Anastasia V. Bereznikova

Biochemical Factors Influencing Measurement of Cardiac Troponin I in Serum

Abstract Troponin I (cTnI), a sensitive and reliable marker of damaged cardiac tissue, is now widely used in clinics. But the existence of different cTnI assays with a wide variety of cut-off values and discrepancies between the results of measurements of one and the same sample by different assays is puzzling for clinicians. The most urgent issue at the moment is the development of the international standard, which can be used for the calibration of different assays, thus decreasing between assay biases. But another important item, which should be considered by manufacturers, is the standardisation of the epitopes of the antibodies used for the assay development. The importance of such standardisation originates from the complicated biochemical nature of cTnI. Here we briefly try to analyse the main factors that can influence antigen recognition by different antibodies and formulate principles of antibody selection for assay development.

Epitope mapping of anti‐troponin I monoclonal antibodies

Two groups of monoclonal antibodies (MAbs) specific to human cardiac troponin I (cTnI) were generated by immunization of mice by isolated cTnI (group I, 16 MAbs) or by the whole troponin complex (group II, 15 MAbs). Two sets of overlapping decapeptides covering the complete sequence of cTnI were prepared and used for epitope mapping by SPOT technique. Majority of MAbs (28 out of 31) interacts with synthetic peptides thus indicating that they recognize liner epitopes. MAbs raised against isolated cTnI preferentially recognize epitopes located at the N‐ or C‐terminal ends of cTnI. Nine out of fifteen MAbs raised against whole troponin complex interact with epitopes located in the N‐terminal part of cTnI. Generation of MAbs recognizing both isolated cTnI and cTnI inside of troponin complex and mapping their epitopes provides reliable detection of TnI in serum of patients with acute myocardial infarction.

New approach to standardisation of human cardiac Troponin I (cTnI)

Six different cTnI assays (5 commercial and one assay under development) were run with either their own calibrators or with standard dilutions of different forms of cTnI (free or complexed). Twenty-one serum samples from 7 AMI patients were analysed using these combinations of calibrators and assay constructs. The range of cTnI concentrations as measured in AMI serum samples by different assays was more than 10-fold using each assays own standards or when using free native or recombinant cTnI. With recombinant or in vitro formed ternary cTnI-cTnT-TnC complexes the differences were smaller (3-fold). The lowest between-manufacturer bias (1.4-fold) was obtained when the heart tissue derived native troponin complex was used for the calibration of the assays. For all assays, calibrated against this troponin I complex, the recalculated cut-off values were within 0.1-0.25 microgram/L. We conclude that to reduce assay-to-assay variation, native troponin complex is presently the preferred alternative for the standardisation of cTnI assays.

Thrombin-Mediated Degradation of Human Cardiac Troponin T

BACKGROUND Cardiac troponin T (cTnT) is an acknowledged biomarker of acute myocardial infarction (AMI) that is known to be prone to proteolytic degradation in serum. Such degradation is usually explained by the action of μ-calpain, although there could be other candidates for that role. In the current study, we explored the hypothesis that thrombin-mediated cTnT cleavage occurs as a result of the serum sample preparation. METHODS cTnT degradation was studied by using immunoblotting and mass spectrometry (MS) analysis. RESULTS The comparison of cTnT isolated from AMI heparin plasma and serum samples showed that cTnT in the plasma samples was mainly present as the full-sized molecule (approximately 35 kDa), while in serum samples it was present as a 29-kDa fragment. The incubation of recombinant cTnT, or native ternary cardiac troponin complex with thrombin or in normal human serum (NHS), resulted in the formation of a 29-kDa product that was similar to that detected in AMI serum samples. No cTnT degradation was observed when thrombin or NHS was pretreated with hirudin, a specific inhibitor of thrombin, or during incubation of troponin in normal heparin plasma. When the products of thrombin-mediated cTnT proteolysis were analyzed by MS, 2 fragments consisting of amino acid residues (aar) 2-68 and 69-288 were identified, which suggests that thrombin cleaves cTnT between R68 and S69. CONCLUSIONS The results of this study suggest that the 29-kDa fragment of cTnT in AMI serum samples mainly appears due to the cleavage by thrombin during serum sample preparation.

Anti–Cardiac Troponin Autoantibodies Are Specific to the Conformational Epitopes Formed by Cardiac Troponin I and Troponin T in the Ternary Troponin Complex

