Human RBP4 (Retinol Binding Protein 4, Plasma) ELISA Kit (HUES02591)

(No reviews yet) Write a Review
SKU:
HUES02591
€539

Description

ELISA Kit Technical ManualMSDS

Human RBP4 (Retinol Binding Protein 4, Plasma) 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 RBP4 . 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 RBP4 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 RBP4, 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 RBP4. The concentration of Human RBP4 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 Range1.56-100 ng/mL
Sensitivity0.94 ng/mL
Sample Volume Required Per Well100uL
Sample TypeSerum, plasma and other biological fluids

Specificity

This kit recognizes Human RBP4 in samples. No significant cross-reactivity or interference between Human RBP4 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
1002.354
2.366
2.362.283
501.65
1.666
1.6581.581
250.929
0.895
0.9120.835
12.50.457
0.477
0.4670.39
6.250.245
0.229
0.2370.16
3.130.186
0.16
0.1730.096
1.560.122
0.132
0.1270.05
00.072
0.082
0.077--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human RBP4 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human RBP4 were tested on 3 different plates, 20 replicates in each plate.

Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (ng/mL)5.4512.0049.935.1512.2750.52
Standard deviation0.360.622.740.350.642.15
C V (%)6.615.175.496.805.224.26

Recovery

The recovery of Human RBP4 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)91-10899
EDTA plasma (n=5)97-111102
Cell culture media (n=5)86-9892

Linearity

Samples were spiked with high concentrations of Human RBP4 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 (%)96-10992-10988-99
Average (%)10110094
1:4Range (%)94-10880-9184-96
Average (%)1008689
1:8Range (%)91-10480-9583-97
Average (%)988689
1:16Range (%)90-10086-9788-101
Average (%)959294

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 RBP4 (Retinol Binding Protein 4, Plasma) ELISA Kit (HUES02591) 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 RBP4 (Retinol Binding Protein 4, Plasma) ELISA Kit (HUES02591) Protein Information

UniProt Protein Function:Function: Delivers retinol from the liver stores to the peripheral tissues. In plasma, the RBP-retinol complex interacts with transthyretin, this prevents its loss by filtration through the kidney glomeruli.
UniProt Protein Details:

Subcellular location: Secreted.

Involvement in Disease: Defects in RBP4 are a cause of retinol-binding protein deficiency (RBP deficiency) [

MIM:180250]. This condition causes night vision problems. It produces a typical 'fundus xerophthalmicus', featuring a progressed atrophy of the retinal pigment epithelium.

Sequence similarities: Belongs to the calycin superfamily. Lipocalin family.

Mass spectrometry: Molecular mass is 21063.46±1.88 Da from positions 17 - 199. Determined by ESI. Ref.10Molecular mass is 20534 Da from positions 19 - 197. Determined by MALDI. Ref.12Molecular mass is 20162 Da from positions 19 - 194. Determined by MALDI. Ref.12

Sequence caution: The sequence AAF69622.1 differs from that shown. Reason: Erroneous initiation.

NCBI Summary:This protein belongs to the lipocalin family and is the specific carrier for retinol (vitamin A alcohol) in the blood. It delivers retinol from the liver stores to the peripheral tissues. In plasma, the RBP-retinol complex interacts with transthyretin which prevents its loss by filtration through the kidney glomeruli. A deficiency of vitamin A blocks secretion of the binding protein posttranslationally and results in defective delivery and supply to the epidermal cells. [provided by RefSeq]
UniProt Code:P02753
NCBI GenInfo Identifier:62298174
NCBI Gene ID:5950
NCBI Accession:P02753.3
UniProt Secondary Accession:P02753,O43478, O43479, Q5VY24, Q8WWA3, Q9P178, D3DR38
UniProt Related Accession:P02753
Molecular Weight:23,010 Da
NCBI Full Name:Retinol-binding protein 4
NCBI Synonym Full Names:retinol binding protein 4, plasma
NCBI Official Symbol:RBP4  
NCBI Protein Information:retinol-binding protein 4; RBP; PRBP; OTTHUMP00000020114; OTTHUMP00000020115; OTTHUMP00000020116; plasma retinol-binding protein; retinol-binding protein 4, interstitial
UniProt Protein Name:Retinol-binding protein 4
UniProt Synonym Protein Names:Plasma retinol-binding protein
Protein Family:Retinol-binding protein
UniProt Gene Name:RBP4  
UniProt Entry Name:RET4_HUMAN
View AllClose

Additional Information

Product type:
ELISA
Reactivity:
Human
ELISA Type:
Sandwich
View AllClose

0 Reviews

View AllClose