The measurement of cardiac marker proteins in plasma of patients with chest pain initially suspected of having an acute myocardial infarction (AMI) is an important parameter for the correct assessment or exclusion of AMI. The aim of the present study is to develop rapid, simple and economical tests for early detection of AMI based on the immunoassay assessment of human heart Fatty Acid-Binding Protein (FABP).
To develop a sensitive immunoassay, monoclonal antibodies with high affinity for FABP have been developed using hybridoma techniques. Seven monoclonal antibodies were characterized for its specific reaction to FABP including affinity and epitope mapping using surface plasmon resonance. The association rates (Kass
) for heart FABP to MAbs were found to be in the range from 3.13x10[to power of 4] to 4.89x10[to power of 5] M[to power of -1] s[to power of -1] and the dissociation rates (Kdiss
) fall within 10[to power of -3] s[to power of -1]. Three separated epitopes have been defined for the seven antibodies. The affinities and epitope map provided important information for the construction of sandwich-type immunoassay for early detection of AMI.
The clinical evaluation of 50 chest pain patients presented to Prince of Wales Hospital (Hong Kong) confirmed that FABP elevated in serum as quickly as 1 - 3 hours after the onset of the first clinical symptoms of AMI. An Enzyme Linked Immunosorbent Assay (ELISA) was used for measurement of FABP in serum. All the other established infarction markers (CPK and LDH) are late markers: they only appear 6 - 8 hours after myocardial infarction in serum. Normally, the FABP concentration in human plasma is l-3 ng/ml. The discriminator value was set at 10 ng FABP/ml. A typical AMI leads to plasma FABP value up to 200 ng/ml or even above. FABP is therefore an outstanding new early infarction marker. Early detection is important for starting early successful treatment. By monitoring FABP in plasma, the assessment or exclusion of an infarction is possible as early as 1 hour after the admission when the patients are admitted within 24 hours after the onset of the first symptoms of AMI.
The alarm system based on displacement immunoassay was established to monitor the increase in FABP on-line. Enzyme-labeled monoclonal antibodies were associated with FABP immobilized in the Sepharose column and displaced by analyte FABP in the sample. The system allowed detecting both physiological (2 - 6 ng/ml) and pathological concentrations (12 - 2000 ng/ml) of FABP in an on-line flow system. The specificity of the assay has been validated by running through a protein equal in size with FABP, lysozyme, which did not show any displacement. The FABP concentration in serum after reaching a peak value following the onset of AMI, decreases due to elimination of the FABP by the kidneys. This behavior allows not only to calculate the severity of AMI and to backdate the acute event, but also it allows to monitor the reinfarction. Therefore, in combination with a ensitive detector - such as an electrochemical sensor for detecting the enzyme activity of the label, this on-line displacement assay has the potential to be a future on-line alarm system for Emergency Units or Coronary Care Unit in hospitals.
For the FABP detection, a so-called "mmunofiltration assay" principle has been developed. It uses a sandwich technique in which capture antibodies were bound to a cellulose membrane and colloidal gold-labeled monoclonal antibodies were used as detectors. The result can be seen after washing as a red spot if FABP is present. If no antigen (FABP) is in the sample, the membrane shows no red spot. The test requires only one to two drops of blood serum and the overall performance time is l-2 min. This amazingly short time contrasts with the longer time needed for migration type dipsticks. Clinically, 242 serial blood samples obtained from 83 patients admitted to the hospital with chest pain (72 AMI, 9 unstable angina, 2 other causes) have been studied and validated against a standard quantitative FABP immunoassay (established sandwich ELISA). Using an upper reference level of 12 ng FABP/mL (83 samples below, 159 samples above the cutoff), the false positive rate of the rapid test was only 4 % and the false negative rate was 14%. This was an amazing first result for a rather unoptimized attempt using no additional device but the experimenter's eyes. For a decision: "yes, AMI" or "no AMI" this has been already a great step forward. A novel CCD camera based Digital Image Analyzer is used as transducer to make quantitative detection of FABP possible. The quantitative immunofiltration test was in the detection range of 10 - 950 ng/ml which covers the pathophysiological FABP values.
Both the qualitative "yes/no" test and the quantitative FABP will hopefully help to make a practical breakthrough in early AMI diagnosis in Hong Kong, China and later Asia, and ultimately the whole world.
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