Desmosine ELISA Kit

For Research Use Only

Desmosine Kit

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B.I.T.S.® Desmosine ELISA Kit

BDESV1

96-well strip plate desmosine conjugate pre-coated

Competition ELISA

Urine

Colorimetric

1h 30 minutes

0.819 ng/mL

0.82-200 ng/mL

Intra-assay CV% < 5.4

Inter-assay CV % < 8.0

Average 104% Range 90-116%

1 in 5, 1 in 10, 1 in 20, 1 in 40 with recovery < 115%

Desmosine from elastin


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The unique B.I.T.S.® ELISA for desmosine has been developed by Mologic to provide fast, accurate assays of desmosine in urine. It’s unique because it’s been tested against DPD and PYD for cross reactivity and because it’s been validated against the exacting and rigorous reference assay of LC/MS-MS. The LC/MS-MS method as described in the literature takes 24-48 hours, depending on how much sample-preparation (mainly acid hydrolysis) is considered necessary. The B.I.T.S.® assay needs no sample work-up.

B.I.T.S.® assays contain fully characterised ovine antibodies for unparalleled performance.

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 anti-desmosine 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 on inflammation and the host response to infection.

In validating the assay, the results were compared with the gold standard of LC/MS-MS, which has always been seen as the only accurate means for assaying desmosine in clinical samples. This highly accurate and sensitive method is costly and relatively time-consuming, even for researchers with ready access to such sophisticated analytical instrumentation. Now, equivalent accuracy for desmosine determinations can be gained through this new ELISA.

The B.I.T.S. desmosine ELISA is the first reliable, rigorously validated assay for urinary desmosine, either reported in the literature or available commercially. This is an important product milestone, which will open up the use of desmosine as a robust biomarker with great utility in inflammatory disease 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.


BITS

PRODUCT OVERVIEW

This is a solidphase competition Enzyme Immunoassay (ELISA) designed to measure desmosine in urine, with a 1.5 hour assay time. The wells of the supplied strip microplate are pre-coated with an ovalbumin-desmosine 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 immediately followed by the addition of an enzyme-linked polyclonal ovine antibody specific for desmosine. After a 1-hour incubation at ambient temperature, the wells are emptied and washed to remove unbound desmosine-antibody reagent. Finally, a chromogenic substrate solution is added to each well and incubated until adequate colour develops in the control wells (typically 20 to 30 minutes). The intensity of the colour is measured with a standard plate reader. The colour intensity (optical density) is indirectly proportional to the desmosine concentration.

Desmosine in different animal species is identical to human desmosine so there is no basis for designating desmosine antibodies or assay kits as specific for any particular species.  However, this B.I.T.S. ELISA has been optimised (in terms of assay range, specificity and interference) for measurement of desmosine in human urine.

Example Desmosine standard curve Example of a desmosine standard curve


ASSAY PROCEDURE

Equilibrate all materials and prepared reagents to room temperature (18-25°C) prior to use. It is recommended to assay all standards, controls and samples 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 then 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 Desmosine standard or sample per well followed by 50µL Alk-Phos conjugate per well. Cover the well 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 microplate as described

6. Add 100µL pNPP substrate solution to each well and incubate for 20-30 minutes or until adequate yellow colour density develops. Protect from light.

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


Microplate Summary of the assay procedure

INFORMATION

Desmosine is a 526 dalton, tetrafunctional, pyridinium ring-containing amino acid that covalently links mature elastin fibres. During leukocyte-mediated inflammation, elastin is degraded by matrix metalloproteinases and neutrophil elastase. When the elastin is degraded, desmosine enters the blood and passes through the kidney into the urine, either as free desmosine, or as variously sized peptide fragments with desmosine still attached. The amount of desmosine that collects in the urine will give an indication of how much leukocyte-driven tissue damage is occurring 1.  Desmosine is therefore an indicator of elevated lung elastin fibre turnover and a marker of the effectiveness of agents with potential to reduce elastin breakdown. Increased excretion in the urine has been associated with a number of chronic diseases including chronic obstructive pulmonary disease (COPD), cystic fibrosis, aortic aneurysms and atherosclerosis 2, 3.

Level of free desmosine in urine of healthy subjects is 4.6±0.8 ng/mg creatinine, and 6.3±1.0 in COPD patients 4.

The anti-desmosine antibody is the defining feature of the Mologic desmosine assay, as the assay performance and diagnostic utility is a consequence of the affinity and specificity of the antibody.


