Product Name:	Human IGF 1 ELISA Kit
Product Code:	HUFI00148
Size:	96 Assays
Alias:	IGF-1, IGF1, IGFI, IGF-I, IGF1A, IGF-IA, IGF-IB, MGF, Somatomedin C
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human IGF-1 concentrations in serum plasma and other biological fluids.
Sensitivity:	37.5pg/ml
Range:	62.5-4000 pg/mL (1ng=1.9mIU)
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Human IGF-1 and the recovery rates were calculated by comparing the measured value to the expected amount of Human IGF-1 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	85-104	98
EDTA plasma(n=5)	90-103	95
UFH plasma(n=5)	85-99	92

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human IGF-1 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample	1:2	1:4	1:8
serum(n=5)	85-101%	89-99%	90-105%
EDTA plasma(n=5)	82-97%	86-98%	88-100%
UFH plasma(n=5)	81-99%	84-100%	82-98%

CV(%):

Intra-Assay: CV<8%
Inter-Assay: CV<10%

Component	Quantity	Storage
ELISA Microplate (Dismountable)	8×12 strips	4°C for 6 months
Lyophilized Standard	2	4°C/-20°C
Sample/Standard Dilution Buffer	20ml	4°C
Biotin-labeled Antibody(Concentrated)	120ul	4°C (Protect from light)
Antibody Dilution Buffer	10ml	4°C
HRP-Streptavidin Conjugate(SABC)	120ul	4°C (Protect from light)
SABC Dilution Buffer	10ml	4°C
TMB Substrate	10ml	4°C (Protect from light)
Stop Solution	10ml	4°C
Wash Buffer(25X)	30ml	4°C
Plate Sealer	5	-

Other materials and equipment required:

Microplate reader with 450 nm wavelength filter
Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
Incubator
Deionized or distilled water
Absorbent paper
Buffer resevoir

Uniprot

P05019

UniProt Protein Function:	IGF1: The insulin-like growth factors, isolated from plasma, are structurally and functionally related to insulin but have a much higher growth-promoting activity. May be a physiological regulator of [1-14C]-2-deoxy-D-glucose (2DG) transport and glycogen synthesis in osteoblasts. Stimulates glucose transport in rat bone-derived osteoblastic (PyMS) cells and is effective at much lower concentrations than insulin, not only regarding glycogen and DNA synthesis but also with regard to enhancing glucose uptake. Defects in IGF1 are the cause of insulin-like growth factor I deficiency (IGF1 deficiency). IGF1 deficiency is an autosomal recessive disorder characterized by growth retardation, sensorineural deafness and mental retardation. Belongs to the insulin family. 3 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:Secreted; Secreted, signal peptide; Motility/polarity/chemotaxis Chromosomal Location of Human Ortholog: 12q23.2 Cellular Component: insulin-like growth factor binding protein complex; extracellular space; plasma membrane; extracellular region Molecular Function:integrin binding; insulin-like growth factor receptor binding; protein binding; growth factor activity; hormone activity; insulin receptor binding Biological Process: muscle development; positive regulation of transcription, DNA-dependent; chondroitin sulfate proteoglycan biosynthetic process; exocrine pancreas development; glycolate metabolic process; water homeostasis; positive regulation of glucose import; positive regulation of fibroblast proliferation; proteoglycan biosynthetic process; inner ear development; positive regulation of DNA binding; muscle hypertrophy; platelet activation; positive regulation of protein import into nucleus, translocation; positive regulation of mitosis; regulation of establishment and/or maintenance of cell polarity; positive regulation of phosphoinositide 3-kinase cascade; cell activation; positive regulation of peptidyl-tyrosine phosphorylation; branching morphogenesis of a tube; insulin-like growth factor receptor signaling pathway; response to heat; regulation of gene expression; positive regulation of transcription from RNA polymerase II promoter; alveolus development; positive regulation of epithelial cell proliferation; negative regulation of apoptosis; positive regulation of insulin-like growth factor receptor signaling pathway; myoblast proliferation; positive regulation of smooth muscle cell proliferation; positive regulation of glycogen biosynthetic process; positive regulation of activated T cell proliferation; signal transduction; positive regulation of smooth muscle cell migration; negative regulation of cell proliferation; platelet degranulation; glial cell differentiation; positive regulation of MAPKKK cascade; mammary gland development; positive regulation of cell proliferation; DNA replication; skeletal development; positive regulation of granule cell precursor proliferation; phosphoinositide-mediated signaling; multicellular organism growth; regulation of multicellular organism growth; myotube cell development; satellite cell compartment self-renewal involved in skeletal muscle regeneration; myoblast differentiation; positive regulation of osteoblast differentiation; positive regulation of protein kinase B signaling cascade; cell proliferation; cellular protein metabolic process; positive regulation of tyrosine phosphorylation of Stat5 protein; positive regulation of glycolysis; Ras protein signal transduction; blood vessel remodeling; positive regulation of Ras protein signal transduction; blood coagulation; cell motility; positive regulation of DNA replication Disease: Insulin-like Growth Factor I Deficiency
NCBI Summary:	The protein encoded by this gene is similar to insulin in function and structure and is a member of a family of proteins involved in mediating growth and development. The encoded protein is processed from a precursor, bound by a specific receptor, and secreted. Defects in this gene are a cause of insulin-like growth factor I deficiency. Several transcript variants encoding different isoforms have been found for this gene.[provided by RefSeq, Mar 2009]
UniProt Code:	P05019
NCBI GenInfo Identifier:	124263
NCBI Gene ID:	3479
NCBI Accession:	P05019.1
UniProt Secondary Accession:	P05019,P01343, Q14620, B2RWM7, E9PD02,
UniProt Related Accession:	P05019
Molecular Weight:	17,762 Da
NCBI Full Name:	Insulin-like growth factor I
NCBI Synonym Full Names:	insulin-like growth factor 1 (somatomedin C)
NCBI Official Symbol:	IGF1
NCBI Official Synonym Symbols:	IGFI; IGF-I; IGF1A
NCBI Protein Information:	insulin-like growth factor I; MGF; IGF-IA; IGF-IB; somatomedin-C; mechano growth factor; insulin-like growth factor IA; insulin-like growth factor IB
UniProt Protein Name:	Insulin-like growth factor I
UniProt Synonym Protein Names:	Mechano growth factor; MGF; Somatomedin-C
Protein Family:	Insulin-like growth factor
UniProt Gene Name:	IGF1
UniProt Entry Name:	IGF1_HUMAN

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37°C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.

Step	Protocol
1.	Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!
2.	Aliquot 0.1ml standard solutions into the standard wells.
3.	Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.
4.	Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.
5.	Seal the plate with a cover and incubate at 37 °C for 90 min.
6.	Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.
7.	Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.
8.	Seal the plate with a cover and incubate at 37°C for 60 min.
9.	Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.
10.	Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min.
11.	Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.
12.	Add 90 µl of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.
13.	Add 50 µl of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.
14.	Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.

When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.

Sample Type	Protocol
Serum	If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.
Plasma	Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.
Urine & Cerebrospinal Fluid	Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.
Cell culture supernatant	Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.
Cell lysates	Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Tissue homogenates	The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.
Tissue lysates	Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Breast Milk	Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.