Overview

  • Product name
    Human HMW Kininogen ELISA Kit, Fluorescent
  • Detection method
    Fluorescent
  • Precision
    Intra-assay
    Sample n Mean SD CV%
    Serum 8 3.9%
    Inter-assay
    Sample n Mean SD CV%
    Serum 3 11.4%
  • Sample type
    Cell culture supernatant, Saliva, Urine, Serum, Heparin Plasma, EDTA Plasma, Citrate Plasma
  • Assay type
    Sandwich (quantitative)
  • Sensitivity
    9.5 pg/ml
  • Range
    9.8 pg/ml - 20000 pg/ml
  • Recovery

    Sample specific recovery
    Sample type Average % Range
    Cell culture supernatant 107 99% - 111%
    Saliva 117 114% - 120%
    Urine 118 115% - 120%
    Serum 89 88% - 90%
    Heparin Plasma 90 84% - 93%
    EDTA Plasma 90 87% - 92%
    Citrate Plasma 88 82% - 91%

  • Assay time
    1h 30m
  • Assay duration
    One step assay
  • Species reactivity
    Reacts with: Human
    Does not react with: Mouse, Rat, Cow, Monkey
  • Product overview

    HMW Kininogen in vitro CatchPoint SimpleStep ELISA (Enzyme-Linked Immunosorbent Assay) kit is designed for the quantitative measurement of HMW Kininogen protein in human serum, plasma, and cell culture supernatants.


    This CatchPoint SimpleStep ELISA kit has been optimized for Molecular Devices Microplate Readers. Click here for a list of recommended Microplate Readers.
    If using a Molecular Devices’ plate reader supported by SoftMax® Pro software, a preconfigured protocol for these CatchPoint SimpleStep ELISA Kits is available with all the protocol and analysis settings at www.softmaxpro.org.


    The CatchPoint 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.  CatchPoint HRP Development Solution containing the Stoplight Red Substrate is added. During incubation, the substrate is catalyzed by HRP generating a fluorescent product. Signal is generated proportionally to the amount of bound analyte and the intensity is measured in a fluorescence plater reader at 530/570/590 nm Excitation/Cutoff/Emission.

  • Notes

    High molecular weight kininogen (HMW kininogen) is a 72kDa highly glycosylated protein with an important role in the assembly of the plasma kallikrein-kinin system and in the blood coagulation process.  It is encoded by the KNG1 gene, which generates both HMWK and low molecular weight kininogen (LMW kininogen) via alternative splicing.  Both HMW kininogen and LMW kininogen share a heavy chain consisting of protein domains 1, 2 and 3 and differ in their light chain.  HMW kininogen contains a 56kDa light chain consisting of domain 5 and 6H, whereas LMW kininogen contains a 4kDa light chain consisting of domain 5L.  Heavy and light chains of HMW kininogen and LMW kininogen are linked via domain 4 which contains the bradykinin nonapeptide.