CORRELATION OF IMMUNOASSAY RESULTS WITH RESULTS FROM THE LC-MS/MS REFERENCE METHOD

The heterogeneity of desmosine in urine is an important factor affecting any immunoassay aimed at testing fresh, unmodified urine samples. Currently, assays used in the laboratory for desmosine are thought to only “see” the free, unattached desmosine. The desmosine still linked to peptide fragments is thought to skew the calibration. For this reason, urine samples are generally pre-treated with an extended, aggressive acid hydrolysis at 108°C (lasting between 24 and 48 hours) to release free desmosine from all the peptide stubs before analysis by isotope-dilution liquid chromatography-tandem mass spectrometry 5. The overall process is very time consuming and cumbersome but highly accurate, such that it now constitutes the gold standard reference assay 6.

The Mologic desmosine assay has been validated against the gold standard LC-MS/MS method with a challenging range of clinical samples. There was a strong correlation between desmosine concentrations determined by the reference assay (LC-MS/MS) and the desmosine concentrations determined by the immunoassay system with Spearman’s rank of 0.8405.


NEGLIGIBLE CROSS-REACTIVITY TO SIMILAR CROSS-LINKING MOLECULES EXPECTED IN THE URINE

One of the biggest problems with desmosine immunoassays is the structural similarity between desmosine and other naturally occurring cross-linking molecules, which are often present at high concentrations. Previously reported antibodies have been highly cross-reactive and this has hampered their use. For this reason, research advances on desmosine as a biomarker of inflammatory disease are almost always based on sophisticated laboratory techniques such as LC-MS/MS. The most important cross-reacting substances are from bone and these are known as deoxypyridinoline (DPD) and pyridinoline (PYD). They are released during the process of bone resorption. Since bone resorption and synthesis are part and parcel of normal bone homeostasis, measurable amounts both DPD and PYD can usually be found in the urine of healthy volunteers. The levels can be very high in individuals with, for example, osteoporosis or other forms of bone disease or collagen breakdown.

NEGLIGIBLE CROSS-REACTIVITY TO SIMILAR CROSS-LINKING MOLECULES EXPECTED IN THE URINE

Structures of desmosine, PYD (pyridinoline) and DPD (deoxypyridinoline) to show their similarity. Many anti-desmosine antibodies are cross reactive with these similar molecules, which means that the assays derived from them are not able to fully discriminate between these very similar, but diagnostically disparate, structures. Desmosine assays constructed with cross-reactive antibodies give falsely elevated results with samples containing high levels of PYD and DPD. Mologic has developed ovine antibodies that can confidently distinguish between them, so the resulting assays are not subject to cross-reactivity, so eliminating any danger of erroneous results derived through interference.

The specificity of the assay was evaluated by measuring the degree of cross-reactivity of various compounds that were used in place of desmosine. The amount of antibody that was bound at each concentration was calculated and the approximate percentage of cross reactivity was calculated from the amount of compound that produced a signal equivalent to 50%.

Cross reactant% cross reactivity
Isodesmosine3.2
Pyridinoline (PYD)0.04
Deoxypyridinoline (DPD)0.08

URINARY DESMOSINE AS A ROBUST DIAGNOSTIC FACTOR IN CONFIRMING THE PRESENCE OF COPD

Samples collected from 20 healthy and 100 COPD subjects gave good discrimination between the two groups with a p value <0.05 and an AUC value of 0.8885.

URINARY DESMOSINE AS A ROBUST DIAGNOSTIC FACTOR IN CONFIRMING THE PRESENCE OF COPD

Analysis of healthy (n=20) and COPD (n=100) groups for desmosine/creatinine ratios by 2 statistical tests; unpaired t-test Mann-Whitney test.

1. CA Lindberg et al. (2012) Total desmosines in plasma and urine correlate with lung function. Eur Respir J, 39: 839-845

2. S Viglio et al. (2000) MEKC of desmosine and Isodesmosine in urine of chronic obstructive lung disease patients. Eur Respir J, 15(6): 1039-1045

3. T Osakabe et al. (1999) Characteristic change of urinary elastin peptides and desmosine in the aortic aneurysm. Biol Pharm Bull, 22(8): 854-857

4. S Ongay et al. (2014) Quantification of free and total desmosine and isodesmosine in human urine by liquid chromatography tandem mass spectrometry: A comparison of the surrogate-analyte and the surrogate-matrix approach for quantitation. J. Chrom. A, 1326: 13-19

5. Albarbarawi et al. (2010) Measurement of urinary total desmosine and isodesmosine using isotope-dilution liquid chromatography-tandem mass spectrometry. Analytical chemistry, 82(9), 3745-50

6. JT Huang et al. (2012) Clinical validity of plasma and urinary desmosine as biomarkers for chronic obstructive pulmonary disease. Thorax, 67(6), 502-8


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