Enzyme-driven Switchable Fluorescent Immunoassay For Complement Activated C1s And Its Application In The Diagnosis Of Coronary Heart Disease

Background and objective:Coronary atherosclerotic heart disease（CHD）is the most common and deadly disease in clinic,which causes angina pectoris,myocardial infarction,and even sudden death.Unfortunately,up to now,there are no effective serum biomarkers in the diagnosis and risk stratification of this disease.Previous studies have shown that complement classic activation play vital roles in the pathogenesis and progression of CHD,and the esterase activity of activated C1s works as the key step in this complement activation.Nevertheless,the functions of activated C1s in the development of CHD are still unclear.Also,there are no available assays for activated C1s detection.Thus,in this project,we firstly prepared C1s specific antibody using phage expression technology,and then developed the specific fluoroimmunoassay to detect the levels of activated C1s in the serum by combining the esterase activity of C1s with the peptide-based fluorescence resonance energy transfer assay.Moreover,the esterase activities of activated C1s in the peripheral blood of the patients with CHD have been analyzed to explore its potential significance in the diagnosis of CHD in clinic.Thus,this project has not only established one immunoassay for activated C1s,but also explored the possbility of activated C1s work as a new potential serum biomarker for the early diagnosis and risk stratification of CHD.Methods:1.Preparation of peptides and antibodies in the fluorescence immunoassay for complement activation C1s enzyme（1）Design and selection of substrate peptide:Three substrate peptides were prepared according to the cleavage sites in C4 and C2 mediated by activated C1s.The N-and C-termini of the polypeptides are further modified with fluorescent group R and fluorescent quenching group Q respectively.Next,the activated C1s was reacted with a substrate peptide,and the K_cat/K_m of each candidate peptide was calculated to screen suitable substrates.Additionally,the catalytic efficiency of the activated C1s to the screened candidate was compared at 25°C or 37°C,and the appropriate reaction conditions were selected for the reaction system.（2）Phage library screening of monoclonal antibodies to activated C1s:（1）BALB/c mice were immunized with activated C1s protein for at least four times,and the immune effects were determined based on serum titer by using ELISA;（2）The splenic tissues were collected for total RNA extraction,followed by the synthesis of c DNA using RT-PCR,which was then used as a template to amplify the heavy chain variable region（VH）and light chain variable region（VL）of antibody for activated C1s;（3）Splicing by overlap extension PCR（SOE-PCR）was used to splice the variable regions of the heavy and light chains of antibody into a single chain antibody fragment（sc Fv）;（4）The sc Fv was inserted into p CANTAB5E phagemid after digestion,followed by directional cloning to construct recombinant phagemid;（5）The recombinant phagemids were transformed into TG1 competent cells to construct an expression library;（6）M13KO7 helper phage was utilized to transfect the TG1 library to construct a phage display library of anti-activated C1s single-chain antibody;（7）The gradient dilution of the phage antibody library was utilized to calculate the capacity of antibody library,together with selection of positive clones and analysis of the phage library diversity;（8）The constructed phage library was amplified,and the inactivated C1s protein was used as the target antigen for reverse screening,followed by positive screening using the activated C1s protein.Afterwards,three rounds of screening were performed through the"adsorption-elution-amplification"steps,and then sequence analysis was performed.