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human C-reactive protein
Instant ELISA
BMS288INST
Enzyme-linked immunosorbent assay for quantitative Not for diagnostic or therapeutic procedures. human C-reactive protein
BMS288INST
TABLE OF CONTENTS
1 Intended Use
The human C-reactive protein Instant ELISA is an enzyme-linked immunosorbent assay for the quantitative detection of human C-reactive protein levels in cel culture supernatants, human serum, plasma or other body fluids. The human C-reactive protein Instant ELISA is for
research use only. Not for diagnostic or therapeutic procedures.
2 Summary
C-reactive protein (CRP) is an acute phase protein exclusively produced in the liver. It has been identified in 1930 in the plasma of pneumonia patients and was named for its ability to bind C-polysaccaride of pneumococcus (1, 2). CRP belongs to the family of alpha globulin composed of five identical subunits resulting in a molecular weight of The physiological roles of CRP are numerous, one of the critical functions is its importance in host defense (1). CRP is synthesized rapidly by hepatocytes in response to cytokines released into circulation by activated leukocytes. Serum and plasma levels of CRP have been shown to rise during response to a wide variety of diseases including bacterial infections, acute phase of rheumatoid arthritis (3), inflammation of the bile duct. High CRP is also found in Guil ain-Barre syndrome patients, in multiple sclerosis, viral infections, tuberculosis, acute infectious hepatitis, necrotic diseases, burned patients and after surgical CRP thus has been shown to be a useful indicator of inflammatory processes (9). CRP levels rise in serum or plasma within 24 to 48 hours after acute tissue damage, peaking at acute stage and decreasing with resolution of inflammation or trauma (1, 10, 11). In addition to being a marker of inflammatory diseases, CRP turned out to be a predictive value as cardiac marker to assess risk of cardio vascular and peripheral 3 Principles of the Test
An anti-human C-reactive protein polyclonal Figure 1 present in the sample or standard binds to antibodies adsorbed to the microwel s; an reactive protein antibody binds to human C- conjugated anti-human C-reactive protein is removed during a wash step and substrate solution reactive with HRP is added to the A coloured product is formed in proportion to Figure 3 protein present in the sample. The reaction human C-reactive protein standard dilutions 4 Reagents Provided
1 aluminium pouch with a Microwell Plate coated with Polyclonal
Antibody (murine) to human C-reactive protein, HRP-Conjugate
(anti human C-reactive protein monoclonal (murine) antibody) and 2 aluminium pouches with a human C-reactive protein Standard
curve (coloured)
1 bottle (25 ml) Wash Buffer Concentrate 20x (phosphate-buffered
1 vial (15 ml) Substrate Solution (tetramethyl-benzidine)
1 vial (5 ml) Assay Buffer (Use when an external predilution of the
1 vial (15 ml) Stop Solution (1M Phosphoric acid)
2 adhesive Plate Covers
5 Storage Instructions
Store ELISA plate and Standard curves or whole kit at -20°C. The plate and the standard curves can also be removed, stored at -20°C, remaining kit reagents can be stored between 2° and 8°C. Expiry of the The expiry of the kit components can only be guaranteed if the components are stored properly, and if, in case of repeated use of one component, the reagent is not contaminated by the first handling. 6 Specimen Collection
Cel culture supernatants, human serum, plasma, or other body fluids are suitable for use in the assay. Remove the serum or plasma from the clot or red cel s as soon as possible after clotting and separation. Samples containing a visible precipitate must be clarified prior to use in the assay. Do not use grossly hemolyzed or lipemic specimens. Samples must be stored frozen at -20°C to avoid loss of bioactive human C-reactive protein. If samples are to be run within 24 hours, they may be stored at 2° to 8°C (for sample stability refer to 13). Avoid repeated freeze-thaw cycles. Prior to assay, frozen serum or plasma should be brought to room temperature slowly and mixed gently. 7 Materials Required But Not Provided
− 5 µl to 1000 µl adjustable single channel micropipettes with − 50 µl to 300 µl adjustable multichannel micropipette with disposable − Beakers, flasks, cylinders necessary for preparation of reagents − Device for delivery of wash solution (multichannel wash bottle or − Microwel strip reader capable of reading at 450 nm (620 nm as − Statistical calculator with program to perform linear regression 8 Precautions for Use
