin. Only a small percentage (less than 2%) of the human AFP shows estrogen-binding properties. Dimeric and trimeric forms have been found in addition to the monomeric form. Plasma. Synthesized by the fetal liver and yolk sac. Belongs to the ALB/AFP/VDB family. |
| UniProt Protein Details: | Protein type:Secreted, signal peptide; Secreted Chromosomal Location of Human Ortholog: 4q13. 3 Cellular Component: extracellular space; cytoplasm Molecular Function:metal ion binding Biological Process: transport; progesterone metabolic process; ovulation from ovarian follicle Disease: Alpha-fetoprotein Deficiency; Alpha-fetoprotein, Hereditary Persistence Of |
| NCBI Summary: | This gene encodes alpha-fetoprotein, a major plasma protein produced by the yolk sac and the liver during fetal life. Alpha-fetoprotein expression in adults is often associated with hepatoma or teratoma. However, hereditary persistance of alpha-fetoprotein may also be found in individuals with no obvious pathology. The protein is thought to be the fetal counterpart of serum albumin, and the alpha-fetoprotein and albumin genes are present in tandem in the same transcriptional orientation on chromosome 4. Alpha-fetoprotein is found in monomeric as well as dimeric and trimeric forms, and binds copper, nickel, fatty acids and bilirubin. The level of alpha-fetoprotein in amniotic fluid is used to measure renal loss of protein to screen for spina bifida and anencephaly. [provided by RefSeq, Jul 2008] |
| UniProt Code: | P02771 |
| NCBI GenInfo Identifier: | 120042 |
| NCBI Gene ID: | 174 |
| NCBI Accession: | P02771. 1 |
| UniProt Secondary Accession: | P02771,B2RBU3, |
| UniProt Related Accession: | P02771 |
| Molecular Weight: | 609 |
| NCBI Full Name: | Alpha-fetoprotein |
| NCBI Synonym Full Names: | alpha-fetoprotein |
| NCBI Official Symbol: | AFP |
| NCBI Official Synonym Symbols: | AFPD; FETA; HPAFP |
| NCBI Protein Information: | alpha-fetoprotein; alpha-fetoglobulin; alpha-1-fetoprotein |
| UniProt Protein Name: | Alpha-fetoprotein |
| UniProt Synonym Protein Names: | Alpha-1-fetoprotein; Alpha-fetoglobulin |
| Protein Family: | AFP |
| UniProt Gene Name: | AFP |
| UniProt Entry Name: | FETA_HUMAN |
As the RLU 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) | RLU | Average | Corrected |
| 20 | 33361 34603 | 33982 | 33950 |
| 10 | 11345 13595 | 12470 | 12438 |
| 5 | 5495 5005 | 5250 | 5218 |
| 2.5 | 2396 2652 | 2524 | 2492 |
| 1.25 | 1445 1321 | 1383 | 1351 |
| 0.63 | 882 852 | 867 | 835 |
| 0.31 | 582 664 | 623 | 591 |
| 0 | 31 33 | 32 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human alphaFP were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human alphaFP 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) | 1.10 | 2.72 | 7.51 | 1.00 | 2.89 | 7.49 |
| Standard deviation | 0.12 | 0.29 | 0.81 | 0.10 | 0.22 | 0.86 |
| C V (%) | 10.91 | 10.66 | 10.79 | 10.00 | 7.61 | 11.48 |
Recovery
The recovery of Human alphaFP 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) | 94-109 | 100 |
| EDTA plasma (n=5) | 99-112 | 106 |
| Cell culture media (n=5) | 87-100 | 92 |
Linearity
Samples were spiked with high concentrations of Human alphaFP 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 (%) | 101-120 | 88-101 | 99-110 |
| Average (%) | 109 | 94 | 105 | |
| 1:4 | Range (%) | 84-95 | 94-106 | 98-111 |
| Average (%) | 90 | 99 | 105 | |
| 1:8 | Range (%) | 92-108 | 87-101 | 85-98 |
| Average (%) | 99 | 94 | 92 | |
| 1:16 | Range (%) | 94-106 | 88-98 | 101-114 |
| Average (%) | 100 | 93 | 108 |
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 CLIA 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 A | 1 vial, 5 mL | 4°C (shading light) |
| Substrate Reagent B | 1 vial, 5 mL | 4°C (shading light) |
| 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 100µl of standard solutions into the standard wells.
- Add 100µl of Sample / Standard dilution buffer into the control (zero) well.
- Add 100µl 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 100µL 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 350µL 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 100µL 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 100µL of Substrate mixture solution to each well. Cover with a new plate seal andincubate for no more than 5 min at 37°C. Protect the plate from light.
- Determine the RLU value of each well immediately.
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