BACKGROUND Autoantibodies to cardiac troponins (TnAAbs) could negatively affect cardiac troponin I (cTnI) measurements by TnAAbs-sensitive immunoassays. We investigated the epitope specificity of TnAAbs and its influence on cTnI immunodetection in patients with acute myocardial infarction (AMI). METHODS The specificity of TnAAbs was studied in immunoassays and gel-filtration experiments. The influence of TnAAbs on endogenous troponin measurements was studied in 35 plasma samples from 15 patients with AMI. RESULTS The inhibitory effect of TnAAbs on the cTnI immunodetection was observed only for the ternary cardiac troponin complex (I-T-C) and not for the binary cardiac troponin complex (I-C) or free cTnI. In the same TnAAbs-containing samples, the immunodetection of cardiac troponin T (cTnT) added in the form of I-T-C (but not free cTnT) was also inhibited in the assays that used monoclonal antibodies (mAbs) specific to the 223-242 epitope. The negative effects of TnAAbs on the measurements of endogenous cTnI in AMI samples were less than on the measurements of isolated I-T-C and decreased with time after the onset of symptoms. Early AMI blood samples might contain a mixture of the I-T-C and I-C complexes with the ratio gradually changing with the progression of the disease in favor of I-C. CONCLUSIONS The investigated TnAAbs are specific to the structural epitopes formed by cTnI and cTnT molecules in the I-T-C complex. AMI blood samples contain a mixture of I-C and I-T-C complexes. The concentrations of total cTnI at the early stage of AMI could be underestimated in approximately 5%-10% of patients if measured by TnAAbs-sensitive immunoassays.

Monoclonal antibodies with equal specificity to D-dimer and high-molecular-weight fibrin degradation products.

Fibrin degradation results in the formation of fibrin degradation products (FDPs) of different molecular weights, which include D-dimer. Commercial D-dimer assays recognize multiple forms of FDP with different specificity. As a result, the absence of an international D-dimer standard and the marked discrepancy in the D-dimer values in the same samples measured by assays from different manufacturers have become the primary problems that clinicians face in the D-dimer determination. We consider that an assay with equal specificity to all FDP forms regardless of their molecular weights could help to solve these problems. We aimed to produce mAbs that could equally recognize high-molecular-weight FDP (HMW FDP) and D-dimer. mAbs against D-dimer were produced. The HMW FDP/D-dimer ratios in plasma samples were analyzed following protein separation by gel filtration using the developed fluoroimmunoassay. A sandwich immunoassay with equal specificity to HMW FDP and D-dimer was developed and applied to determine HMW FDP/D-dimer ratios in patients with different diseases. Although the HMW FDP levels prevailed in thrombotic patients, the FDP and D-dimer levels were comparable in septic patients. Meanwhile, the D-dimer levels often exceeded the HMW FDP levels in patients who had undergone surgery. The ‘D-dimer’ levels that were detected by different assays also varied greatly depending on the assay specificities to FDP and D-dimer. Our findings show that the introduction of assays with equal specificities to FDP and D-dimer in clinical practice is a possible way of standardizing D-dimer measurements.

New Protein Vector ApE1 for Targeted Delivery of Anticancer Drugs

A new chimeric gene ApE1 encoding the receptor-binding domain of the human alpha-fetoprotein fused to a sequence of 22 glutamic acid residues was constructed. A new bacterial producer strain E. coli SHExT7 ApE1 was selected for ApE1 production in a soluble state. A simplified method was developed to purify ApE1 from bacterial biomass. It was shown that the new vector protein selectively interacts with AFP receptors on the tumor cell surface and can be efficiently accumulated in tumor cells. In addition, ApE1 was shown to be stable in storage and during its chemical modification. An increased number of carboxyl groups in the molecule allows the production of cytotoxic compound conjugates with higher drug-loading capacity and enhanced tumor targeting potential.

Epitope Specificity of Anti–Cardiac Troponin I Monoclonal Antibody 8I-7

To the Editor: The monoclonal antibody (MAb)1 8I-7 (International Point of Care Inc.) has been considered a sensitive tool for the detection of human cardiac troponin I (cTnI) for at least 15 years (1). It has been used in several cTnI assays and in different cTnI studies (1–5). According to the manufacturers data, 8I-7 is specific to the epitope comprising amino acid residues 137–148 of the cTnI molecule. In the March issue of Clinical Chemistry , Savukoski et al. (2) presented a study dedicated to evaluating the epitope specificity of autoantibodies to cTnI (cTnAAbs). In this study, commercial anti-cTnI MAbs specific to different epitopes were used as capture antibodies in sandwich immunoassays with an anti–troponin C MAb used as a tracer. The authors estimated the inhibition of signal after spiking in cTnAAb-positive and cTnAAb-negative (as a control) samples with a ternary troponin complex. The study showed that the anti-TnI MAbs specific to the region located between residues 65 and 158 …

Application of recombinant antibody fragments for troponin I measurements.

The cardiac isoform of troponin I is a reliable biomarker of damaged cardiomyocytes that accompanies such severe cardiovascular diseases as myocardial infarction. Monoclonal antibody 19C7 recognizes troponin I in the blood-stream with high affinity and specificity. Recombinant antibodies can be used to improve detection systems based on monoclonal antibodies produced with hybridoma technology. In the present study, we compare the properties of monoclonal anti-body 19C7 and its recombinant fragments. It is shown that the recombinant antibody fragments demonstrate similar affinity values as monoclonal antibodies and can be applied for troponin I detection.