Overview

  • Product name

    Human HMW Kininogen ELISA Kit
    See all Kininogen kits
  • Detection method

    Colorimetric
  • 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

    11.5 pg/ml
  • Range

    39.1 pg/ml - 2500 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

    As of May 7, 2019, Human HMW Kininogen SimpleStep ELISA® kit has been re-developed. We have identified new recombinant monoclonal antibodies to provide improved performance and consistency when quantifying HMW Kininogen protein in serum, plasma and cell culture supernatants.


    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

    High molecular weight kininogen (HMWK) 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 (LMWK) via alternative splicing. Both HMWK and LMWK share a heavy chain consisting of protein domains 1, 2 and 3 and differ in their light chain. HMWK contains a 56kDa light chain consisting of domain 5 and 6H, whereas LMWK contains a 4kDa light chain consisting of domain 5L. Heavy and light chains of HMWK and LMWK are linked via domain 4 which contains the bradykinin nonapeptide.

    HMWK is mainly secreted by the liver and helps position optimally prekallikrein and factor XI next to factor XII. Positioning of prekallikrein in contact with factor XII results in activation and cleavage of factor XII into factor XIIa. This leads to a positive feedback mechanisms of contact activation and cleavage of HMWK with the release of bradykinin. Active bradykinin affects smooth muscle contraction, induces hypotension, natriuresis, diuresis, decreases blood glucose level, mediates inflammation by releasing prostaglandins, increases vascular permeability and stimulates nociceptors. Furthermore, HMWK also inhibits the thrombin- and plasmin-induced aggregation of thrombocytes.

    HMWK deficiency is an autosomal recessive coagulation defect known as Fitzgerald trait, Flaujeac trait, Fujiwara trait, Reid trait or Williams’s trait. Patients with low levels of HMWK exhibit abnormal surface-mediated activation of fibrinolysis noted by prolonged partial thromboplastin time (PTT), normal prothrombin time (PT) and normal thrombin time typically found during a preoperative screening workup in asymptomatic individuals. A SNPs in KNG1 gene (rs710446) has been significantly associated with shortened activated partial thromboplastin time (aPTT) increasing the risk of venous thrombosis.

  • Tested applications

    Suitable for: Sandwich ELISAmore details
  • Platform

    Microplate

Properties

  • Storage instructions

    Store at +4°C. Please refer to protocols.
  • Components 1 x 96 tests
    10X Human HMW Kininogen Capture Antibody 1 x 600µl
    Human HMW Kininogen Detector Antibody (Lyophilized) 2 vials
    10X Wash Buffer PT (ab206977) 1 x 20ml
    Antibody Diluent 5BR 1 x 6ml
    Human HMW Kininogen Lyophilized Purified Protein 2 vials
    Plate Seals 1 unit
    Sample Diluent NS 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

    (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
    • BDK
    • BK
    • BK, included
    • bradykinin
    • bradykinin, included
    • Fitzgerald factor
    • Fitzgerald factor, included
    • Flaujeac factor, included
    • High molecular weight kininogen
    • High molecular weight kininogen, included
    • HMWK
    • HMWK, included
    • Ile-Ser-Bradykinin
    • Kallidin I
    • Kallidin II
    • kininogen 1
    • KNG
    • KNG1
    • KNG1_HUMAN
    • LMWK, included
    • Low molecular weight growth-promoting factor
    • Low molecular weight kininogen, included
    • Williams factor, included
    • Williams-Fitzgerald-Flaujeac factor
    see all
  • Database links

Applications

Our Abpromise guarantee covers the use of ab189574 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.

     

Protocols

References

This product has been referenced in:

  • Gutierrez S & Boada MD Neuropeptide-induced modulation of carcinogenesis in a metastatic breast cancer cell line (MDA-MB-231LUC+). Cancer Cell Int 18:216 (2018). Read more (PubMed: 30598641) »
See 1 Publication for this product

Customer reviews and Q&As

1-2 of 2 Abreviews or Q&A

Abreviews
The clinical starting material used was peripheral blood mononuclear cells (PBMCs) prepared from patients with chronic lymphocytic leukaemia (CLL).
Cells were defrosted and rested for 2 hours whilst a cell count was performed. All lysates were extracted from 2,000,000 cells in 1 ml of 1x Cell Extraction Buffer PTR. Protein lysates were extracted in accordance with the kit instructions. The ELISA was performed in accordance with the kit instructions. All samples and standards were tested in duplicate.
There is only 1 plate of 96 wells per kit but you can use the strips separately. We initially used some strips of wells to test the standard curve and to assess the best sample dilution.
The standard curve was reproducible. The level of HMW kininogen in the 20 CLL samples tested was calculated to range from 0 to 2537 pg/ml (based on 2,000,000 starting cells per ml of lysate).
Our AbTrial code is: ABTRIAL-EBR67.

