Overview

  • Product name

    Human Agrin ELISA Kit
    See all Agrin kits
  • Detection method

    Colorimetric
  • Precision

    Intra-assay
    Sample n Mean SD CV%
    Serum 3 4.2%
    Inter-assay
    Sample n Mean SD CV%
    Serum 5 5.7%
  • Sample type

    Cell culture supernatant, Urine, Serum, Cell culture extracts, Tissue Extracts, Heparin Plasma, EDTA Plasma, Citrate Plasma, Cerebral Spinal Fluid
  • Assay type

    Sandwich (quantitative)
  • Sensitivity

    4.6 pg/ml
  • Range

    28.13 pg/ml - 1800 pg/ml
  • Recovery

    Sample specific recovery
    Sample type Average % Range
    Cell culture supernatant 119 116% - 121%
    Urine 106 101% - 114%
    Serum 97 87% - 113%
    Cell culture extracts 98 90% - 103%
    Tissue Extracts 118 115% - 122%
    Heparin Plasma 108 101% - 112%
    EDTA Plasma 106 104% - 109%
    Citrate Plasma 113 97% - 121%
    Cerebral Spinal Fluid 106 86% - 126%

  • Assay time

    1h 30m
  • Assay duration

    One step assay
  • Species reactivity

    Reacts with: Human
    Does not react with: Mouse, Rat, Cow
  • Product overview

    Agrin in vitro SimpleStep ELISA® (Enzyme-Linked Immunosorbent Assay) kit is designed for the quantitative measurement of Agrin protein in human serum, plasma, cerebrospinal fluid, urine, cell culture supernatants, and cell and tissue extracts.


    The SimpleStep ELISA® employs an affinity tag labeled capture antibody and a reporter conjugated detector antibody which immunocapture the sample analyte in solution. This entire complex (capture antibody/analyte/detector antibody) is in turn immobilized via immunoaffinity of an anti-tag antibody coating the well. To perform the assay, samples or standards are added to the wells, followed by the antibody mix. After incubation, the wells are washed to remove unbound material. TMB substrate is added and during incubation is catalyzed by HRP, generating blue coloration. This reaction is then stopped by addition of Stop Solution completing any color change from blue to yellow. Signal is generated proportionally to the amount of bound analyte and the intensity is measured at 450 nm. Optionally, instead of the endpoint reading, development of TMB can be recorded kinetically at 600 nm.


     

  • Notes

    Agrin is a heparan sulfate basal lamina glycoprotein that plays a central role in the formation and the maintenance of the neuromuscular junction (NMJ) and directs key events in postsynaptic differentiation. Agrin is a component of the AGRN-LRP4 receptor complex that induces the phosphorylation and activation of MUSK. The activation of MUSK in myotubes induces the formation of NMJ by regulating different processes including the transcription of specific genes and the clustering of AChR in the postsynaptic membrane. Calcium ions are required for maximal AChR clustering. Agrin function in neurons is highly regulated by alternative splicing, glycan binding and proteolytic processing. Agrin modulates calcium ion homeostasis in neurons, specifically by inducing an increase in cytoplasmic calcium ions. Agrin functions differentially in the central nervous system (CNS) by inhibiting the alpha(3)-subtype of Na+/K+-ATPase and evoking depolarization at CNS synapses.

  • Tested applications

    Suitable for: Sandwich ELISAmore details
  • Platform

    Pre-coated microplate (12 x 8 well strips)

Properties

  • Storage instructions

    Store at +4°C. Please refer to protocols.
  • Components 1 x 96 tests
    10X Human Agrin Capture Antibody 1 x 600µl
    10X Human Agrin Detector Antibody 1 x 600µl
    10X Wash Buffer PT (ab206977) 1 x 20ml
    50X Cell Extraction Enhancer Solution (ab193971) 1 x 1ml
    5X Cell Extraction Buffer PTR (ab193970) 1 x 10ml
    Antibody Diluent 4BI 1 x 6ml
    Human Agrin Lyophilized Recombinant Protein 2 vials
    Plate Seals 1 unit
    Sample Diluent NS (ab193972) 1 x 50ml
    SimpleStep Pre-Coated 96-Well Microplate (ab206978) 1 unit
    Stop Solution 1 x 12ml
    TMB Development Solution 1 x 12ml
  • Research areas

