Detection Of Cardiac Troponin I Autoantibodies And Its Negative Interference On Cardiac Troponin I Immunoassays

Cardiac troponin is a myocardial regulatory protein.One of Its subunit is cardiac troponin I which is a polypeptide of 210 amino acid with relative molecular weight of 24000 and only resides in the atrial and ventricular myocardium.Cardiac troponin I has well cardiac specificity because the N-terminal of TnI in myocardium has more 31 amino acids than in skeletal muscle,and the differential of amino acid in two TnI is 40 percent.The forms of cTnI released into the peripheral blood have different complex when myocardial injuryed,including cTnI-C,cTnI-T and cTnI -T-C and so on.Now it has been confirmed that 90%form of cTnI in the blood of patients with acute myocardial infraction is the binary cTnI-C complex.cTnI is released into bloodstream when myocardial necrosis and they are sensitive and specific biochemical marker for detection of acute coronary syndrome.The first report that cTnI for diagnosis of myocardial infraction was published in 1987 by Cummins et al.Since then,cTnI has been shown not only the primary diagnosis markers of myocaidial infraction,but also important serum biomarker which can be used to determine the infarct size and prognostic indicator independent of the electrocardiogram(ECG).At the same time,cTnI can also be used for evaluation the death risk of unstable angina and myocardial infraction patients with coronary heart disease,and the only biomarker which can be used to judge wether to accept the treatment of anticoagulant drugs such as platelet glycoproteinâ…¡b/â…¢.In recent years,cTnI begains to be used as diagnosis of viral myocarditis and judgement of myocardial damage extent caused by drugs and surgery trauma.Antibody-competitive RIA and competitie ELISA are used for detection of cTnI. At first,the detection antibodies anginst cTnI are polyclonal antibodies which responsed to a lot of cross-reactions.Four decades having passed since immunoassays were first introduced,numerous improvement in assay design, detection systems,and instrumentation had emerged.With cTnI monoclonal antibodies and detection system continues to be optimized,the sensitivity of cTnI immunoassays greatly improved.Most sequences of cTnI have antigenicity.Now more than sixteen cTnI monoclonal antibodies are approved for detection antibodies in cTnI immunoassays.Moreover,the use of antibodies against epitopes in the central part of cTnI has been generally recommended because the N-and C-terminal ends are susceptible to proteolytic degradation both in vivo and in vival.In 2007, national academy of clinical biochemistry and IFCC committee for standardization of markers of cardiac damage laboratory medicine practice guidelines point out that epitopes located on the stable part of the cTnI molecule should be a poriority.But further study showed that many commercial cTnI immunoassays are prone to be negative or positive interfered because use of antibodies directed against the most stable region of cTnI(amino acides 30-110) is recommended.Although state-of-the-art immunoassays can quantify very small amounts of analytes in complex biological fluids such as serum and whole blood.However,immunoassays are still not faultless,because several interfering substances may compromise their accuracy.The interference can be either positive or negative,thus causing either falsely high or low test results.Interference caused by antibodies,such as heterophilic antibodies and human anti-species antibodies,is a well-recognized phenomenon,and this interference can usually be eliminated by introduction of blocking agents.Because the true concentration of the analyte in a sample is rarely known,detection of interference could be extremely difficult.During evaluation of an immunoassay,interference can be detected by comparing different assays,testing for linearity,recovery,or commonly known interferents,e.g.,lipemia,icterus, hemolysis,and drugs.For immunoassays in routine use,there is usually no indication of interference other than the result does not fit the clinical picture. When discrepancies between clinical and biochemical data occur,clinicians should be aware of the possibility of interference in immunoassays.In the physiological state,cTnI is the hidden antigen in isolated immune system.In certain pathological conditions,cTnI is released into the bloodstream and may trigger the body's immune system to produce cTnI antoantibodies.In 1996,Jurgen Bohner et al.reported the phenomenon of the negative interference caused by cTnI qutoantibodies.Subsequent study found that many commercial cTnI immunoassays might be prone to be negative interfered because use of detection antibodies directed against the most stable region of cTnI(amino acides 30-110) was recommended.In 2005,Eriksson found cTnI recovery＜10%in 3.2%of patients with chest pain.The serum from these patients might contain cTnI autoantibodies that prevents the recognition of endogenous cardiac troponin.Since serum cTnI autoantibodies may cause such a big interference,but these negative interferences in AMI and myocaiditis patients are rarely reported.What is the positive rate of cTnI autoantibodies in myocardial injury patients,such as AMI and myocaiditis patients? Whether or not cTnI autoantibody titers are linked to the severity of interference? How severe do the cTnI autoantibodies negatively interfere the cTnI immunoassays? Answering these questions may provide beneficial enlightenment for the cTnI clinical application and the improvement of cTnI immunoassays.Partâ… :Expression and purification ofhuman cardiac Troponin I-C in E. coilThe purpose of this part is to prepare coating antigen for eatablishment of cTnI autoantibodies ELISA.The full-length of cTnI was linked with TnC by a short DNA sequence coding for 19 neutral amino acid residues.An expression construct for cTnI-C was engineered