Abcam user community

Verified customer

Submitted Jan 14 2015

Answer

We have not tested this kit on cell lysates and therefore we do not have data to send you to show what the typical ranges are for this sample type.

The kit should however work on isolated platelets, neutrophils or PBMC. I would not test kininogen on whole blood as this won’t allow you to distinguish between soluble kininogen (present in serum/plasma) from intracellular or cell associated kininogen (present in platelets and neutrophils).

The lab have recommend the following when using this kit on cell lysates:

For isolated cell preparations, I would recommend the customer to perform a titration series of the cell extract starting at 100ug/mL with a 1:4 dilution series in order to find the dynamic range of the assay for that particular cell population.

Running cell extracts with the kit will require the customer to prepare the 1X cell extraction buffer PTR (5X PTR and 50x enhancer solution are included with the kit). The solution contains aprotonin (a protease inhibitor), but if the customer is interested in neutrophils I would recommend to add to the 1x PTR a cocktail of protease inhibitors as neutrophils have high concentrations of proteases.

1X Cell Extraction Buffer PTR (For cell and tissue extracts only)

Prepare 1X Cell Extraction Buffer PTR by diluting 5X Cell Extraction Buffer PTR and 50X Cell Extraction Enhancer Solution to 1X with deionized water. To make 10 mL 1X Cell Extraction Buffer PTR combine 7.8 mL deionized water, 2 mL 5X Cell Extraction Buffer PTR and 200 µL 50X Cell Extraction Enhancer Solution Mix thoroughly and gently. If required protease inhibitors can be added.

Alternative – Enhancer may be added to 1X Cell Extraction Buffer PTR after extraction of cells or tissue. Refer to note in the Troubleshooting section.

Isolated platelets/neutrophils or simply PBMCs should be prepared as follows =

Preparation of extracts from cell pellets
Collect non-adherent cells by centrifugation or scrape to collect adherent cells from the culture flask. Typical centrifugation conditions for cells are 500 x g for 5 minutes at 4ºC.
Rinse cells twice with PBS.
Solubilize pellet at 2x107 cell/mL in chilled 1X Cell Extraction Buffer PTR.
Incubate on ice for 20 minutes.
Centrifuge at 18,000 x g for 20 minutes at 4°C.
Transfer the supernatants into clean tubes and discard the pellets.
Assay samples immediately or aliquot and store at ‑80°C. The sample protein concentration in the extract may be quantified using a protein assay.
Dilute samples to desired concentration in 1X Cell Extraction Buffer PTR.

The standard protein should be resuspended in 1X PTR and the standard curve should be run with 1X PTR instead of with the sample diluents included in the kit.


To help you further with determining the normal range of HMWK in lysate, I would suggest performing a literature search to see what information is already available.

As you can see on page 19 of our protocol booklet we show that the mean level of HMW Kininogen in serum is 58.2 μg/mL with a range of 42.8 – 69.5 μg/ml. In the following publication they determine the amount of HMWK in plasma is 670 nmol/l:

Cleaved high molecular weight kininogen binds directly to the integrin CD11b/CD18 (Mac-1) and blocks adhesion to fibrinogen and ICAM-1. June 15, 2000; Blood: 95 (12).

The additional publications below may also help you with your literature search:

Human neutrophils contain and bind high molecular weight kininogen. J Clin Invest. Jul 1989; 84(1): 28–35.
High-molecular weight kininogen. A secreted platelet protein. J Clin Invest. May 1983; 71(5): 1477–1489.

In addition, if you would be willing to send us an abreview for this product using our Abreview system on the following webpage:

https://www.abcam.com/abreviews

I would be happy to give you a testing discount for the value of the product tested which you will be able to use on a future order.

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