Kalon High Sensitivity CRP ELISA

What is CRP?

CRP, or to use the full name, C-reactive protein is a pentameric protein (comprising five identical subunits of a non-glycosylated polypeptide), which is produced in the liver and circulates in solution in the plasma.

CRP was first discovered by its ability to bind to C-polysaccharide which is a component of the pneumoccus bacterium (Streptococcus pneumoniae), a major cause of pneumonia, meningitis and other infections. CRP was later found to bind to a variety of other substances present not only in bacteria but also the host, for example phosphocholine and phospholipids. These are substances which become exposed when cells are damaged. When CRP binds to these substrates they are said to be "opsonised" and are easily recognised by phagocytic cells and in the normal course of things this event is followed by phagocytosis and removal from the site of infection or cell damage. In the human host, CRP bound in this way can also bind C1q, the first component of the complement cascade, and thereby promote cell lysis.

In these respects CRP is an important component of the innate immune response.

A notable feature of CRP is the very wide range in plasma concentration to be found between healthy and diseased states. While the plasma (or serum) concentration in a normal, healthy person is less than 2 mg/L, the level can rise to more than 100 mg/L or even 1000 mg/L when ill. In the case of an acute illness, the rise in level is very rapid (within hours of the onset of symptoms) and similarly the decline is rapid on recovery. In the case of chronic illness an elevated level can be sustained over a long period and this is indicative of an ongoing inflammatory response.

Conditions causing large increases in plasma CRP include bacterial infections, myocardial infarction, arthropathy, trauma and surgery. Viral infections typically lead to a smaller increase in CRP than those due to bacteria. This can be used to distinguish viral from bacterial meningitis.

In recent years it has become apparent that even small increases in CRP are clinically significant. For example, inflammation plays an important part in the progression of atherosclerosis (plaque formation in the arteries). Serum CRP, being a marker of inflammation, has been shown to predict the severity of the ischemic heart disease and the likelihood of the condition progressing to unstable angina, myocardial infarction or sudden death from coronary causes. Several large studies have gone one step further and shown that elevated CRP is predictive of future cardiovascular disease in apparently healthy adults.

A serum level of 3 mg/L is outside the normal range of healthy individuals and has been suggested as decision point for intervention.

Traditional assay methodologies, such as latex agglutination and nephelometry, were designed to detect gross changes in CRP concentration but more sensitive immunoassays are required to accurately detect the smaller rises in CRP seen in these newer studies. The Kalon High Sensitivity CRP Assay (hsCRP) was designed to accurately measure normal levels (around 1 mg/L) and the transition to elevated levels.

References

  • MB Pepys. The acute phase response and C-reactive protein.
    In: Oxford Textbook of Medicine 3rd Edition. Oxford University Press 1996
  • FH Epstein
    Acute-phase proteins and other systemic responses to inflammation.
    New England Journal of Medicine 1999;340:448454
  • Thomson SG et al.
    Hemostatic factors and the risks of myocardial infarction or sudden death in patients with angina pectoris.
    New England Journal of Medicine 1995;332:635-640.
  • Haverkate F et al.
    Production of C-reactive protein and risk of coronary events in stable and unstable angina.
    Lancet 1997;349(9050):462-466.
  • Ridker PM et al.
    Inflammation, aspirin and the risk of cardiovascular disease in apparently healthy men.
    New England Journal of Medicine 1997;336:973-979.
  • Koenig W et al.
    C-reactive protein, a sensitive marker of inflammation, predicts future risk of coronary heart disease in initially healthy middle-aged men. Results from the MONICA Augsburg Cohort Study, 1984 to 1992.
    Circulation 1999;99:237-242.
  • Ridker PM et al.
    C-reactive protein and other markers of inflammation in the prediction of cardiovascular disease in women.
    New England Journal of Medicine 2000;342:836-843.

Overview of the CRP ELISA

The Kalon High Sensitivity CRP assay (hs-CRP ELISA) is an enzyme-linked immunosorbant assay (ELISA) kit that is based on the ubiquitous 96-well microplate so that CRP levels can be conveniently determined in a normal laboratory without the need for a large clinical chemistry analyser.

This assay is therefore a convenient way measure CRP levels in plasma and serum samples for research studies.

The assay is based on the double antibody sandwich format with an alkaline phosphatase enzyme tracer. To run an assay the unknown serum samples and quality-control samples must be diluted in the diluent buffer provided.

A dilution of 1/1000 is both appropriate and convenient for the majority of samples lying in the range 1 to 50 mg/L but the assay will accommodate a very wide range in sample dilution. For example a dilution anywhere from 1/20 to 1/400,000 could be used be used to bring a sample onto the best range of the standard curve.