− Al chemicals should be considered as potential y hazardous. We therefore recommend that this product is handled only by those persons who have been trained in laboratory techniques and that it is used in accordance with the principles of good laboratory practice. Wear suitable protective clothing such as laboratory overal s, safety glasses and gloves. Care should be taken to avoid contact with skin or eyes. In the case of contact with skin or eyes wash immediately with water. See material safety data sheet(s) and/or safety − Reagents are intended for research use only and are not for use in − Do not mix or substitute reagents with those from other lots or other − Do not use kit reagents beyond expiration date on label. − Do not expose kit reagents to strong light during storage or − Do not eat or smoke in areas where kit reagents or samples are − Avoid contact of skin or mucous membranes with kit reagents or − Rubber or disposable latex gloves should be worn while handling kit − Avoid contact of substrate solution with oxidizing agents and metal. − Avoid splashing or generation of aerosols. − In order to avoid microbial contamination or cross-contamination of reagents or specimens which may invalidate the test use disposable − Use clean, dedicated reagent trays for dispensing substrate reagent. − Glass-distil ed water or deionized water must be used for reagent − Substrate solution must be at room temperature prior to use. − Decontaminate and dispose specimens and al potential y contaminated materials as they could contain infectious agents. The preferred method of decontamination is autoclaving for a minimum of − Liquid wastes not containing acid and neutralized waste may be mixed with sodium hypochlorite in volumes such that the final mixture contains 1.0% sodium hypochlorite. Al ow 30 minutes for effective decontamination. Liquid waste containing acid must be neutralized prior to the addition of sodium hypochlorite. 9 Preparation of Reagents and Samples
9.1 Wash Buffer
If crystals have formed in the Wash Buffer Concentrate, warm it gently Pour entire contents (25 ml) of the Wash Buffer Concentrate into a clean 500 ml graduated cylinder. Bring to final volume to 500 ml with glass- distil ed or deionized water. Mix gently to avoid foaming. The pH of the Transfer to a clean wash bottle and store at 2° to 25°C. Please note that 9.2 Assay Buffer
Pour the entire contents (5ml) of the Assay Buffer Concentrate into a
clean 100 ml graduated cylinder. Bring to final volume of 100 ml with 10 Test Protocol
− Use plate immediately after removal from -20°C!
− Do not wait until pellets have completely dissolved before
applying samples - the binding reaction in the standard strips
starts immediately after addition of water!
− Do not try to dissolve pellets by pipetting up and down in the
wells - some parts of the pellet could stick to the tip creating
high variation of results
− Perform the washing step with at least 400 µl of washing buffer
as stated in the manual or fill the wells completely - otherwise
any pellet residues sticking to the rim of the well will not be
removed and create high variation of results
− Allow the washing buffer to sit in the wells for a few seconds
before aspiration
− Remove covers of the standard strips carefully in order that all
the lyophilised pellets remain in the wells
a. Prepare your samples before starting with the test procedure. Dilute serum or plasma samples 1:500 with Assay Buffer according to the II) 50 µl prediluted sample + 450 µl Assay Buffer b. Determine the number of microwel Strips required to test the desired number of samples plus microwel Strips for blanks and standards (coloured). Each sample, standard, blank, and optional control sample should be assayed in duplicate. Remove extra microwel Strips from holder and store in foil bag with the desiccant provided at -20°C sealed tightly. Place microwel strips containing the standard curve in position A1/A2 to H1/H2 (see Table 1). c. Add 50 µl of distil ed water to the sample wel s. d. Add distil ed water to al standard and blank wel s as indicated on the label of the standard strips (A1, A2 to H1, H2). Table depicting an example of the arrangement of blanks, standards e. Add 100 µl of each 1:500 prediluted Sample, in duplicate, to the
f. Cover with a Plate Cover and incubate at room temperature
(18ºC to 25ºC) for 2 hours, if available on a microplate shaker at g. Remove Plate Cover and empty wel s. Wash the microwel strips 3
times with approximately 400 µl Wash Buffer per wel with thorough aspiration of microwel contents between washes. Take care not to After the last wash, tap microwel strips on absorbent pad or paper towel to remove excess Wash Buffer. Use the microwel strips immediately after washing or place upside down on a wet absorbent paper for no longer than 15 minutes. Do not al ow wel s to dry. h. Pipette 100 µl of TMB Substrate Solution to al wel s, including the
i. Incubate the microwel strips at room temperature (18° to 25°C) for about 10 min. Avoid direct exposure to intense light.
