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useful for the measurement of Human HP-FGA in EDTA-plasma and culture supernatant

Prolyl 4-Hydroxylated Human α-Fibrinogen ELISA Kit

Background

Fibrinogen is one of major component of plasma, and is a hexamer composed of pairs of three polypeptides (α-, β- and γ-chain). Prolyl hydroxylation is known to be essential for folding, secretion, and stability of collagen triple helix.
In the powerful study using 2-Dimensional Image Converted Analysis of Liquid chromatography and mass spectrometry (2DICAL), it is found that proline 565 and 530 residues in α-fibrinogen is hydroxylated by the Prolyl hydroxylase P4HA1. In addition, the plasma level of prolyl 4-hydroxylated α-fibrinogen (HP-FGA) was increased in individuals who suffer pancreatic cancer. The significant difference between the said group (n=43) and healthy subjects (n=43) was observed in the 2-DICAL analysis.
This product is a kit that measures Prolyl 4-Hydroxylated Human α-Fibrinogen (Human HP-FGA) of EDTA-plasma and culture supernatant by the ELISA method.

Test Principle

This assay kit employs the quantitative sandwich ELISA technique based on two mouse monoclonal antibodies against to different epitope of HP-FGA with hydroxylation on its proline 565 residue. One of antibody specifically binds the region which contains hydoxylated proline 565 residue on HP-FGA.
Standard solutions and samples (culture medium and clinical sample, e.g. plasma) are applied into the wells and incubated, HP-FGA is captured by the coated antibody. Following, the microplate is incubated with HRP-conjugated antibody. Finally, a chromogen (TMB) is applied to each test well. The strength of coloring is in proportion to the quantities of Human HP-FGA.

Performancer Characteristics

< Example of standard curve >

Measurement Range : 2.73 - 175 ng/mL
Sensitivity : 0.43 ng/mL

*The sensitivity for this kit was determined using the guidelines under the National Committee for Clinical Laboratory Standards (NCCLS) Evaluation Protocols.(National Committee for Clinical Laboratory Standards Evaluation Protocols, SC1, (1989) Villanova, PA: NCCLS.)

< Specificity >
Substance Cross-Reactivity
Human HP-FGA 100 %
Human Serum Albumin < 0.1 %
Human Transferrin < 0.1 %
Human IgG < 0.1 %

< Dilution linearity >

< Added Recovery Assay >
Specimen Theoretical
Value (ng/mL)
Measured
Value (ng/mL)
%
10%FCS added
RPMI-1640 (x2)
43.75 40.59 92.8
21.88 19.73 90.2
112.66 111.32 98.8
Human Plasma (EDTA) (x3,000) 90.78 90.37 99.5
43.75 40.59 92.8
21.88 19.73 90.2

Kit Contents

Product List

Product Name Cat# Quantity Price

Prolyl 4-Hydroxylated (ALPHA)-Fibrinogen ELISA Kit

KAL-KG612 1KIT

¥ 100,000
$ 1334
€ 1000

1 Precoated plate (Anti-Human FGA Mouse IgG MoAb Affinity Purify) 96Well x 1
2 Labeled antibody Conc. : (30X) HRP conjugated Anti- Human HP-FGA Mouse IgG Fab' Affinity Purify 0.4mL x 1
3 Standard (Human HP-FGA) 0.5mL x 2
4 EIA buffer (1% BSA, 0.05% Tween20 in PBS) 30mL x 1
5 Solution for Labeled antibody (1% BSA, 0.05% Tween20 in PBS) 12mL x 1
6 Chromogen (TMB solution) 15mL x 1
7 Stop solution (1N H2SO4) 12mL x 1
8 Wash buffer Conc ((40X) 0.05% Tween20 in phosphate buffer) 50mL x 1

REFERENCES
1. Ono M, Matsubara J, Honda K, Sakuma T, Hashiguchi T, Nose H, Nakamori S, Okusaka T, Kosuge T, Sata N, Nagai H, Ioka T, Tanaka S, Tsuchida A, Aoki T, Shimahara M, Yasunami Y, Itoi T, Moriyasu F, Negishi A, Kuwabara H, Shoji A, Hirohashi S, Yamada T. Prolyl 4-hydroxylation of alpha-fibrinogen: a novel protein modification revealed by plasma proteomics. J Biol Chem. 2009 Oct 16;284(42):29041-9.

To be used for research only. DO NOT use for human gene therapy or clinical diagnosis.