（3）Recombination and performance verification of activated C1s antibody:（1）PCR was conducted to amplify the DNA fragments in VH and VL of activated C1s antibody;（2）PCR products were purified using electrophoresis,and then the cloned target sequences were transformed and recombined;（3）Positive colonies were identified by PCR,electrophoresis and sequencing;（4）Plasmids were transfected into HEK293 cells and cultured for one week;（5）The supernatant was collected,and Protein A affinity chromatography was utilized to purify the antibody;（6）Use SEC-HPLC was used to detect the purity of the activated C1s antibody;（7）ELISA was performed to determine the binding status of antibody with activated C1s,inactivated C1s,C1,and C1r,respectively;（8）To prevent the potential negtive effects of C1s antibody on the enzymatic activity of activated C1s,different concentrations of C1s antibody were firstly used to incubate with activated C1s.Then,the substrate peptide was added,followed by detecting the cleavage effects of activated C1s on the substrate.（4）Preparation and screening of C1s antibody-coupled magnetic beads:（1）Carboxyl magnetic beads purchased from different manufacturers,which with particle sizes between 0.8-2.7μm,were used to conjuated with the activated C1s antibody.（2）The C1s conjugated magnetic beads were used to react with the activated C1s,and then the cleaving power of bead captured activated C1s on the substrate peptide was detected;（3）To avoid the activation of the inactivated C1s in the blood,EDTA anticoagulated plasma were used as samples to compared the efficiencies of different immunoassay which established by four different magnetic beads.2.Establishment of a fluorescent immunoassay based on anti-C1s capture for complement activation C1s enzyme（1）Establishment of C1s enzyme activity dependent fluorescence immunoassay:The anti-C1s conjugated magnetic beads were pretreated and mixed with the samples.After being incubated and washed,the substrate peptide was added,and the signals of fluorescence were monitored with a microplate reader.（2）Evaluation of C1s enzyme activity dependent fluorescence immunoassay:（1）The optimal reaction time was fixed by comparing the effects of different reaction times on signals of fluorescence;（2）The lowest detection limit was determined by testing the zero-value calibrator for at least 20 times;（3）The recovery rate was calculated by adding different concentrations of activated C1s to samples of known concentration;（4）The intra-and inter-batch precision examination was done by repeating the test for 10 times using low,medium and high value samples from same or different batches;（5）The cross-reaction rate was detected by adding inactivated C1s,activated C1r,MASP1,and MASP2 to samples with known concentrations;（6）Bilirubin,chyle and hemoglobin were added to samples with known concentrations to detect the anti-interference effects of this method;（7）Serum and various anticoagulant（EDTA,citrate,heparin）treated plasma were tested in this immunoassay for candidate sample selection;（8）To test the effects of freeze time on the immunoassay results,serum samples were stored at-80°C,followed by testing at 3,6,and 12 months,respectively;（9）The reference value range was fixed by enrolling 306individuals.3.Effects of EDTA on enzymatic activity of activated C1s（1）Comparing the enzymatic activity of activated C1s between serum and EDTA treated plasma samples;（2）Comparing effects of EDTA treatment on the enzymatic activity of activated C1s using different concentrations of EDTA（0,25,50,and 100m M）;（3）Comparing the effects of Ca Cl₂（1,5,10 m M）,Mg Cl₂（1,6.25,12.5 m M）,Na₃PO₄（5,10,25,50 m M）on the enzymatic activity of activated C1s in samples which treated with EDTA.4.Detection and diagnostic values of activated C1s in CHD patients（1）Subjects:169 CHD patients from Taizhou People’s Hospital were recruited into the experiment,including 47 patients with stable angina pectoris（SAP）,76patients with unstable angina pectoris（UAP）,and 46 cases with acute myocardial infarction（AMI）.In addition,46 non-CHD control group（NCHDC）which including31 X syndrome and 15 myocardial bridge,and 76 healthy control were recruited.The demographic data from each volunteer were also collected.（2）The levels of activated C1s enzymatic activity in serum of the CHD and the control were measured using this immunoassay.ELISA was utilized to detect the levels of C1s,C3,C3a,C4,C4a,and C5a in serum.