CK-MB QUANTITATIVE DETERMINATION IN HUMAN SERUM OR PLASMA BY THE ACCESS IMMUNOASSAY SYSTEM Principle Principle of Procedure The Access

CK-MB assay is a two-site immunoenzymatic (sandwich) assay. A sample is added to a reaction vessel with mouse monoclonal anti-human CK-MB antibody-alkaline phosphatase conjugate and paramagnetic particles coated with mouse monoclonal anti-human CK-BB antibody. Human serum or plasma (EDTA) CK-MB binds to the immobilized anti-CK-BB on the solid phase by the sub-unit B epitopes (common to CK-BB and CK-MB isoforms), while the mouse anti-CK-MB conjugate reacts specifically with serum or plasma (EDTA) CK-MB (no reaction with CK-MM or CK-BB isoforms). After the incubation, separation in a magnetic field and washing removes material not bound to the solid phase. A chemiluminescent substrate, Lumi-Phos* 530, is added to the reaction vessel and light generated by the reaction is measured with a luminometer. The photon production is proportional to the amount of CKMB in the sample. The amount of analyte is determined by means of a stored, multi-point calibration curve. http://pathlabmed.uchc.edu/pdf/uid515.pdf

Method Explanation The i-STAT CK-MB test cartridge uses a two-site enzyme-linked immunosorbant assay (ELISA) method. Antibodies specific for an epitope unique to the CK-MB subunit, that therefore do not bind CK-MM or CK-BB, are located on an electrochemical sensor fabricated on a silicon chip. Also deposited in another location on the sensor silicon chip is an antibody/alkaline phosphatase enzyme conjugate specific to an epitope on the B subunit of creatine kinase. The specificity of the conjugate antibody to the B subunit allows this conjugate to recognize CK-MB and CK-BB, but not CK-MM. The whole blood or plasma sample is brought into contact with the sensors allowing the enzyme conjugate to dissolve into the sample. The CK-MB within the sample becomes labeled with alkaline phosphatase and is captured onto the surface of the electrochemical sensor during an incubation period of approximately three minutes. The sample is washed off the sensors, as well as excess enzyme conjugate. Within the wash fluid is a substrate for the alkaline phosphatase enzyme. The enzyme bound to the antibody/antigen/antibody sandwich cleaves the substrate releasing an electrochemically detectable product. The electrochemical (amperometric) sensor measures this enzyme product which is proportional to the concentration of CK-MB within the sample.

AxSYM CK-MB is a Microparticle Enzyme Immunoassay (MEIA) for the quantitative measurement of the MB isoenzyme of creatine kinase (CK-MB) in human serum or plasma. The assay is used to assist in the diagnosis of acute myocardial infarction (AMI). The CK-MB binds to the Anti-CK-MB Coated Microparticles forming an antibody-antigen complex. An aliquot of Assay Diluent is transferred to the matrix cell followed by an aliquot of the reaction mixture containing the antibody-antigen complex bound to the microparticles. The microparticles bind irreversibly to the glass fiber matrix. The matrix cell is washed to remove unbound materials. The Anti-CK-MM (Goat): Alkaline Phosphatase Conjugate is dispensed onto the matrix cell and binds to the antibody-antigen complex. The matrix cell is washed to remove unbound materials. The substrate, 4-Methylumbelliferyl Phosphate, is added to the matrix cell and the fluorescent product is measured by the MEIA optical assembly. http://www.promtest.am/tests/cardiac/CK-MB.pdf CK-MB Enzyme Immunoassay The CK-MB ELISA test is based on the principle of a solid phase enzyme-linked immunosorbent assay . The assay system utilizesa monoclonal antibody directed against a distinct antigenic determinant on the CK-MB molecule is used for solid phase immobilization (on the microtiter wells). A goat anti-CK-MM antibody conjugated to horseradish peroxidase (HRP) is in the antibodyenzyme conjugate solution. The test sample is allowed to react simultaneously with the two antibodies, resulting in the CK-MBmolecules being sandwiched between the solid phase and enzymelinked antibodies. After a 1 hour incubation at room temperature, thewells are washed with water to remove unbound labeled antibodies.A solution of TMB Reagent is added and incubated at roomtemperature for 20 minutes, resulting in the development of a blue color. The color development is stopped with the addition of Stop Solution changing the color to yellow. The concentration of CK-MB is directly proportional to the color intensity of the test sample. Absorbance is measured spectrophotometrically at 450 nm.

