Human Immunology ELISA Kits 1
Human HP (Haptoglobin) CLIA Kit (HUES00855)
- SKU:
- HUES00855
- Product Type:
- ELISA Kit
- ELISA Type:
- CLIA Kit
- Size:
- 96 Assays
- Sensitivity:
- 1.88ng/mL
- Range:
- 3.13-200ng/mL
- ELISA Type:
- Sandwich
- Synonyms:
- Hpt, BP, Hp2-Alpha, HPA1S
- Reactivity:
- Human
- Sample Type:
- Serum, plasma and other biological fluids
- Research Area:
- Immunology
Description
| Assay type: | Sandwich |
| Format: | 96T |
| Assay time: | 4.5h |
| Reactivity: | Human |
| Detection method: | Chemiluminescence |
| Detection range: | 3.13-200 ng/mL |
| Sensitivity: | 1.88 ng/mL |
| Sample volume: | 100µL |
| Sample type: | Serum, plasma and other biological fluids |
| Repeatability: | CV < 15% |
| Specificity: | This kit recognizes Human HP in samples. No significant cross-reactivity or interference between Human HP 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 HP. 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 HP 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 HP, 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 HP. The concentration of Human HP in the samples can be calculated by comparing the RLU of the samples to the standard curve.
| UniProt Protein Function: | HP: Haptoglobin combines with free plasma hemoglobin, preventing loss of iron through the kidneys and protecting the kidneys from damage by hemoglobin, while making the hemoglobin accessible to degradative enzymes. Defects in HP are the cause of anhaptoglobinemia (AHP). AHP is a condition characterized by the absence of the serum glycoprotein haptoglobin. Serum levels of haptoglobin vary among normal persons: levels are low in the neonatal period and in the elderly, differ by population, and can be influenced by environmental factors, such as infection. Secondary hypohaptoglobinemia can occur as a consequence of hemolysis, during which haptoglobin binds to free hemoglobin. Belongs to the peptidase S1 family. |
| UniProt Protein Details: | Protein type:Secreted, signal peptide; Secreted Chromosomal Location of Human Ortholog: 16q22. 2 Cellular Component: extracellular space; extracellular region Molecular Function:antioxidant activity; protein binding; hemoglobin binding; catalytic activity Biological Process: receptor-mediated endocytosis; response to hydrogen peroxide; immune system process; metabolic process; defense response to bacterium; negative regulation of oxidoreductase activity; acute-phase response; defense response Disease: Anhaptoglobinemia |
| NCBI Summary: | This gene encodes a preproprotein, which is processed to yield both alpha and beta chains, which subsequently combine as a tetramer to produce haptoglobin. Haptoglobin functions to bind free plasma hemoglobin, which allows degradative enzymes to gain access to the hemoglobin, while at the same time preventing loss of iron through the kidneys and protecting the kidneys from damage by hemoglobin. Mutations in this gene and/or its regulatory regions cause ahaptoglobinemia or hypohaptoglobinemia. This gene has also been linked to diabetic nephropathy, the incidence of coronary artery disease in type 1 diabetes, Crohn's disease, inflammatory disease behavior, primary sclerosing cholangitis, susceptibility to idiopathic Parkinson's disease, and a reduced incidence of Plasmodium falciparum malaria. The protein encoded also exhibits antimicrobial activity against bacteria. A similar duplicated gene is located next to this gene on chromosome 16. Multiple transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Oct 2014] |
| UniProt Code: | P00738 |
| NCBI GenInfo Identifier: | 123508 |
| NCBI Gene ID: | 3240 |
| NCBI Accession: | P00738. 1 |
| UniProt Secondary Accession: | P00738,P00737, Q0VAC4, Q0VAC5, Q2PP15, Q3B7J0, Q6LBY9 Q9UC67, B0AZL5, |
| UniProt Related Accession: | P00738 |
| Molecular Weight: | |
| NCBI Full Name: | Haptoglobin |
| NCBI Synonym Full Names: | haptoglobin |
| NCBI Official Symbol: | HP |
| NCBI Official Synonym Symbols: | BP; HPA1S; HP2ALPHA2 |
| NCBI Protein Information: | haptoglobin; zonulin; binding peptide; haptoglobin alpha(1S)-beta; haptoglobin alpha(2FS)-beta; haptoglobin, beta polypeptide; haptoglobin, alpha polypeptide |
| UniProt Protein Name: | Haptoglobin |
| UniProt Synonym Protein Names: | ZonulinCleaved into the following 2 chains:Haptoglobin alpha chain; Haptoglobin beta chain |
| Protein Family: | Haptoglobin |
| UniProt Gene Name: | HP |
| UniProt Entry Name: | HPT_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 |
| 200 | 44019 44127 | 44073 | 44035 |
| 100 | 16751 19671 | 18211 | 18173 |
| 50 | 8296 8116 | 8206 | 8168 |
| 25 | 3900 3970 | 3935 | 3897 |
| 12.5 | 2117 1847 | 1982 | 1944 |
| 6.25 | 1132 972 | 1052 | 1014 |
| 3.13 | 581 615 | 598 | 560 |
| 0 | 36 40 | 38 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human HP were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human HP 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) | 10.03 | 31.34 | 88.62 | 9.59 | 31.31 | 85.18 |
| Standard deviation | 1.15 | 2.79 | 6.66 | 1.15 | 3.24 | 7.15 |
| C V (%) | 11.47 | 8.90 | 7.52 | 11.99 | 10.35 | 8.39 |
Recovery
The recovery of Human HP 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) | 99-110 | 105 |
| EDTA plasma (n=5) | 84-97 | 90 |
| Cell culture media (n=5) | 91-103 | 97 |
Linearity
Samples were spiked with high concentrations of Human HP 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 (%) | 96-114 | 84-98 | 86-98 |
| Average (%) | 104 | 90 | 91 | |
| 1:4 | Range (%) | 85-97 | 95-109 | 91-108 |
| Average (%) | 90 | 101 | 98 | |
| 1:8 | Range (%) | 99-111 | 89-105 | 87-99 |
| Average (%) | 105 | 96 | 94 | |
| 1:16 | Range (%) | 92-105 | 86-97 | 100-117 |
| Average (%) | 99 | 91 | 107 |
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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