Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Human
Detection Method:	Colormetric
Detection Range:	0.16-10 ng/mL
Sensitivity:	0.10 ng/mL
Sample Volume Required Per Well:	100µL
Sample Type:	Serum, plasma and other biological fluids
Specificity:	This kit recognizes Human SIRPA in samples. No significant cross-reactivity or interference between Human SIRPA and analogues was observed.

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

UniProt Protein Function:	SHPS1: Immunoglobulin-like cell surface receptor for CD47. Acts as docking protein and induces translocation of PTPN6, PTPN11 and other binding partners from the cytosol to the plasma membrane. Supports adhesion of cerebellar neurons, neurite outgrowth and glial cell attachment. May play a key role in intracellular signaling during synaptogenesis and in synaptic function. Involved in the negative regulation of receptor tyrosine kinase-coupled cellular responses induced by cell adhesion, growth factors or insulin. Mediates negative regulation of phagocytosis, mast cell activation and dendritic cell activation. CD47 binding prevents maturation of immature dendritic cells and inhibits cytokine production by mature dendritic cells. Binds PTPN11 when tyrosine-phosphorylated, except in macrophages, where it primarily binds PTPN6. Binds GRB2 in vitro. Binds FGR. Binds JAK2 irrespective of its phosphorylation status and forms a stable complex. Binds SCAP1 and/or SCAP2. The resulting complex recruits FYB. Binds PTK2B. Ubiquitous. Highly expressed in brain. Detected on myeloid cells, but not T-cells. Detected at lower levels in heart, placenta, lung, testis, ovary, colon, liver, small intestine, prostate, spleen, kidney, skeletal muscle and pancreas. 3 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:Cell surface; Motility/polarity/chemotaxis; Cell adhesion; Receptor, misc. ; Membrane protein, integral Chromosomal Location of Human Ortholog: 20p13 Cellular Component: membrane; integral to membrane; plasma membrane Molecular Function:SH3 domain binding Biological Process: blood coagulation; cell adhesion; leukocyte migration
NCBI Summary:	The protein encoded by this gene is a member of the signal-regulatory-protein (SIRP) family, and also belongs to the immunoglobulin superfamily. SIRP family members are receptor-type transmembrane glycoproteins known to be involved in the negative regulation of receptor tyrosine kinase-coupled signaling processes. This protein can be phosphorylated by tyrosine kinases. The phospho-tyrosine residues of this PTP have been shown to recruit SH2 domain containing tyrosine phosphatases (PTP), and serve as substrates of PTPs. This protein was found to participate in signal transduction mediated by various growth factor receptors. CD47 has been demonstrated to be a ligand for this receptor protein. This gene and its product share very high similarity with several other members of the SIRP family. These related genes are located in close proximity to each other on chromosome 20p13. Multiple alternatively spliced transcript variants have been determined for this gene. [provided by RefSeq, Jul 2008]
UniProt Code:	P78324
NCBI GenInfo Identifier:	91105764
NCBI Gene ID:	140885
NCBI Accession:	NP_001035111. 1
UniProt Secondary Accession:	P78324,O00683, O43799, Q8N517, Q8TAL8, Q9H0Z2, Q9UDX2 Q9UIJ6, Q9Y4U9, A2A2E1, A8K411, B2R6C3,
UniProt Related Accession:	P78324
Molecular Weight:	54,967 Da
NCBI Full Name:	tyrosine-protein phosphatase non-receptor type substrate 1
NCBI Synonym Full Names:	signal-regulatory protein alpha
NCBI Official Symbol:	SIRPA
NCBI Official Synonym Symbols:	BIT; MFR; P84; SIRP; MYD-1; SHPS1; CD172A; PTPNS1
NCBI Protein Information:	tyrosine-protein phosphatase non-receptor type substrate 1; myd-1 antigen; inhibitory receptor SHPS-1; macrophage fusion receptor; CD172 antigen-like family member A; tyrosine phosphatase SHP substrate 1; brain-immunoglobulin-like molecule with tyrosine-b
UniProt Protein Name:	Tyrosine-protein phosphatase non-receptor type substrate 1
UniProt Synonym Protein Names:	Brain Ig-like molecule with tyrosine-based activation motifs; Bit; CD172 antigen-like family member A; Inhibitory receptor SHPS-1; Macrophage fusion receptor; MyD-1 antigen; Signal-regulatory protein alpha-1; Sirp-alpha-1; Signal-regulatory protein alpha-2; Sirp-alpha-2; Signal-regulatory protein alpha-3; Sirp-alpha-3; p84
Protein Family:	Nonribosomal peptide synthetase
UniProt Gene Name:	SIRPA
UniProt Entry Name:	SHPS1_HUMAN

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
10	2.318 2.34	2.329	2.257
5	1.53 1.538	1.534	1.462
2.5	0.94 0.922	0.931	0.859
1.25	0.391 0.407	0.399	0.327
0.63	0.259 0.247	0.253	0.181
0.32	0.18 0.158	0.169	0.097
0.16	0.12 0.124	0.122	0.05
0	0.069 0.075	0.072	--

Precision

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

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human SIRPA 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)	0.47	1.43	3.70	0.48	1.56	3.78
Standard deviation	0.03	0.06	0.11	0.03	0.07	0.16
C V (%)	6.38	4.20	2.97	6.25	4.49	4.23

Recovery

The recovery of Human SIRPA 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)	95-106	100
EDTA plasma (n=5)	93-107	100
Cell culture media (n=5)	87-97	92

Linearity

Samples were spiked with high concentrations of Human SIRPA 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 (%)	98-110	94-109	88-98
1:2	Average (%)	105	99	93
1:4	Range (%)	88-100	83-94	83-96
1:4	Average (%)	95	88	89
1:8	Range (%)	93-105	86-97	85-96
1:8	Average (%)	99	92	90
1:16	Range (%)	88-104	85-98	87-101
1:16	Average (%)	96	92	93

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 ×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	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

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 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.
Add 50 µL of Stop Solution to each well. Note: Adding the stop solution should be done inthe same order as the substrate solution.
Determine the optical density (OD value) of each well immediately with a microplate readerset at 450 nm.