（3）The differences in the above indicators between the CHD group and the control group were compared,and the correlations in these indicators were further analyzed.Logistic regression analysis was performed to evaluate the risk factors of CHD.Receiver operating characteristic（ROC）curve was depicted as the diagnostic values of activated C1s（model 1）and C1s-based（model 2）models for CHD.5.Correlation between the levels of activated C1s in serum and coronary artery stenosisIn this study,we recruited 215 patients from the Department of Cardiovascular Medicine,Taizhou People’s Hospital.All patients in both CHD and non-CHD control were confirmed by using coronary angiography.Based on major vascular stenosis（MVS）,CHD patients were further classified into non-stenosis control（NSC）,MVS-group（with a MVS of less than 75%）and MVS+group（with a MVS of 75%or more）,followed by Gensini scoring.Kruskal-Wallis H test was used to compare the differences of complement-related indicators among three groups.Spearman correlation analysis was used to analyze the correlation between complement-related indicators and Gensini score.Logistic regression was used to analyze the influence factors in coronary artery stenosis.Results:1.Optimization of raw materials used in the fluorescent detection method of complement C1s enzymatic activities:（1）Design and screening of substrate peptides:（1）The K_cat/K_m value of peptide 3was significantly higher than that of substrate peptide 1（P<0.05）and substrate peptide 2（P<0.05）;（2）The K_catvalue of C1s enzymatic activity at 37°C was significantly higher than that at 25°C（P<0.05）.（2）Screening,recombination and functional evaluation of activated C1s monoclonal antibody:（1）A phage library with a library capacity of 9.6×10⁹colony-forming units（c.f.u）was finally established,which with an insertion rate of 90%and good sequence diversity.Moreover,two sequences of anti-activated C1s antibody were screened and constructed for further experiments in this library;（2）The recombinant expression of the activated C1s antibody was performed in HEK293 cell,and the results showed that the purity of antibody was up to 95.5%by using SEC-HPLC;（3）The recombinant C1s antibody had a strong binding ability with the activated C1s protein(EC₅₀:4.112×10^-5 m M),but weak binding ability with inactivated C1s and C1 proteins(EC₅₀:7.324×10^-3 m M,EC₅₀:4.538×10^-3 mmol/L),and non-binding with C1r protein(EC₅₀:814.9 m M);（4）There were no obvious inhibitory effects on the enzymatic activity of activated C1s on target peptide 3various,when various concentrations of activated C1s antibody（5,10,20,40,80 and160μg/m L）were added into the samples（P>0.05）.（3）Preparation of C1s antibody-conjugated magnetic beads:（1）Changes in fluorescence intensity of the Zecen magnetic beads reached the highest,and followed by Merck magnetic beads,when use 10μg/m L activated C1s standard for testing;（2）When Zecen magnetic beads were used,the fluorescence intensity in the plasma was significantly higher than that in the plasma control（P<0.05）.2.Establishment and evaluation of a fluorescent immunoassay based on antibody-captured C1s enzymatic activities.（1）In this assay,the enzymatic reaction was close to the plateau when the reaction time up to 60 min,which with the R² value close to 1.The standard curve fitting equation was Y=49.418X²+1120.4X-42.3（R²=0.9985）.The linear range was less than 10μmol·min^-1·m L^-1.（2）The lowest detection limit was 0.096μmol·min^-1·m L^-1.（3）The recoveries of the detection method were 91.21%,102.75%,and 90.35%,and the recovery were in a range of 90%to 110%,when various concentrations of activated C1s were added into the serum samples with three concentrations.（4）The intra-assay CV values of low,medium and high concentration C1s activity serum samples were 8.91%,6.11%,and 6.68%,respectively.The inter-assay CVs were 9.40%,9.36%,and 8.48%,respectively.Both the coefficients of variation of the intra-assay and the inter-assay precision were less than 10.0%.（5）The cross-reactivity rates of inactivated C1s,activated C1r,MASP1,and MASP2 in this method were 0.16%,0.15%,0.04%,and 0.04%,respectively.Thus,all cross-reactivity rates were less than 0.5%.（6）Low,medium and high concentration activated C1 samples,which were added with high-concentration of bilirubin（0.20 mg/m L）,chyle mixture（2000 FTU）,and hemoglobin（5 mg/m L）respectively,were detected using this assay.The data showed that all results were within the allowable range of measurement deviation（CV<10%）.