by inserting the corresponding cDNA into a pet28 plasmid. Recombinant plasmid was then transformed into E.coli cells,and protein expression was induced by isopropyl-β-D-thiogalactopyranoside(IPTG).Solubility expression of cTnI-C in prokaryotic system was successfully obtained.Fusion protein which had an N-terminal His-tag sequence which could be purified by affinity chromatography on a Ni²⁺-Sepharose column.The identification result of purified product showed that there is a clearly expression protein band in 43KD by 12%SDS-PAGE.The purity of protein is 86.9% by grayscale scanning detecting.Western Blot by using cTnI monoclonal of showed that there was a single blot in 43KD and no degradation.Fusion protein was preserved in -80℃.The fusion protein was stable in all experimental course when Access-2 chemiluminescence immuneassay was used to analyze.Partâ…¡:Establishment of anti-cTnI ELISAby solubility cTnI-C fusion proteinThis experiment is to eatablish anti-cTnI ELISA by using solubility cTnI-C fusion protein.96 maxisorp microtitration plates were coated with cTnI-C fusion protein,which are capable of binding antibodies against cTnl.80 patients with AMI from changhai hospital were screened blindly.The serum with pesk absorbance at 450nm wl was positive contrast which could specifcally bind with 43KD protein. The mixing serum of 40 healthy individuals was negative contrast.Through Chessboard titration experiments,the concentration of coating cTnI protein was confirmed 10μg/L.Serum samples were fiftyfold diluted with PBS buffer.HRP-IgG was eight-thousands diluted with PBS.A_450nm of posotive contrast serum was 1.52.A_450nm of 40 healthy individuals was 0.19 and SD was 0.12.So the cutoff value of anti-cTnI antibody was 0.55.In this part,testing cTnI autoantibodies coating antigen is used in cTnI-C fusion protein,not cTnI monomer.Whether there are TnC autoantibody in human circulating?Theoretically judgement,TnC widely exist in skeletal muscle,such as in caidiac,so TnC has not caidiac-specific and should not be concealed antigen.But there are not reported in the literature whether TnC autoantibodies appear in human circulating.So we prokaryotic expression of the reorganization of TnC(170 amino acids) as a coating antigen to examine the TnC autoantibodies.The result showed that both normal and AMI patients,in particular,15 patients with cTnI autoantibodies are not TnC autoantibodies.Therefore,the results can completely rule out the possibility TnC autoantibodies interference in our experiments.Partâ…¢:Screening and confirming the positive serumwith cTnI autoantibodies in AMI and myocarditis patientsAnti-cTnI antibody ELISA was enralled for assessment of sera obtained from 121 patients with AMI,24 with myocarditis and 210 healthy subjects.Binding specificity of cTnI antibody from positive sera by ELISA was confirmed with Western Blot.Thirteen of the 121 AMI patients(10.47%) and 2 of the 24 myocarditis patients(8.2%) had positive anti-cTnI antibody compared with none in the healthy subjects.The recovery of cTnI by adding cTnI-C fusion protein corresponding to final cTnI concentration of 0.625～100μg/L to sample with anti-cTnI antibody was inhibited.The Spearman correlation coefficient was 0.934,r wad 0.005.Partâ…£:Assessment of negative interferenceto routine cTnI immunoassaysThe recovery of cTnI studies was employing to evaluating the effects of cTnI autoantibodies to routine 5 cTnI immunoassays,including Access-2(Backman Coulter,clinical diagnosis value is 0.1μg/L);Vidas(Biomerieux,clinical diagnosis value is 0.1μg/L);Architect i2000(Abbott,clinical diagnosis value is 0.3μg/L); Axsym(Abbott clinical diagnosis value is 2.3μg/L);Dimension X Pand(Dade Behring,clinical diagnosis value is 0.15μg/L).Recovery 0～50%is low recovery. Recovery 50～80%is secondary recovery.Recovery 80%～is normal recovery. The cTnI-C complex corresponding to 20μg/L cTnI was added to 13 AMI patients with anti-cTnI antibodys(Adimission 6～24h).There are 2 patients lower than the value of clinical diagnosis on Architect i2000,3 patients lower than the value of clinical diagnosis on Axsym,Dimension X Pand and Vidas.All the 13 AMI patients are above the values of the clinical diagnosis.There are 1 low cTnI recovery on Access-2,1 secondary cTnI recovery and 1 low cTnI recovery on Architect i2000,2 secondary cTnI recovery and 1 low cTnI recovery on Axsym,3 secondary cTnI recovery and 2 low cTnI recovery on Dimension X Pand,1 secondary cTnI recovery and 4 low cTnI recovery on Vidas,respectively.Nonparametric Test were performed with the SPSS11.0,P=0.0018(Kuskal-Wallis Test).The recovery of 5 cTnI immunoassays are significant difference.R² of Access-2,Architect i2000 (Abbott),Axsym(Abbott),Dimension X Pand(Dade Behring),Vidas (Biomerieux) are0.841,0.808,0.772,0.707 and 0.424,respective.In this study,Anti-cTnI antibody ELISA was employed for assess sera obtained from 121 patients with AMI,24 with myocarditis and 210 healthy subjects.Binding specificity of cTnI antibody from positive sera by ELISA was confirmed with Western Blot.The recovery of cTnI studies was employing to evaluating the effects of cTnI autoantibodies on cTnI immunoassays.When cTnI-C complex corresponding to 20μg/L cTnI was added,5 of the 13 serums with anti-cTnI antibody were found with the inhibition of recovery.So autoantibodies to cTnI increased in a significant number of patients with AMI and myocarditis shown that these autoantibodies could interfere negatively the cTnI immunoassay.Thus,More attention of negative interference should be paid.In this study,the recovery of cTnI was employed to evaluating the effects of cTnI autoantibdodies on cTnI immunoassays.The assessment includes most cTnI immunoassays in our countary.But.what kind of patient's cTnI autoantibodies improved suddenly,and wherther this phenomenon connect with disease progression and prognosis? All of these need further study in future.