fMLP ELISA Kit

For Research Use Only

Our new ELISA kit provides fast, accurate results for levels of fMLP in human urine, in a simple, robust, easily implemented format. To our knowledge, this is the first commercial assay for fMLP. We hope that it will facilitate the exploitation of fMLP as a biomarker of inflammatory disease.

fMLP is a potent polymorphonuclear leucocyte chemotactic factor implicated in a wide range of inflammatory conditions (Pugin 2012; Marasco 1984). It is a tripeptide produced by many enteric bacteria, including Escherichia coli, as a by-product of protein synthesis. In addition to being secreted, fMLP is also found in the surface membranes of many types of bacteria (Carlson 2006).

Naccache (1977) suggests that it has a pro-inflammatory effect. It has been implicated in the pathogenesis of Crohn’s disease, ulcerative colitis and pouchitis (Anton 1989; Buyse 2002; Nast 1988). It has been shown to induce colonic inflammation in animal models (Nast 1988) and to increase expression of major histocompatibility complex class I molecules in intestinal epithelial cells (Merlin 2001).

Typical physiological levels of fMLP, e.g. in the colon, are ~100nM (Carlson 2006).

fMLPKit1Web

 

 

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fMLP (N-formyl-Met-Leu-Phe) competition ELISA Kit.

BFMLPV1

96-well strip plate, fMLP-conjugate pre-coated

Competition ELISA

Human Urine

Colorimetric

1h 45 minutes

0.15 ng/mL

0.8-50 ng/mL

Intra-assay CV% <4.3

Inter-assay CV % <8.0

Average 104% Range 85-112%

1 in 5, 1 in 10, 1 in 20, 1 in 40 with recovery 96-100%

No significant cross-reactants.


To purchase our Kits, contact our distributors, S-Curve Scientific.

S-CurveScientific

07557 657056

Info@scurvescientific.co.uk

http://www.scurvescientific.co.uk

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fMLPKit3Web

The unique B.I.T.S.® ELISA fMLP assay kit is a solid-phase competition Enzyme-Linked Immunosorbent Assay (ELISA) designed to measure fMLP in human urine, with a 1.5h assay time. It is for research use only. The wells of the supplied strip microplate are pre-coated with an fMLP-ovalbumin conjugate to form the competitive capture surface.  The first step of the assay is to wash the wells and then pipette the standards and samples into appropriate wells.  This is followed by the addition of an enzyme-linked polyclonal ovine antibody specific for fMLP. After a 1h incubation at ambient temperature, the wells are emptied and washed to remove unbound fMLP-antibody reagent. Finally, a chromogenic substrate solution is added to each well and incubated until adequate colour develops in the control wells (typically 40-50 min). The intensity of the colour is measured with a standard plate reader. The colour intensity (optical density) is inversely proportional to the fMLP concentration.

fMLPAssay

Assay Procedure

 

The simple procedure makes it compatible with any research laboratory with basic EIA/ELISA instrumentation.

Refined and simplified step-wise procedure minimises operator error and significantly reduces time between sampling and results. Time saved in executing the assay will give you more time to do more research.

Kits manufactured to the highest quality by Mologic scientists who have developed these assays from scratch to ensure stability and batch to batch reproducibility are better than industry standards. Despite this commitment to quality, the price of the kits is in line with the competition – quality without compromise.

Mologic has invested time and resources over the past 5 years into the task of producing antibodies of the highest possible quality, with tailor-made specificity and sensitivity to ensure that the assay gives the right sensitivity and specificity for use in research.

B.I.T.S.® Imunoassays – a great new resource for Biomedical and life sciences researchers working in inflammation and host immune response to infection!

B.I.T.S.® – our assay range will be expanding throughout 2015.

ASSAY PROCEDURE

Equilibrate all materials and prepared reagents to room temperature (18-25˚C) prior to use. It is recommended that all standards, controls and samples are tested in duplicate.

1. Prepare all reagents, working standards, and samples as directed in the previous sections.

2. Remove excess microplate strips from the plate frame and return them immediately to the foil pouch containing the desiccant and reseal.

3. Wash plate four times with 300µL per well of 1X wash buffer manually or three times with 300µL of 1X wash buffer if using a plate washer. Tap the microplate 4-5 times on absorbent paper towel to completely remove the liquid.

4. Add 50µL of fMLP standard or diluted sample per well followed by 50µL Alk-Phos conjugate per well. Cover the plate with a plate sealer and incubate for one hour at room temperature on a shaker. Start the timer after the last sample addition.

5. Wash plate as previously described.

6. Add 100µL pNPP substrate solution to each well and incubate for 40-50 minutes to allow yellow colour to develop. Protect from light

7. Read the absorbance on a microplate reader at a wavelength of 405nm.

fMLPCurve


BITS

PRODUCT OVERVIEW

The unique B.I.T.S.® ELISA fMLP assay kit is a solid-phase competition Enzyme-Linked Immunosorbent Assay (ELISA) designed to measure fMLP in human urine, with a 1.5h assay time. It is for research use only. The wells of the supplied strip microplate are pre-coated with an fMLP-ovalbumin conjugate to form the competitive capture surface.  The first step of the assay is to wash the wells and then pipette the standards and samples into appropriate wells.  This is followed by the addition of an enzyme-linked polyclonal ovine antibody specific for fMLP. After a 1h incubation at ambient temperature, the wells are emptied and washed to remove unbound fMLP-antibody reagent. Finally, a chromogenic substrate solution is added to each well and incubated until adequate colour develops in the control wells (typically 20-30 min). The intensity of the colour is measured with a standard plate reader. The colour intensity (optical density) is inversely proportional to the fMLP concentration.


REFERENCES

P A Anton (1989). Increased Neutrophil Receptors for and response to the pro-inflammatory bacterial peptide Formyl-Methionyl-Leucyl-Phenylalanine in Crohn’s Disease. Gastroenterology, 97(1): 20-28.

M Buyse (2002). PepT1-mediated fMLP transport induces intestinal inflammation in vivo.
Am. J. Physiol. Cell Physiol, 283: C1795-C1800.

R M Carlson (2006). fMLP induces Hsp27 expression, attenuates NF-κB activation, and confers intestinal epithelial cell protection. Am. J. Physiol. Gastrointest Liver Physiol, 292: G1070-G1078.

W A Marasco et al. (1984). Purification and identification of formylmethionyl-leucyl-phenlalanine as the major peptide neutrophil chemotactic factor produced by Escherichia coliJ. Biol. Chem., 259: 5430-5439.

D Merlin et al. (2001). Colonic epithelial hPepT1 expression occurs in inflammatory bowel disease: transport of bacterial peptides influences expression of MHC Class 1 molecules. Gastroenterology, 120: 1666–1679.

P H Naccache et al. (1977). Transport of sodium, potassium, and calcium across rabbit polymorphonuclear leukocyte membranes. Effect of chemotactic factor. Journal Cell Biol., 73(2): 428-444.

C C Nast (1988). Chemotactic peptides. Mechanisms, functions, and possible role in inflammatory bowel disease. Digestive Diseases Sci., 33(3): Suppl. 50S-57S.

J Pugin (2012). How tissue injury alarms the immune system and causes a systemic inflammatory response syndrome. Annals of Intensive Care, 2(27): 1-6.h

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