The colour development on the plate should be monitored and
the substrate reaction stopped (see point j. of this protocol)
before positive wells are no longer properly recordable.
Determination of the ideal time period for colour development
has to be done individually for each assay.
It is recommended to add the stop solution when the highest standard has developed a dark blue colour. The colour development can be monitored by the ELISA reader at 620 nm. The substrate reaction should be stopped as soon as Standard 1 has reached an j. Stop the enzyme reaction by quickly pipetting 100 µl of Stop
Solution into each wel , including the blank wel s. It is important that
the Stop Solution is spread quickly and uniformly throughout the microwel s to completely inactivate the enzyme. Results must be read immediately after the Stop Solution is added or within one hour if the microwel strips are stored at 2 - 8°C in the dark. k. Read absorbance of each microwel on a spectro-photometer using 450 nm as the primary wave length (optional y 620 nm as the reference wave length; 610 nm to 650 nm is acceptable). Blank the plate reader according to the manufacturer's instructions by using the blank wel s. Determine the absorbance of both the samples and the Note: In case of incubation without shaking the obtained O.D.
values may be lower than indicated below. Nevertheless the
results are still valid.
11 Calculation of Results
− Calculate the average absorbance values for each set of duplicate standards and samples. Duplicates should be within 20 per cent of − Create a standard curve by plotting the mean absorbance for each standard concentration on the ordinate against the human C-reactive protein concentration on the abscissa. Draw a best fit curve through − To determine the concentration of circulating human C-reactive protein for each sample, first find the mean absorbance value on the ordinate and extend a horizontal line to the standard curve. At the point of intersection, extend a vertical line to the abscissa and read the corresponding human C-reactive protein concentration. − *Samples have been diluted 1:500, thus the concentration read
from the standard curve must be multiplied by the dilution
factor (x 500).
− It is suggested that each testing facility establishes a control sample of known human C-reactive protein concentration and runs this additional control with each assay. If the values obtained are not within the expected range of the control, the assay results may be − A representative standard curve is shown in Figure 4. This curve cannot be used to derive test results. Every laboratory must prepare a standard curve for each group of microwel strips assayed. * N.B: There is a common dilution factor for samples due to the conjugate which must then be included in the calculation. The samples contribute 100 µl to the final volume per wel . These 100 µl are composed of 100 µl of the 1:500 prediluted sample. This is a 1:500 The remaining 50 µl to give 150 µl are due to the addition of 50 µl 50 µl conjugate results in 50 µl reconstitution volume, addition of 100 µl 1:500 prediluted sample ( = 1:500 dilution) Representative standard curve for human C-reactive protein Instant ELISA. Human C-reactive protein was diluted in serial 2-fold steps in Assay Buffer, each symbol represents the mean of 3 paral el titrations. Do not use this standard curve to derive test results. A standard curve must be run for each group of microwel strips assayed. bsorption 450 nm
A
Concentration (pg/ml)
Typical data using the human C-reactive protein INSTANT ELISA The OD values of the standard curve may vary according to the conditions of assay performance (e.g. operator, pipetting technique, washing technique or temperature effects). Furthermore shelf life of the kit may affect enzymatic activity and thus colour intensity. Values 12 Limitations