The CRP calibrators are supplied ready-to-use and do not require any further dilution.

The microplate is supplied pre-coated with affinity purified anti-CRP antibodies.

CRP calibrators, diluted samples and controls are added to the microtitre wells and incubated at ambient temperature for 1 hour and then washed. The tracer (affinity purified anti-CRP enzyme conjugate) is next added to the wells which are incubated for another 1 hour and then washed. A chromogenic enzyme substrate solution is added to the wells and incubated at ambient temperature. After 30 minutes the enzyme reaction is stopped by the addition of a "stop solution." The optical density of the chromogen in each microwell is then read in a microplate photometer.

The photometer should be set to read the optical density at 405 nm, which is the OD max for paranitrophenolphosphate.

The amount of colour produced in each well is dependent on the amount of enzyme attached to the microtitre well which relates to the level of CRP in the original sample.

hsCRP Standard Curve

The optical density of each CRP calibrator (standard) should be used to construct a standard curve. It is recommended to fit the data to the 4-parameter logistic equation by means of curve fitting software.

Next the level of the quality control samples should be determined. The information gathered so far should be used to determine whether the assay is valid.

Provided this is the case, the CRP level in the unknown samples can be calculated. To do this the CRP concentration of the diluted sample in each well is first read off the standard curve. Next, this level is corrected for the sample dilution prepared to find the CRP level in the original sample. Finally, the mean (average) level for the sample is calculated from any sample replicates run in the assay.

CRP ELISA Performance

Expected values

The CRP level was determined at a sample dilution of 1/1000 in 32 serum samples from volunteer blood donations

Median 1.61 mg/L range 0.20 to 5.48 (2.5th to 97.5th percentiles i.e. 95% of population)

Reproducibility

Below is a summary of our in-house data on the reproducibility we have obtained for three quality-control samples. This is "real" data collected in the course of running the hsCRP ELISA over a period of five years using many batches of ELISA kits.

Reproducibility. The averaged variation in controls values seen within an assay and from day to day over the course of five years and many batches of ELISA kits.
  Control 1 Control 2 Control 3
Mean CRP (mg/L) 2.8 12.7 72.8
Within run CV (RMS CV) 5.2% 8.7% 15%
Day to day CV 12% 8.4% 16%
Runs 44 27 23

Limit of detection and limit of quantitation

Kalon hsCRP ELISA - Linearity on Sample Dilution. Click for larger image

The determination of the limit of detection (LOD) and limit of quantitation (LOQ) can be problematic for assays with a non-linear response and in consequence there are as almost as many methods used to derive these parameters as there are CRP assays, so care should be exercised in comparing the figures from one assay to another. These depend on the local methods and equipment used and are therefore laboratory-specific.

Linearity on dilution

Three samples were diluted using a wide range of dilutions sufficient to bring the response onto the standard curve (0 to 100 µg/L)

For all practical purposes the dilution-response was parallel to the standard curve in the range 1/20 to 1/400,000.

Kalon hsCRP ELISA - Linearity on Sample Dilution. Click for larger image

While a dilution of 1/1000 is appropriate for most samples the accuracy of low CRP levels might be improved by using a dilution of 1/100 and the accuracy of high levels can be improved by using dilutions greater than 1/1000 as appropriate.

The percentage recovery for samples A and B are presented as examples the tables below.

Recovery on dilution of sample A. The recovery on dilution of a sample with a CRP concentration of 73.7 mg/L.
Sample
Dilution
Expected
Dose (µg/L )
Measured
Dose (µg/L)
Recovery
(%M/E)
Dilution corrected
CRP (mg/L)
1/800 91 102 111% 82.2
1/1600 45.7 42.8 94% 69.1
1/3,200 23.1 21.3 92% 68.2
1/6,400 11.5 11.5 100% 73.5
1/13,400 5.41 5.54 102% 75.5
1/25,600 2.84 2.83 99% 73.3

 

Recovery on dilution of sample A. The recovery on dilution of a sample with a CRP concentration of 1.24 mg/L.
Sample
Dilution
Expected
Dose (µg/L )
Measured
Dose (µg/L)
Recovery
(%M/E)
Dilution corrected
CRP (mg/L)
1/20 61.8 61.3 99% 1.2
1/40 31.0 29.9 96% 1.2
1/79 15.6 14.6 94% 1.2
1/159 7.79 7.73 99% 1.2
1/321 3.85 3.92 102% 1.3
1/649 1.90 2.01 106% 1.3
1/1,291 0.96 0.99 103% 1.3

Publications

The Kalon hsCRP ELISA is sold for research use, so you might expect results based on the use of the assay to appear in peer-reviewed journals. Here is a selection.