  • Function

    Isoform 1: heparan sulfate basal lamina glycoprotein that plays a central role in the formation and the maintenance of the neuromuscular junction (NMJ) and directs key events in postsynaptic differentiation. Component of the AGRN-LRP4 receptor complex that induces the phosphorylation and activation of MUSK. The activation of MUSK in myotubes induces the formation of NMJ by regulating different processes including the transcription of specific genes and the clustering of AChR in the postsynaptic membrane. Calcium ions are required for maximal AChR clustering. AGRN function in neurons is highly regulated by alternative splicing, glycan binding and proteolytic processing. Modulates calcium ion homeostasis in neurons, specifically by inducing an increase in cytoplasmic calcium ions. Functions differentially in the central nervous system (CNS) by inhibiting the alpha(3)-subtype of Na+/K+-ATPase and evoking depolarization at CNS synapses. This secreted isoform forms a bridge, after release from motor neurons, to basal lamina through binding laminin via the NtA domain.
    Isoform 2: transmembrane form that is the predominate form in neurons of the brain, induces dendritic filopodia and synapse formation in mature hippocampal neurons in large part due to the attached glycosaminoglycan chains and the action of Rho-family GTPases.
    Isoform 1, isoform 4 and isoform 5: neuron-specific (z+) isoforms that contain C-terminal insertions of 8-19 AA are potent activators of AChR clustering. Isoform 5, agrin (z+8), containing the 8-AA insert, forms a receptor complex in myotubules containing the neuronal AGRN, the muscle-specific kinase MUSK and LRP4, a member of the LDL receptor family. The splicing factors, NOVA1 and NOVA2, regulate AGRN splicing and production of the 'z' isoforms.
    Isoform 3 and isoform 6: lack any 'z' insert, are muscle-specific and may be involved in endothelial cell differentiation.
    Agrin N-terminal 110 kDa subunit: is involved in regulation of neurite outgrowth probably due to the presence of the glycosaminoglcan (GAG) side chains of heparan and chondroitin sulfate attached to the Ser/Thr- and Gly/Ser-rich regions. Also involved in modulation of growth factor signaling.
    Agrin C-terminal 22 kDa fragment: this released fragment is important for agrin signaling and to exert a maximal dendritic filopodia-inducing effect. All 'z' splice variants (z+) of this fragment also show an increase in the number of filopodia.
  • Tissue specificity

    Expressed in basement membranes of lung and kidney. Muscle- and neuron-specific isoforms are found. Isoforms (y+) with the 4 AA insert and (z+8) isoforms with the 8 AA insert are all neuron-specific. Isoforms (z+11) are found in both neuronal and non-neuronal tissues.
  • Involvement in disease

    Myasthenic syndrome, congenital, 8
  • Sequence similarities

    Contains 4 EGF-like domains.
    Contains 9 Kazal-like domains.
    Contains 2 laminin EGF-like domains.
    Contains 3 laminin G-like domains.
    Contains 1 NtA (N-terminal agrin) domain.
    Contains 1 SEA domain.
  • Domain

    The NtA domain, absent in TM-agrin, is required for binding laminin and connecting to basal lamina.
    Both laminin G-like 2 (G2) and laminin G-like 3 (G3) domains are required for alpha-dystroglycan/DAG1 binding. G3 domain is required for C-terminal heparin, heparan sulfate and sialic acid binding.
  • Post-translational
    modifications