− Since exact conditions may vary from assay to assay, a standard curve must be established for every run. − Bacterial or fungal contamination of either screen samples or reagents or cross-contamination between reagents may cause − Disposable pipette tips, flasks or glassware are preferred, reusable glassware must be washed and thoroughly rinsed of al detergents − Improper or insufficient washing at any stage of the procedure wil result in either false positive or false negative results. Empty wel s completely before dispensing fresh wash solution, fil with Wash Buffer as indicated for each wash cycle and do not al ow wel s to sit − The use of radioimmunotherapy has significantly increased the number of patients with human anti-mouse IgG antibodies (HAMA). HAMA may interfere with assays utilizing murine monoclonal antibodies leading to both false positive and false negative results. Serum samples containing antibodies to murine immunoglobulins can stil be analysed in such assays when murine immunoglobulins (serum, ascitic fluid, or monoclonal antibodies of irrelevant specificity) 13 Performance Characteristics
13.1 Sensitivity
The limit of detection of human C-reactive protein defined as the analyte concentration resulting in an absorbance significantly higher than that of the dilution medium (mean plus 2 standard deviations) was determined to be 3 pg/ml (mean of 6 independent assays). 13.2 Reproducibility
13.2.1 Intra-assay
Reproducibility within the assay was evaluated in 3 independent experiments. Each assay was carried out with 6 replicates of 7 serum samples containing different concentrations of human C-reactive protein. 2 standard curves were run on each plate. Data below show the mean human C-reactive protein concentration and the coefficient of variation for each sample (see Table 3). The calculated overal intra- assay coefficient of variation was 6.9%. The mean human C-reactive protein concentration and the coefficient of 13.2.2 Inter-assay
Assay to assay reproducibility within one laboratory was evaluated in 3 independent experiments by 3 technicians. Each assay was carried out with 6 replicates of 7 serum samples containing different concentrations of human C-reactive protein. 2 standard curves were run on each plate. Data below (see Table 4) show the mean human C-reactive protein concentration and the coefficient of variation calculated on 18 determinations of each sample. The calculated overal coefficient of The mean human C-reactive protein concentration and the coefficient of variation calculated on 18 determinations of each sample. 13.3 Spike Recovery
The spike recovery was evaluated by spiking 4 levels of human C- reactive protein into normal human serum. Recoveries were determined in 3 independent experiments. The unspiked serum was used as blank in these experiments. The overal mean recovery was 103%. 13.4 Dilution Parallelism
4 serum samples with different levels of human C-reactive protein were analysed at serial 2 fold dilutions (1:500 – 1:2000) with 4 replicates each. The recovery ranged between 84% and 109% with an overal 13.5 Sample Stability
13.5.1 Freeze-Thaw Stability
Aliquots of serum samples (unspiked or spiked) were stored at -20°C and thawed several times, and the human C-reactive protein levels determined. There was loss of human C-reactive protein immunoreactivity by freezing and thawing. 13.5.2 Storage Stability
Aliquots of serum samples (spiked or unspiked) were stored at -20°C, 2- 8°C, room temperature (RT) and at 37°C, and the human C-reactive protein level determined after 24, 48 and 96 h. There was no significant loss of human C-reactive protein immunoreactivity during storage under 13.6 Specificity
To define the specificity of this ELISA several proteins were tested for cross reactivity. There was no cross reactivity observed. 13.7 Expected Values
A panel of 8 sera from randomly selected healthy donors (males and females) was tested for human C-reactive protein. The detected human C-reactive protein levels ranged between 136 and 800 ng/ml with a mean level of 381 ng/ml and a standard deviation of 214 ng/ml. 14 Bibliography
1) “Clinical Guide to Laboratory Tests”. Edited by N.W. Tietz, 3rd Edition. W.B. Saunders Company, Philadelphia, P.A. 19106 (1995). 2) Dixon, J.S., et al.: C-reactive protein in the serial assessment of disease acitivity in rheumatoid arthritis. Scand J Rheum, 13: 39-44, 3) Dowling, P., and Cook, S. : Immune events in demyelinating disease. In Wolfgang, F., El ison, G.W., Stevens J.G., and Andrew, J.M. (eds.): Multiple sclerosis. Academic Press Inc., New York, 4) Hedlund, P.: Clinical and experimental studies on C-reactive protein (acute phase protein). Thesis Acta Med Scand, 128 (Suppl, 5) Hedlund, P.: The appearance of acute phase protein in various diseases. Acta Med Scand, 128, (Suppl, 196): 579-601, 1947. 6) Hind, C.R.H., and Pepys, M.B.: The role of serum C-reactive (CRP) measurement in clinical practice. Int Med. 5: 112-151, 1984. 