（7）The results from different types of samples showed that the C1s enzymatic activity levels in EDTA treated plasma was significant lower than that in serum,and heparin or citrate treated plasma（P<0.001）.The C1s enzymatic activity in serum was significant higher than that in heparin treated plasma（P<0.05）.There were no statistical differences in the C1s activity in citrate treated plasma and heparin treated plasma（P>0.05）.（8）After the designated freezing period,the samples were thawed and C1s enzymatic activity measured again.The data showed that there no difference in C1s activity between the fresh and cryopreserved samples,which were stored at-80°C for3,6,and 12 months（P>0.05）.（9）The data from the serum samples of 306 healthy individuals showed that the levels of C1s enzymatic activity from 94.77%（290/306）of healthy volunteers were in a range of（0.26-1.90）μmol·min^-1·m L^-1.3.Effects of EDTA anticoagulant on the enzymatic activity of complement C1s.（1）The enzymatic activity of C1s in EDTA treated plasma was significantly lower than that in serum samples（P<0.001）.（2）The effects of EDTA on the enzymatic activity of C1s in samples were explored.In the absence of EDTA,the enzymatic activity of activated C1s was defined as 100%,and then the activities of C1s were further defined as 78.70%,48.67%,and 10.59%,which in the presence of EDTA concentration at 25 m M,50m M,and 100 m M,respectively.These data indicated that with the EDTA concentration increased,the activity of activated C1s decreased significantly in a dose-dependent manner.（3）To study the effects of Ca Cl₂ on C1s activity in EDTA treated samples,the sample was treated with 25 m M EDTA and then Ca Cl₂with a concentration of 0,1,5,and 10 m M,respectively.Firstly,the enzymatic activity of C1s in sample without EDTA was defined as 100%.And then,the data showed that in the presence of 25m M EDTA,the enzymatic activities of activated C1s in samples were 78.70%,46.94%,22.63%,and 4.04%respectively,when it treated with Ca Cl₂at concentration of 0,1,5,and 10 m M.Thus,it indicated tht the activities of activated C1s in EDTA treated samples decreased with the increased Ca Cl₂ concentration.（4）To explore the effects of Mg Cl₂on EDTA treated samples,the enzymatic activity of C1s in samples without EDTA was defined as 100%,and then the activities of C1s in both 25 m M EDTA and different concentration Mg Cl₂treated samples,The data showe that the enyzmatic activities of C1s in EDTA samples were 78.70%,54.99%,23.38%,and 3.42%,which were treated with Mg Cl₂at concentration of 0,1,6.25,and 12.5 m M.So,these data declared that the enzymatic abilities of C1s in EDTA treated samples were decreased with the concentration of Mg Cl_2.（5）To investigate the effects of Na₃PO₄ on C1s activity in EDTA treated samples,Na₃PO₄ with different concentration were added into samples with EDTA at 100m M.As the activity of C1s in sample without EDTA was defined as 100%,the data showed that the enzymatic activities of 100 m M treated samples were 10.59%,17.87%,22.94%,36.44%,and 64.08%,which simultaneously treated with Na₃PO₄at concentration of 0,5,10,25,and 50 m M,respectively.Thus,it indicated that the enzymatic activities of activated C1s in EDTA treated samples were increased significantly with the concentration of Na₃PO₄.4.The diagnostic value of serum activated C1s in CHD patients（1）Comparison of complement-related indicators in HC,NCHD,SAP,UAP,and AMI groups.Compared with HC group,both levels of enzymatic activities of activated C1s and C1s protein in serum of NCHDC,SAP,UAP,and AMI groups were significant higher（P<0.05）.In addition,the enzymatic activity of activated C1s in serum of AMI patients was significantly higher than that in NCHDC,SAP and UAP groups（P<0.05）.（2）Correlation between complement-related indicators and other laboratory indicators was also analyzed,and the data showed that the levels of activated C1s were positively correlated with C3,TC,TG,and LDL-c（P<0.05）,and negatively correlated with the levels of C5a（P<0.05）.The protein levels of C1s was positively correlated with Hb A1c（P<0.05）,and was negatively correlated with HDL-c（P<0.05）.C4a levels were positively correlated with C5a and TG levels（P<0.05）.C3 was correlated with C3a（P<0.05）.