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ELISAGenie/Human CETP (Cholesteryl Ester Transfer Protein) ELISA Kit (HUES01874)/Cholesteryl Ester Transfer Protein) ELISA Kit (HUES01874/HUES01874

Description
Additional Information

Description

Human CETP (Cholesteryl Ester Transfer Protein) 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 CETP . 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 CETP 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 CETP, 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 CETP. The concentration of Human CETP in samples can be calculated by comparing the OD of the samples to the standard curve.

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	100uL
Sample Type	Serum, plasma and other biological fluids

Specificity

This kit recognizes Human CETPin samples. No significant cross-reactivity or interference between Human CETPand 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
20	2.4792.499	2.489	2.425
10	1.6441.67	1.657	1.593
5	1.0180.994	1.006	0.942
2.5	0.450.476	0.463	0.399
1.25	0.260.256	0.258	0.194
0.63	0.1750.161	0.168	0.104
0.31	0.1160.118	0.117	0.053
0	0.0610.067	0.064	--

Precision

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

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human CETPwere 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)	1.01	2.37	9.20	1.03	2.44	10.07
Standard deviation	0.06	0.11	0.32	0.06	0.14	0.43
C V (%)	5.94	4.64	3.48	5.83	5.74	4.27

Recovery

The recovery of Human CETPspiked 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)	83-95	90
EDTA plasma (n=5)	84-99	90
Cell culture media (n=5)	86-99	93

Linearity

Samples were spiked with high concentrations of Human CETPand 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 (%)	84-98	97-110	95-111
1:2	Average (%)	90	103	102
1:4	Range (%)	89-105	82-96	84-96
1:4	Average (%)	96	88	89
1:8	Range (%)	89-103	81-94	86-100
1:8	Average (%)	95	87	92
1:16	Range (%)	91-107	80-93	82-94
1:16	Average (%)	98	86	88

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 Standard	2 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 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 (25X)	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

Human CETP (Cholesteryl Ester Transfer Protein) ELISA Kit (HUES01874) 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 CETP (Cholesteryl Ester Transfer Protein) ELISA Kit (HUES01874) Protein Information

UniProt Protein Function:	CETP: Involved in the transfer of insoluble cholesteryl esters in the reverse transport of cholesterol. Defects in CETP are the cause of hyperalphalipoproteinemia type 1 (HALP1). Affected individuals show high levels of alpha-lipoprotein (high density lipoprotein/HDL). Belongs to the BPI/LBP/Plunc superfamily. BPI/LBP family. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:Lipid-binding; Secreted; Secreted, signal peptide; Vesicle Chromosomal Location of Human Ortholog: 16q21 Cellular Component: extracellular space; extracellular region; vesicle Molecular Function:lipid transporter activity; phospholipid transporter activity; cholesterol transporter activity; cholesterol binding; phosphatidylcholine binding; lipid binding; triglyceride binding Biological Process: cholesterol metabolic process; receptor-mediated endocytosis; cholesterol transport; lipid homeostasis; lipoprotein metabolic process; lipid transport; phosphatidylcholine metabolic process; cholesterol homeostasis; reverse cholesterol transport; triacylglycerol metabolic process; phospholipid transport; triacylglycerol transport; transmembrane transport; phospholipid homeostasis Disease: Hyperalphalipoproteinemia 1
NCBI Summary:	The protein encoded by this gene is found in plasma, where it is involved in the transfer of cholesteryl ester from high density lipoprotein (HDL) to other lipoproteins. Defects in this gene are a cause of hyperalphalipoproteinemia 1 (HALP1). Two transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Oct 2013]
UniProt Code:	P11597
NCBI GenInfo Identifier:	71153497
NCBI Gene ID:	1071
NCBI Accession:	P11597.2
UniProt Secondary Accession:	P11597,Q13987, Q13988, Q53YZ1,
UniProt Related Accession:	P11597
Molecular Weight:	47,787 Da
NCBI Full Name:	Cholesteryl ester transfer protein
NCBI Synonym Full Names:	cholesteryl ester transfer protein, plasma
NCBI Official Symbol:	CETP
NCBI Official Synonym Symbols:	BPIFF; HDLCQ10
NCBI Protein Information:	cholesteryl ester transfer protein; lipid transfer protein I; BPI fold containing family F
UniProt Protein Name:	Cholesteryl ester transfer protein
UniProt Synonym Protein Names:	Lipid transfer protein I
Protein Family:	Cholesteryl ester transfer protein
UniProt Gene Name:	CETP
UniProt Entry Name:	CETP_HUMAN

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Additional Information

Reactivity:	Human
ELISA Type:	Sandwich