7) Kindmark, C.O.: The Concentration of C-reactive protein in Sera from Healthy Individuals. Scand J Clin Lab Invest, 29: 407-411, 8) Kushner, I.: “C-reactive protein in rheumatology,” Arthritis Rheum 9) Kushner, I., Rzewnicki, D.L.: The acute phase response: General aspects. Bail iere’s Clinical Rheumatology 8: 513-530, 1994. 10) Macy, E.M., Hayes, T.E., and Tracy, R.P.: Variability in the measurement of C-reactive protein in healthy subjects: implications for reference interval and epidemiological applications. Clin Chem, 11) Morley, J.J., Kushner, I.: Serum C-reactive Protein Levels. In: Kushner, I., Volanakis, J.E., and Gerwutz, H., eds., C-Reactive Protein and the Plasma Protein Response to Tissue Injury. Annals 12) Powel , L., Roantree, R.J., and Rantz, L.A.: C-reactive Protein. A review. Clinical experince with the C-reactive protein test. Am J 13) Ridker, P.M., et. al.: Inflammation, aspirin, and the risk of cardiovascular disease in apparently healthy men. N Engl J Med, 14) Ridker, P.M., et. al.: Inflammation, Pravastatin, and the risk of coronary events after myocardial infarction in patients with average cholesterol levels. Circulation, 98: 839-844, 1998. 15) Ridker, P.M., et. al.: Plasma concentration of C-reactive protein and the risk of developing peripheral vascular disease. Circulation, 97: 16) Ridker, P.M., et. al.: Propestive study of C-reactive protein and the risk of future cardiovascular events among apparently healthy 17) Ridker, P.M., Glynn, R.J., and Hennekens, C.H.: C-reactive protein adds to the predictive value of total and HDL cholesterol in determining risk of first myocardial infarction. Circulation, 97: 2007- 18) Roberts, W.L., et. al.: Evaluation of Four Automated High- Sensitivity C-Reactive Protein Methods: Implications for Clinical and Epidemiological Applications. Clin Chem 46: 4, 461-468, 2000. 19) Schultz, D.R., and Arnold P.I.: “Properties of four acute phase proteins: C-reactive protein, serum amyloid A protein, glycoprotein, and fibrinogen.” Seminars in Arthritis and Rheumatism 20: 129-147, 20) Shine, B., de Beer, F.C., and Pepys, M.B.: Solid phase radiommunoassay for human C-reactive protein. J Lab Clinica 21) Taubes, G.: “Does Inflammation Cut to The Heart of the Matter?”. 22) Tracy, R.P., et. al.: Relationship of C-reactive protein to risk of cardiovascular disease in the elderly: results for the Cardiovascular Health Study and the Rural Health Promotion Project. Arter Thromb 23) USA Center for Disease Control/National Institute of Health Manual, “Biosafety in Microbiological and Biomedical Laboratories”, 24) Van Leeuwen, M., and Van Rijswijk, M.H.: Acute phase proteins in monitoring inflammatory disorders. Bail iere’s Clinical 25) Votila, M., et. El. : Immunol Methods, 42 : 11, 1981. 26) Yudkin, J.S., et. al. : C-reactive Protein in Healthy Subjects : Association with Obesity, Insulin Resistance, and endothelial dysfunction. A potential role for cytokines originating from adipose tissue? Arterioscler Thromb Vasc Biol 19: 972-8, 1999.
For literature update refer to www.bendermedsystems.com/44.html
15 Ordering Information
Europe-Headquarters
For technical information please contact: www.bendermedsystems.com
Cat.No. BMS288INST human C-reactive protein INSTANT ELISA
16 Reagent Preparation Summary
16.1 Wash Buffer
Add Wash Buffer Concentrate 20 x (25 ml) to 475 ml distil ed water
16.2 Assay Buffer
Add Assay Buffer Concentrate 20 x (5 ml) to 95 ml distil ed water
17 Test Protocol Summary
− Predilute sample with Assay Buffer 1:500 − Place standard strips in position A1/A2 to H1/H2. − Add 50 µl distilled water to sample wel s.
− Add distilled water, in duplicate, to al standard and blank wel s as
indicated on the label of the standard strips. − Add 100 µl Sample to designated wel s.
− Cover microwel strips and incubate 2 hours at room temperature (18° to 25°C) on a microplate shaker at 100 rpm. − Empty and wash microwel strips 3 times with 400 µl Wash Buffer.
− Add 100 µl of TMB Substrate Solution to al wel s including blank
− Incubate the microwel strips for about 10 minutes at room − Add 100 µl Stop Solution to al wel s including blank wel s.
− Blank microwel reader and measure colour intensity at 450 nm. Note: Samples have been diluted 1:500, thus the concentration
read from the standard curve must be multiplied by the dilution
factor (x 500).

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