（3）The complement-related indicators between CHD and the control were analyzed,and the data declared tht the levels of activated C1s,C1s,and C4 in the CHD group were significantly higher than those in the control（P<0.05）.The levels of C4a in CHD was significantly lower than that of the control group（P<0.05）.（4）Risk factors for CHD were analyzed,and the data showed that Using the occurrence of CHD as dependent variable,univariate Logistic regression analysis was performed using the indicators with significant statistical differences in the comparison of baseline data and complement-related indicators between the two groups.Indices except C4 were considered as the factors affecting CHD.Then multivariate Logistic regression analysis was carried out using the index of P<0.05 as the independent variable（Model 1:based on activated C1s;Model 2:based on C1s）.In these two models,the history of hypertension,smoking,family history of CHD,and Hb Alc were independent risk factors for CHD,while HDL-c and C4a were independent protective factors for CHD.（5）Diagnostic values of activated C1s and C1s in CHD:The values of activated C1s and C1s alone for CHD diagnosis were limited.Among them,the areas under the curve（AUC）of activated C1s and C1s were 0.69（0.63-0.75）and 0.68（0.62-0.75）,respectively.Then,the diagnostic values of Model 1 and Model 2 that were established based on the activated C1 and C1 were further respectively evaluated,and the data showed that the diagnostic value of Model 1 was higher than that of Model 2[0.91（0.88-0.95）vs 0.89（0.85-0.93）].5.Correlation between activated C1s in serum and coronary artery stenosis.（1）The complement-related indicators in MVS+,MVS-and NSC groups were Comparied and the data showed that there were significant statistic differences in several target items among the three groups,including activated C1s,C4a,and C5a（P<0.05）.Among them,the enzymatic activity of activated C1s in MVS+group was significantly higher than that in both MVS-and NSC groups（P<0.05）.The levels of C5a in MVS+group were significantly lower than that in NSC group（P<0.05）,and the levels of C4a in MVS-group were significantly lower than that in NSC group（P<0.05）.（2）Spearman correlation analysis showed that Gensini score was positively correlated with activated C1s enzymatic activity（P<0.001）and the level of C4a（P=0.020）.In contrast,it was negatively correlated with C5a level（P<0.05）.（3）Hierarchical Logistic regression was further performed with the degree of coronary stenosis（NSC=0,MSV-=1,MVS+=2,grade data）which defined as the dependent variable,and the general demographic characteristics,laboratory indicators and complement-related indicators with statistical differences as the independent variables.The results showed that age（OR=1.04,95%CI:1.01-1.08）,activated C1s（OR=1.94,95%CI:1.29-2.93）,history of hypertension（OR=2.06,95%CI:1.13-3.77）,and smoking history（OR=2.11,95%CI:1.08-4.12）were independent risk factors for vascular stenosis,while the level of C4a（OR=0.997,95%CI:0.995-0.999）was an independent protective factor for vascular stenosis.Conclusions:1.We established a fluorescence immunoassay for activated C1s detection,which with high sensitivity,high specificity and strong anti-interference effects.Thus,it was suitable for the detection of clinical serum samples.Also,this method can be used to explore the contribution of the activated initiated complement classical activation pathway in the pathogenesis,diagnosis,and treatment of various diseases.2.EDTA,citrate,and heparin can inhibit the enzymatic activities of activated C1s.Among them,besides its function in the chelation with calcium ions,EDTA had the strongest inhibitory function might depend on other potential mechanisms in this process.Therefore,serum samples are suitable for the detection of the enzymatic acitivity of activated C1s in clinic.3.The enzyme activity of activated C1s can be used in the diagnosis of CHD and the assessment of the severity of coronary artery stenosis.Our data showed that the activation of activated C1s plays an important role in the pathogenesis and development of CHD.Thus,these results also can provide a new target and theoretical basis for the treatment of CHD.