  • 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
    100X Stoplight Red Substrate 1 x 120µl
    10X Human HMW Kininogen Capture Antibody 1 x 600µl
    10X Human HMW Kininogen Detector Antibody 2 vials
    10X Wash Buffer PT (ab206977) 1 x 20ml
    500X Hydrogen Peroxide (H2O2, 3%) 1 x 50µl
    Antibody Diluent 5BI 1 x 6ml
    Human HMW Kininogen Lyophilized Purified Protein 2 vials
    Plate Seals 1 unit
    Sample Diluent NS 1 x 50ml
    SimpleStep Pre-Coated Black 96-Well Microplate 1 unit
    Stoplight Red Substrate Buffer 1 x 12ml
  • Research areas
  • Function
    (1) Kininogens are inhibitors of thiol proteases; (2) HMW-kininogen plays an important role in blood coagulation by helping to position optimally prekallikrein and factor XI next to factor XII; (3) HMW-kininogen inhibits the thrombin- and plasmin-induced aggregation of thrombocytes; (4) the active peptide bradykinin that is released from HMW-kininogen shows a variety of physiological effects: (4A) influence in smooth muscle contraction, (4B) induction of hypotension, (4C) natriuresis and diuresis, (4D) decrease in blood glucose level, (4E) it is a mediator of inflammation and causes (4E1) increase in vascular permeability, (4E2) stimulation of nociceptors (4E3) release of other mediators of inflammation (e.g. prostaglandins), (4F) it has a cardioprotective effect (directly via bradykinin action, indirectly via endothelium-derived relaxing factor action); (5) LMW-kininogen inhibits the aggregation of thrombocytes; (6) LMW-kininogen is in contrast to HMW-kininogen not involved in blood clotting.
  • Tissue specificity
    Secreted in plasma. T-kinin is detected in malignant ovarian, colon and breast carcinomas, but not in benign tumors.
  • Involvement in disease
    Defects in KNG1 are the cause of high molecular weight kininogen deficiency (HMWK deficiency) [MIM:228960]. HMWK deficiency is an autosomal recessive coagulation defect. Patients with HWMK deficiency do not have a hemorrhagic tendency, but they exhibit abnormal surface-mediated activation of fibrinolysis.
  • Sequence similarities
    Contains 3 cystatin domains.
  • Post-translational
    modifications
    Bradykinin is released from kininogen by plasma kallikrein.
    Hydroxylation of Pro-383 occurs prior to the release of bradykinin.
    Phosphorylation sites are present in the extracelllular medium.
    N- and O-glycosylated. O-glycosylated with core 1 or possibly core 8 glycans.
  • Cellular localization
    Secreted > extracellular space.
  • Information by UniProt
  • Alternative names
    • Alpha 2 thiol proteinase inhibitor
    • Alpha-2-thiol proteinase inhibitor
    • BDK
    • BK
    • Bradykinin
    • Fitzgerald factor
    • High molecular weight kininogen
    • HMWK
    • Ile-Ser-Bradykinin
    • Kallidin I
    • Kallidin II
    • Kininogen 1
    • KNG
    • KNG1
    • KNG1_HUMAN
    • Low molecular weight growth-promoting factor
    • Williams-Fitzgerald-Flaujeac factor
    see all
  • Database links

Applications

Our Abpromise guarantee covers the use of ab229438 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

  • ELISA Protocol Summary
  • The HMW Kininogen standard curve was prepared as described in Section 10. Raw data generated on SpectraMax M4 Multi-Mode Microplate Reader is shown in the table. Background-subtracted data values (mean +/- SD) are graphed.

  • The concentrations of HMW Kininogen were measured in duplicates, interpolated from the HMW Kininogen standard curves and corrected for sample dilution. Undiluted samples are as follows: serum 1:80,000, plasma (citrate) 1:320,000, plasma (heparin) 1:320,000 and plasma (EDTA) 1:80,000. The interpolated dilution factor corrected values are plotted (mean +/- SD, n=2). The mean HMW Kininogen concentration was determined to be 163 µg/mL in serum, 202 µg/mL in plasma (citrate), 151 µg/mL in plasma (EDTA), and 173 µg/mL in plasma (heparin).

  • The concentrations of HMW Kininogen were measured in duplicates, interpolated from the HMW Kininogen standard curves and corrected for sample dilution. Undiluted samples are as follows: urine 5%, saliva 50%, and HepG2 cell supernatant 25%. The interpolated dilution factor corrected values are plotted (mean +/- SD, n=2). The mean HMW Kininogen concentration was determined to be 38,903 pg/mL in urine, 2,857 pg/mL in saliva, and 7,454 pg/mL in HepG2 cell supernatant.

  • Interpolated dilution factor corrected values are plotted (mean +/- SD, n=2). The mean HMW Kininogen concentration was determined to be 79 µg/mL with a range of 62-112 µg/mL.

Protocols

References

ab229438 has not yet been referenced specifically in any publications.

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