Human LAT (Linker For Activation of T-cell) CLIA Kit (HUES00568)

Name: Human LAT (Linker For Activation of T-cell) CLIA Kit (HUES00568)
Brand: Human Immunology ELISA Kits 1
SKU: HUES00568
Availability: OutOfStock

Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Human
Detection method:	Chemiluminescence
Detection range:	62.50-4000 pg/mL
Sensitivity:	37.50 pg/mL
Sample volume:	100µL
Sample type:	Serum, plasma and other biological fluids
Repeatability:	CV < 15%
Specificity:	This kit recognizes Human LAT in samples. No significant cross-reactivity or interference between Human LAT 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 LAT. 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 LAT 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 LAT, 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 LAT. The concentration of Human LAT in the samples can be calculated by comparing the RLU of the samples to the standard curve.

UniProt Protein Function:	Required for TCR (T-cell antigen receptor)- and pre-TCR-mediated signaling, both in mature T-cells and during their development. Involved in FCGR3 (low affinity immunoglobulin gamma Fc region receptor III)-mediated signaling in natural killer cells and FCER1 (high affinity immunoglobulin epsilon receptor)-mediated signaling in mast cells. Couples activation of these receptors and their associated kinases with distal intracellular events such as mobilization of intracellular calcium stores, PKC activation, MAPK activation or cytoskeletal reorganization through the recruitment of PLCG1, GRB2, GRAP2, and other signaling molecules.
NCBI Summary:	The protein encoded by this gene is phosphorylated by ZAP-70/Syk protein tyrosine kinases following activation of the T-cell antigen receptor (TCR) signal transduction pathway. This transmembrane protein localizes to lipid rafts and acts as a docking site for SH2 domain-containing proteins. Upon phosphorylation, this protein recruits multiple adaptor proteins and downstream signaling molecules into multimolecular signaling complexes located near the site of TCR engagement. Alternative splicing results in multiple transcript variants encoding different isoforms. [provided by RefSeq, Jul 2008]
UniProt Code:	O43561
NCBI GenInfo Identifier:	14194891
NCBI Gene ID:	27040
NCBI Accession:	O43561. 1
UniProt Secondary Accession:	O43561,O43919, B7WPI0, C7C5T6, G5E9K3,
UniProt Related Accession:	O43561
Molecular Weight:	27,774 Da
NCBI Full Name:	Linker for activation of T-cells family member 1
NCBI Synonym Full Names:	linker for activation of T-cells
NCBI Official Symbol:	LAT
NCBI Official Synonym Symbols:	LAT1; pp36
NCBI Protein Information:	linker for activation of T-cells family member 1
UniProt Protein Name:	Linker for activation of T-cells family member 1
UniProt Synonym Protein Names:	36 kDa phospho-tyrosine adapter protein; pp36; p36-38
Protein Family:	Latexin
UniProt Gene Name:	LAT
UniProt Entry Name:	LAT_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 (pg/mL)	RLU	Average	Corrected
4000	54401 63761	59081	59046
2000	25523 30195	27859	27824
1000	14595 12373	13484	13449
500	6151 7059	6605	6570
250	3313 3173	3243	3208
125	1692 1470	1581	1546
62.50	740 770	755	720
0	34 36	35	--

Precision

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

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human LAT 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 (pg/mL)	199.35	458.22	1759.83	196.53	442.23	1641.21
Standard deviation	18.30	43.03	137.79	23.92	43.82	135.24
C V (%)	9.18	9.39	7.83	12.17	9.91	8.24

Recovery

The recovery of Human LAT 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)	101-115	106
EDTA plasma (n=5)	86-99	92
Cell culture media (n=5)	95-109	102

Linearity

Samples were spiked with high concentrations of Human LAT 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 (%)	87-100	90-102	97-110
1:2	Average (%)	94	97	104
1:4	Range (%)	87-102	101-117	100-116
1:4	Average (%)	94	109	106
1:8	Range (%)	86-98	92-103	101-114
1:8	Average (%)	91	98	106
1:16	Range (%)	100-111	97-110	89-105
1:16	Average (%)	106	103	96

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.