Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Human
Detection method:	Chemiluminescence
Detection range:	0.94-60 ng/mL
Sensitivity:	0.56 ng/mL
Sample volume:	100µL
Sample type:	Serum, plasma and other biological fluids
Repeatability:	CV < 15%
Specificity:	This kit recognizes Human CD4 in samples. No significant cross-reactivity or interference between Human CD4 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 CD4. 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 CD4 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 CD4, 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 CD4. The concentration of Human CD4 in the samples can be calculated by comparing the RLU of the samples to the standard curve.

UniProt Protein Function:	CD4: Accessory protein for MHC class-II antigen/T-cell receptor interaction. May regulate T-cell activation. Induces the aggregation of lipid rafts. Associates with LCK. Binds to HIV-1 gp120 and to P4HB/PDI and upon HIV-1 binding to the cell membrane, is part of P4HB/PDI- CD4-CXCR4-gp120 complex. Interacts with HIV-1 Envelope polyprotein gp160 and protein Vpu. Interacts with Human Herpes virus 7 capsid proteins. Interacts with PTK2/FAK1; this interaction requires the presence of HIV-1 gp120.
UniProt Protein Details:	Protein type:Membrane protein, integral; Cell surface Chromosomal Location of Human Ortholog: 12p13. 31 Cellular Component: T cell receptor complex; endoplasmic reticulum membrane; endoplasmic reticulum lumen; early endosome; plasma membrane; integral to membrane; external side of plasma membrane; lipid raft Molecular Function:protein binding; enzyme binding; transmembrane receptor activity; protein homodimerization activity; zinc ion binding; extracellular matrix structural constituent; coreceptor activity; receptor activity; glycoprotein binding; protein kinase binding; MHC class II protein binding Biological Process: maintenance of cellular protein localization; viral reproduction; positive regulation of interleukin-2 biosynthetic process; cytokine production; T cell selection; positive regulation of calcium-mediated signaling; defense response to Gram-negative bacterium; signal transduction; induction by virus of cell-cell fusion in host; T cell receptor signaling pathway; enzyme linked receptor protein signaling pathway; regulation of defense response to virus by virus; positive regulation of peptidyl-tyrosine phosphorylation; cell surface receptor linked signal transduction; positive regulation of protein kinase activity; T cell costimulation; protein palmitoleylation; innate immune response; entry into host cell; immune response; transmembrane receptor protein tyrosine kinase signaling pathway; T cell differentiation; regulation of T cell activation Disease: Okt4 Epitope Deficiency
NCBI Summary:	This gene encodes a membrane glycoprotein of T lymphocytes that interacts with major histocompatibility complex class II antigenes and is also a receptor for the human immunodeficiency virus. This gene is expressed not only in T lymphocytes, but also in B cells, macrophages, and granulocytes. It is also expressed in specific regions of the brain. The protein functions to initiate or augment the early phase of T-cell activation, and may function as an important mediator of indirect neuronal damage in infectious and immune-mediated diseases of the central nervous system. Multiple alternatively spliced transcript variants encoding different isoforms have been identified in this gene. [provided by RefSeq, Aug 2010]
UniProt Code:	P01730
NCBI GenInfo Identifier:	116013
NCBI Gene ID:	920
NCBI Accession:	P01730. 1
UniProt Secondary Accession:	P01730,Q4ZGK2, Q5U066, Q9UDE5, B2R737, D3DUS5,
UniProt Related Accession:	P01730
Molecular Weight:	458
NCBI Full Name:	T-cell surface glycoprotein CD4
NCBI Synonym Full Names:	CD4 molecule
NCBI Official Symbol:	CD4
NCBI Official Synonym Symbols:	CD4mut
NCBI Protein Information:	T-cell surface glycoprotein CD4; CD4 receptor; CD4 antigen (p55); T-cell surface antigen T4/Leu-3
UniProt Protein Name:	T-cell surface glycoprotein CD4
UniProt Synonym Protein Names:	T-cell surface antigen T4/Leu-3; CD_antigen: CD4
Protein Family:	CD40 ligand
UniProt Gene Name:	CD4
UniProt Entry Name:	CD4_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
60	36173 38153	37163	37138
30	14556 17402	15979	15954
15	7941 6801	7371	7346
7.5	3322 3804	3563	3538
3.75	1856 1710	1783	1758
1.88	932 916	924	899
0.94	492 512	502	477
0	24 26	25	--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human CD4 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human CD4 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)	3.31	5.61	26.06	3.57	5.71	24.07
Standard deviation	0.33	0.62	2.56	0.33	0.52	2.48
C V (%)	9.97	11.05	9.82	9.24	9.11	10.30

Recovery

The recovery of Human CD4 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)	84-97	91
EDTA plasma (n=5)	85-100	92
Cell culture media (n=5)	91-107	98

Linearity

Samples were spiked with high concentrations of Human CD4 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 (%)	87-104	88-99	94-107
1:2	Average (%)	95	93	102
1:4	Range (%)	92-104	87-98	94-110
1:4	Average (%)	97	92	101
1:8	Range (%)	88-103	98-111	87-98
1:8	Average (%)	95	105	93
1:16	Range (%)	102-118	90-102	101-116
1:16	Average (%)	109	95	109

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.