2. Describe other methods for CK-MB assays.

Two-tube immunochemical method

A new commercial kit (Impres-MB; International Immunoassay Labs.) recently was introduced for measuring the MB
isoenzyme of creatine kinase (CK-MB) based on the use of monoclonal antibodies. After antibodies to CK-MM isoenzyme
are added to precipitate the CK-MM, antibodies to CK-M monomer are added to precipitate the M-subunit isoenzymes of
CK. Subtracting the enzymatic activity of the second supernate from the residual activity in the first yields the activity of
CK-MB. Results are not affected by CK-BB, mitochondrial CK, or adenylate kinase. However, the anti-CK-MM antibodies
precipitated only about 98% of serum CK-MM and may have partly precipitated CK-MB isoenzyme (average analytical
recovery of CK-MB, 86.6%). Comparison between Impres-MB (y) and electrophoresis (x) yielded the following linearregression equation: y = 0.79x + 3 (r = 0.982, n = 97). Data for CK-MB temporal kinetics, obtained from patients with
myocardial infarction, correlated significantly in both methods; however, peak activity values of CK-MB were significantly
different, confirming that the difference between the new method and the electrophoretic method average 20%.
i-STAT CK-MB test
The i-STAT CK-MB test is an in vitro diagnostic test for the quantitative measurement of creatine kinase MB mass in whole
blood or plasma samples. CK-MB measurements can be used as an aid in the diagnosis and treatment of myocardial
infarction (MI). 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
CKMB within the sample
Wuerzburg et al. and Gerhardt et al. Method
While these procedures can be useful, newer methods have recently been introduced by Wuerzburg et al. and Gerhardt et al.
Their procedure employs a polyclonal antibody to the CK-M monomer to completely inhibit the activity of CK-MM and
one-half the activity of CK-MB. The activity of the non-inhibited CK-B monomer can then be measured. The present
reagent includes the polyclonal antibody in a reagent which measures CK activity. Inhibition of CK-M occurs during the lag
phase of the CK assay system, leaving only CK-B activity to be measured. The CK measuring reagent is based on a
modification of the IFCC recommended formulation.
The sample is incubated in the CK-MB reagent which includes the anti-CK-M antibody. The activity of the noninhibited
CK-B is then determined using the following series of reactions:
CK
Creatinine phosphate +ADP ---------> creatine + ATP
HK
ATP + Glucose --------> ADP + Glucose-6-phosphate G-6-PDH
G-6-P + NAD+ ------------> 6-Phosphogluconate + NADH + H+
The remaining CK-B catalyzes the reversible phosphorylation of ADP in the presence of creatine phosphate to form ATP and
creatine. The auxiliary enzyme hexokinase (HK) catalyzes the phosphorylation of glucose by the ATP formed, to produce
ADP and glucose-6-phosphate (G-6-P). The G-6-P is oxidized to 6-phosphogluconate with the concomitant production of
NADH. The rate of NADH formation, measured at 340nm, is directly proportional to serum CK-B activity.

Immunoinhibition
CK-MB is composed of the two moieties CK-M and CK-B. A specific antibody inhibits the CK-M moiety. This antibody
will bind to and inhibit the activity of the M subunit of CK-MB. This means that only the activity of the B subunit in serum
is measured by the NAC-activated creatine kinase reaction. If the activity is multiplied by a factor of 2, it will give the
activity of CKMB in serum.
Atypical macro CK may result in falsely elevated CK-MB results. For this reason, this assay is primarily used as a screening
test to eliminate negative CK-MBs, and all positive CK-MBs by immunoinhibition are further investigated.
Mass measurement
The mass assay for CK-MB is an immunometric sandwich assay. Either a polyclonal, or more widely used today,
monoclonal antibody is linked to the CK-MB moiety, the CK-MM or the CK-BB is also covalently linked to a poly or
monoclonal antibody bound to a solid surface or paramagnetic particles, then eluted by a series of washes, and finally, CKMB is measured through production of fluorogenic substrate (e.g. Stratus, Dade Division, IMX, Abbott Laboratories) or
using chemiluminescence (ACS:180, Ciba Corning).

REFERENCE:
http://www.chronolab.com/point-of-care/index.php?option=com_content&view=article&id=418&Itemid=67
http://www.ncbi.nlm.nih.gov/pubmed/2178803
http://www.i-stat.com/products/ctisheets/716675-00G.pdf