  • Dose-response for inhaled fluticasone on airway and systemic inflammation in COPD.
    P.A. Williamson, D. Menzies, K.L. Clearie, S. Vaidyanathan and B.J. Lipworth
    European Respiratory Journal 2010;37:206-209
  • Cord blood telomere length, telomerase activity and inflammatory markers in pregnancies in women with diabetes or gestational diabetes.
    JA Cross, RC Temple, JC Hughes, NC Dozio, C Brennan, K Stanley, HR Murphy, D Fowler, DA Hughes, MJ Sampson.
    Diabetic Medicine. 2010;27:1264-1270
  • Biochemical and Vascular Aspects of Pediatric Chronic Fatigue Syndrome.
    G Kennedy, F Khan, A Hill, C Underwood, JJF Belch.
    Archives of Pediatrics and Adolescent Medicine. 2010;164:817-823
  • Validation of novel diet related biomarkers of colorectal cancer risk.
    JC Mathers, IT Johnson, D Turnbull.
    "Arguably the most interesting aspect of the multivariate modelling analysis was the observation that all three techniques achieved statistically significant predictions for plasma CRP, which is a blood-borne, acute phase protein, and a marker of systemic inflammatory activity. This is potentially very important because it implies a mechanistic relationship between the composition of the colonic mucosal proteome and the inflammatory status of the individual".
    Food Standards Agency 01-Oct-2010.
  • A high sensitivity assay for the inflammatory marker C-Reactive protein employing acoustic biosensing.
    JD McBride and MA Cooper
    Journal of Nanobiotechnology 2008;6:5
  • Low-grade inflammation and arterial wave reflection in patients with chronic fatigue syndrome.
    VA Spence, G Kennedy, JJF Belch, A Hill & F Khan
    Clinical Science. 2008;114:561-566
  • Cytokine activation is predictive of mortality in Zambian patients with AIDS-related diarrhoea.
    I Zulu, G Hassan, L Njobvu RN, W Dhaliwal, S Sianongo and P Kelly.
    BMC Infectious Diseases 2008;8:156.
  • Effect of aspirin, clopidogrel and dipyridamole on soluble markers of vascular function in normal volunteers and patients with prior ischaemic stroke.
    L Zhao, L Gray, J Leonardi-Bee, CS Weaver, S Heptinstall, PMW Bath.
    Platelets. 2006;17:100-104
  • Monocyte Telomere Shortening and Oxidative DNA Damage in Type 2 Diabetes.
    MJ Sampson, MS Winterbone, JC Hughes, N Dozio, DA Hughes.
    Diabetes Care, 2006;29:283-289 link
  • Respiratory Symptoms, Pulmonary Function, and Markers of Inflammation Among Bar Workers Before and After a Legislative Ban on Smoking in Public Places.
    D Menzies, A Nair, PA Williamson, S Schembri, MZH Al-Khairalla, M Barnes, TC Fardon, L McFarlane, GJ Magee, BJ Lipworth.
    Journal of the American Medical Association. 2006;296:1742-1748.
  • Granulocyte-Colony-Stimulating Factor Mobilizes Bone Marrow Stem Cells in Patients With Subacute Ischemic Stroke The Stem Cell Trial of Recovery EnhanceMent After Stroke (STEMS) Pilot Randomized, Controlled Trial (ISRCTN 16784092).
    N Sprigg, PM Bath, L Zhao, MR Willmot, LJ Gray, MF Walker, MS Dennis & N Russell.
    Stroke. 2006;37:2979-2983
  • Systemic and Upper and Lower Airway Inflammation at Exacerbation of Chronic Obstructive Pulmonary Disease. John R. Hurst, Wayomi R. Perera, Tom M. A. Wilkinson, Gavin C. Donaldson and Jadwiga A. Wedzicha.
    American Journal of Respiratory and Critical Care Medicine. 2006;173:71-78.
  • Serum amyloid A in autoimmune thrombosis.
    S Sodin-Semrla, P Zigona, S Cucnika, T Kvedera, A Blincb, M Tomsica & B Rozmana.
    Autoimmunity Reviews 2006;6:21-27.
  • Investigation of platelet glycoprotein IIIa polymorphism using flow cytometry in patients with rheumatoid arthritis M McLaren, A Waring, B Galarraga, A Rudd, K Morley, JJF Belch
    Scandavian Journal of Rheumatology. 2005;34:437-440.
  • Allopurinol reduces B-type natriuretic peptide concentrations and haemoglobin but does not alter exercise capacity in chronic heart failure.
    AD Gavin, AD Struthers.
    Heart. 2005;91:749-753

Instructions for use

Click here to see PDF file of Kit Insert

Ordering

Kits can be purchased in single plate and multiplate packs from Kalon Biological Ltd contact us for more information.