Immunoenzymometric assay (TYPE 3): The essential reagents required for an immunoenzymometric assay include high affinity and specificity antibodies (enzyme conjugated and immobilized), with different and distinct epitope recognition, in excess, and native antigen. In this procedure, the immobilization takes place during the assay at the surface of a microplate well through the interaction of streptavidin coated on the well and exogenously added biotinylated monoclonal anti-CKMB antibody. Upon mixing biotin labeled monoclonal antibody, the enzyme-labeled antibody and a serum containing the native antigen reaction results between the native antigen and the antibodies, without competition or stearic hindrance, to form a soluble sandwich complex. Simultaneously, the complex is deposited to the well through the high affinity reaction of streptavidin and biotinylated antibody. After equilibrium is attained, the antibody-bound fraction is separated from unbound antigen by decantation oraspiration. The enzyme activity in the antibodybound fraction is directly proportional to the native antigen concentration. By utilizing several different serum references of known antigen values, a dose response curve can be generated from which the antigen concentration of an unknown can be ascertained. PRINCIPLES OF PROCEDURE Creatine kinase (CK), present in the sample, catalyzes the transfer of a high energy phosphate group from creatine phosphate to ADP. The ATP produced in this reaction is subsequently used to phosphorylate glucose to produce glucose-6-phosphate (G-6-P) in the presence of hexokinase. G-6-P is then oxidized by glucose-6-phosphate dehydrogenase (G-6-PDH) with the concomitant reduction of nicotinamide adenine dinucleotide phosphate (NADP) to nicotinamide adenine dinucleotide phosphate reduced (NADPH). The rate of formation of NADPH is monitored at 340 nm and is proportional to the activity of CK in the sample. These reactions occur in the presence of N-acetyl-L-cysteine (NAC) which is present as an enzyme reactivator. Methodology: NAC (N-acetyl-L-cysteine) CREATINE KINASE (CK-Nac) http://www.ilexmedical.com/files/PDF/CK_ARC_CHEM.pdf Kinetic determination of the creatine kinase based upon IFCC and DGKC recommendations. Creatine kinase (CK) catalyses the reversible transfer of a phosphate group from phosphocreatine to ADP. This reaction is coupled to those catalysed by

hexokinase (HK) and glucose-6-phosphate dehydrogenase (G6P-DH): Creatine phosphate + CK Creatine + ATP ATP + DHK ADP + Glucose-6-phosphate G6P + NADP+

-Phosphogluconate + NADPH + H+ The rate of NADPH formation, measured photometrically, is proportional to the catalytic concentration of CK present in the sample1,2. http://www.toprakmedikal.com/documents/NAC-017.pdf CK-MB REAGENTSET (UV-KINETIC) This procedure involves measurement of CK activity in the presence of an antibody to CK-M momomer. This antibody completely inhibits the activity of CK-MM and half of of the activity of CK-MB while not accecting the B subunit activity of CK-MB and CK-BB. Then we use the CK method to quantitatively determine the CK-B activity. The CK-MB activity is obtained by multiplying the CK-B activity by 2. http://sst-intl.com/wp-content/uploads/2013/02/CK-MB.pdf A number of methods are available for the determination of CK-MB activity. This reagent utilises an immunoinhibition method employing a polyclonal antibody to the CK-M monomer. It inhibits completely the activity of CK-MM and one-half the activity of CK-MB. The activity of the non inhibited CK-B monomer is then assayed. Creatine kinase catalyses the reaction between creatine phosphate and ADP (adenosine5-diphosphate) with formation of creatine and ATP (adenosine-5-triphosphate). The latter compound phosphorylates glucose to G-6-P (glucose-6-phosphate) in the presence of HK (hexokinase). G-6-P is oxidised to 6-PG (gluconate-6-phosphate) and in the same time an equimolar amount of NADP (nicotinamide adenine dinucleotide phosphate) is reduced to

NADPH in a reaction catalysed by G-6P-DH (glucose-6-phosphate dehydrogenase). The increase in absorbance at 340 nm, resulting from the formation of NADPH, is proportional to the activity of creatine kinase in the sample. (3)