    Contains heparan and chondroitin sulfate chains and alpha-dystroglycan as well as N-linked and O-linked oligosaccharides. Glycosaminoglycans (GAGs), present in the N-terminal 110 kDa fragment, are required for induction of filopodia in hippocampal neurons. The first cluster (Gly/Ser-rich) for GAG attachment contains heparan sulfate (HS) chains and the second cluster (Ser/Thr-rich), contains chondroitin sulfate (CS) chains. Heparin and heparin sulfate binding in the G3 domain is independent of calcium ions. Binds heparin with a stoichiometry of 2:1. Binds sialic acid with a stoichiometry of 1:1 and binding requires calcium ions.
    At synaptic junctions, cleaved at two conserved sites, alpha and beta, by neurotrypsin. Cleavage at the alpha-site produces the agrin N-terminal 110-kDa subunit and the agrin C-terminal 110-kDa subunit. Further cleavage of agrin C-terminal 110-kDa subunit at the beta site produces the C-terminal fragments, agrin C-terminal 90 kDa fragment and agrin C-terminal 22 kDa fragment. Excessive cleavage at the beta-site releases large amounts of the agrin C-terminal 22 kDa fragment leading to destabilization at the neuromuscular junction (NMJ).
  • Cellular localization

    Cell junction, synapse. Cell membrane and Secreted, extracellular space, extracellular matrix. Synaptic basal lamina at the neuromuscular junction.
  • Information by UniProt
  • Alternative names

    • AGRIN
    • Agrin C-terminal 22 kDa fragment
    • Agrin proteoglycan
    • AGRIN_HUMAN
    • Agrn
    • C22
    • C90
    • FLJ45064
    • OTTHUMP00000044043
    see all
  • Database links

Applications

Our Abpromise guarantee covers the use of ab216945 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
Sandwich ELISA Use at an assay dependent concentration.

Images

  • SimpleStep ELISA technology allows the formation of the antibody-antigen complex in one single step, reducing assay time to 90 minutes. Add samples or standards and antibody mix to wells all at once, incubate, wash, and add your final substrate. See protocol for a detailed step-by-step guide.

     

  • Background-subtracted data values (mean +/- SD) are graphed.

  • Background-subtracted data values (mean +/- SD) are graphed.

  • Background-subtracted data values (mean +/- SD) are graphed.

  • The concentrations of Agrin were measured in duplicates, interpolated from the Agrin standard curves and corrected for sample dilution. Undiluted samples are as follows: serum 25%, plasma (citrate) 25%, plasma (heparin) 25% and plasma (EDTA) 25%. The interpolated dilution factor corrected values are plotted (mean +/- SD, n=2). The mean Agrin concentration was determined to be 3,719 pg/mL in neat serum, 3,562 pg/mL in neat plasma (citrate), 3,526 pg/mL in neat plasma (heparin), and 3,445 pg/mL in neat plasma (EDTA).

  • The concentrations of Agrin were measured in duplicates, interpolated from the Agrin standard curves and corrected for sample dilution. Undiluted samples are as follows: Urine 12.5%, cerebrospinal fluid 6.25%, HepG2 cell culture supernatant 100% and HeLa cell culture supernatant 100%. The interpolated dilution factor corrected values are plotted (mean +/- SD, n=2). The mean Agrin concentration was determined to be 9,142 pg/mL in neat urine, 18,572 pg/mL in neat cerebrospinal fluid, 1,365 pg/mL in HepG2 cell culture supernatant, and 711.7 pg/mL in HeLa cell culture supernatant.

  • Interpolated concentrations of native Agrin in human brain tissue extract based on a 300 µg/mL extract load, human liver tissue extract based on a 1000 µg/mL extract load, SH-SY5Y cell extract based on a 500 µg/mL extract load, and HepG2 cell extract based on a 100 µg/mL extract load. The concentrations of Agrin were measured in duplicate and interpolated from the Agrin standard curve and corrected for sample dilution. The interpolated dilution factor corrected values are plotted (mean +/- SD, n=2). The mean Agrin concentration was determined to be 540 pg/mL in brain tissue extract, 740 pg/mL in liver tissue extract, 252 pg/mL in SH-SY5Y cell extract, and 796 pg/mL in HepG2 cell extract.

  • Interpolated dilution factor corrected values are plotted (mean +/- SD, n=2). The mean Agrin concentration in neat samples was determined to be 3909 pg/mL with a range of 2879 – 5073 pg/mL.

Protocols

References

ab216945 has not yet been referenced specifically in any publications.

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