Human GIF (Gastric Intrinsic Factor) ELISA Kit (HUES01607)

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HUES01607
€539

Description

ELISA Kit Technical ManualMSDS

Human GIF (Gastric Intrinsic Factor) ELISA Kit

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 GIF . 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 GIF 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 GIF, 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 GIF. The concentration of Human GIF in samples can be calculated by comparing the OD of the samples to the standard curve.

Assay typeSandwich
Format96T
Assay time4.5h
ReactivityHuman
Detection MethodColormetric
Detection Range3.13-200 ng/mL
Sensitivity1.88 ng/mL
Sample Volume Required Per Well100uL
Sample TypeSerum, plasma and other biological fluids

Specificity

This kit recognizes Human GIF in samples. No significant cross-reactivity or interference between Human GIF and analogues was observed.

Typical Data

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
2002.437
2.449
2.4432.364
1001.602
1.628
1.6151.536
500.919
0.893
0.9060.827
250.432
0.45
0.4410.362
12.50.257
0.245
0.2510.172
6.250.183
0.161
0.1720.093
3.130.127
0.127
0.1270.048
00.079
0.079
0.079--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human GIF were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human GIF were tested on 3 different plates, 20 replicates in each plate.

Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (ng/mL)10.8523.7481.209.9823.5776.16
Standard deviation0.581.213.780.671.063.15
C V (%)5.355.104.666.714.504.14

Recovery

The recovery of Human GIF 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)92-10499
EDTA plasma (n=5)91-10799
Cell culture media (n=5)88-10495

Linearity

Samples were spiked with high concentrations of Human GIF 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)
1:2Range (%)96-11295-11394-107
Average (%)104103101
1:4Range (%)92-10684-9884-99
Average (%)998990
1:8Range (%)92-10680-9085-96
Average (%)998591
1:16Range (%)86-9886-9885-99
Average (%)929191

Kit Components & Storage

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 X12 strips-20'C, 6 months
Reference Standard2 vials
Concentrated Biotinylated Detection Ab (100X)1 vial, 120 uL
Concentrated HRP Conjugate (100X)1 vial, 120 uL-20'C(shading light), 6 months
Reference Standard & Sample Diluent1 vial, 20 mL4'C, 6 months
Biotinylated Detection Ab Diluent1 vial, 14 mL
HRP Conjugate Diluent1 vial, 14 mL
Concentrated Wash Buffer (25X)1 vial, 30 mL
Substrate Reagent1 vial, 10 mL4'C(shading light)
Stop Solution1 vial, 10 mL4'C
Plate Sealer5 pieces
Product Description1 copy
Certificate of Analysis1 copy

Human GIF (Gastric Intrinsic Factor) ELISA Kit (HUES01607) Assay procedure

    1. 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.
    2. Aliquot 100µl of standard solutions into the standard wells.
    3. Add 100µl of Sample / Standard dilution buffer into the control (zero) well.
    4. Add 100µl of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids) into test sample wells.
    5. Cover the plate with the sealer provided in the kit and incubate for 90 min at 37°C.
    6. 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.
    7. 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.
    8. Add 100µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37°C.
    9. Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
    10. Add 90µL 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.
    11. Add 50 µL of Stop Solution to each well. Note: Adding the stop solution should be done inthe same order as the substrate solution.
    12. Determine the optical density (OD value) of each well immediately with a microplate readerset at 450 nm.

Human GIF (Gastric Intrinsic Factor) ELISA Kit (HUES01607) Protein Information

UniProt Protein Function:GIF: Promotes absorption of the essential vitamin cobalamin (Cbl) in the ileum. After interaction with CUBN, the GIF-cobalamin complex is internalized via receptor-mediated endocytosis. Defects in GIF are the cause of hereditary intrinsic factor deficiency (IFD); also known as congenital pernicious anemia. IFD is an autosomal recessive disorder characterized by megaloblastic anemia. Belongs to the eukaryotic cobalamin transport proteins family. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:

Protein type:Secreted; Secreted, signal peptide

Chromosomal Location of Human Ortholog: 11q13

Cellular Component: extracellular space; lysosomal lumen; microvillus; apical plasma membrane; extracellular region; endosome

Molecular Function:cobalamin binding

Biological Process: vitamin metabolic process; cobalamin metabolic process; cobalamin transport; cobalt ion transport; water-soluble vitamin metabolic process

Disease: Intrinsic Factor Deficiency

NCBI Summary:This gene is a member of the cobalamin transport protein family. It encodes a glycoprotein secreted by parietal cells of the gastric mucosa and is required for adequate absorption of vitamin B12. Vitamin B12 is necessary for erythrocyte maturation and mutations in this gene may lead to congenital pernicious anemia. [provided by RefSeq, Jul 2008]
UniProt Code:P27352
NCBI GenInfo Identifier:62906845
NCBI Gene ID:2694
NCBI Accession:P27352.2
UniProt Related Accession:P27352
Molecular Weight:
NCBI Full Name:Cobalamin binding intrinsic factor
NCBI Synonym Full Names:cobalamin binding intrinsic factor
NCBI Official Symbol:CBLIF  
NCBI Official Synonym Symbols:IF; GIF; INF; IFMH; TCN3  
NCBI Protein Information:cobalamin binding intrinsic factor
UniProt Protein Name:Gastric intrinsic factor
UniProt Synonym Protein Names:Intrinsic factor; IF; INF
Protein Family:Gastric intrinsic factor
UniProt Gene Name:GIF  
UniProt Entry Name:IF_HUMAN
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Additional Information

Reactivity:
Human
ELISA Type:
Sandwich
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