| Research background and purpose:CRP is by far the most valuable acute chronotropic response protein,forming immune complexes by binding to multiple or exogenous ligands and specifically recognising C1 q,which then participates in non-specific immunity of the body by activating the classical complement pathway,thus its changes can assist the physician in determining the pathological status of inflammation-related diseases.It has been shown that CRP is not only a non-specific marker of inflammation,but that diseases such as atherosclerotic thrombotic disease,venous thromboembolism and coronary arteriosclerosis directly cause changes in CRP concentrations.Normal serum CRP concentrations are minimal,essentially less than 10 mg/L.However,when inflammation occurs in the body,CRP concentrations rise extremely rapidly and fall to normal levels as inflammation recovers.CRP as a marker is not affected by radiotherapy,chemotherapy,corticosteroid treatment,etc.Therefore,the detection of CRP is important for early diagnosis,post-operative detection,efficacy observation and prognosis of the disease.Clinical testing for CRP is currently based on non-homogeneous immunoassays such as enzyme-linked immunoassays,immunoturbidimetric assays and colloidal gold immunochromatographic assays.Non-homogeneous immunoassay assays mean that once the immune reaction has reached equilibrium,bound markers must be separated from unbound markers before detection,which requires extended reaction times during the assay due to separation and washing,and may add some error,resulting in reduced sensitivity and accuracy.The Light Initiated Chemiluminescent Assay(LICA)is a homogeneous immunoassay that utilises the energy transfer of single linear oxygen molecules and eliminates the need for separation and washing during the assay.The advantages of this technique are a wide detection range,high sensitivity,high specificity,rapidity and stability,and a small test volume.At present,this technique has not been widely used in China,mainly because the main reagents(photoreceptor microspheres)are imported,and the raw materials of the kit are expensive.The system is based on CRP monoclonal antibody,CRP recombinant antigen,luminescent microspheres and biotin.Materials and Methods:1.Photographic nanoparticles were prepared from phthalocyanine bis(trihexylsilyloxy)silane compounds and carboxylated polystyrene microspheres using a high temperature solvation and adsorption method,on the basis of which microspheres were prepared by coupling streptavidin via a carboxy-amino reaction,and the synthesized photographic microspheres were characterized using a Malvern particle size potential analyzer.2.To establish the assay system for serum CRP photoexcitation chemiluminescence assay based on LICA technology,confirm the optimal assay mode and optimize the reaction conditions,including antibody combination,antibody coating amount,dilution ratio of biotinylated antibody to luminescent microspheres,sample volume of photoreceptor microspheres,reaction time and assay buffer,etc.3.The methodological evaluation of the assay system for the photoexcitation chemiluminescence analysis of serum CRP established in this study included the examination of the linear detection range,HOOK effect,accuracy,sensitivity,precision of the method and its detection in real samples.Results:1.By high-temperature solvation and adsorption method,we have successfully prepared controlled particle size photoreceptor microspheres within 331.3±3.8 nm with a zeta potential of-13.9±0.75 and a Pd I of 0.265±0.014,i.e.they can maintain a stable colloidal state in aqueous solution with good dispersion and can be applied to photoexcited chemiluminescence detection.2.The dual antibody sandwich assay is the best mode of analysis for the detection of serum CRP by LICA.The assay system consists of CRP monoclonal antibody-coated luminescent microspheres,biotin-labelled CRP monoclonal antibody and specific photoreceptor microspheres.The assay conditions were optimised using 0.1 M,p H 8.0Tris-HCl buffer as the assay buffer,a biotin-Ab(CRP-11CC)+ luminescent microspheres-Ab(CRP-1CC)antibody combination,an optimal antibody coating volume of 1 mg,a dilution of 1:200 for the biotinylated antibody and 1:20 for the luminescent microspheres,a sample volume of 175 μL for the photoreceptor microspheres,and a first-stage reaction time of 1:20 for the luminescent microspheres.175 μL,a reaction time of 10 min for the first stage and 5 min for the second stage were used as the optimal working conditions for the assay system.3.The assay system of serum CRP photoexcitation chemiluminescence assay initially established in this study has good detection performance.The analytical sensitivity of the method was 0.77 ng/m L and the functional sensitivity was 1.17 ng/m L;the spiked recoveries of the recovery experiments were 88%～106%;the intra-batch coefficients of variation of high,medium and low value serum samples were no more than 5.71% and no more than 8.34% between batches,all of which were less than 10%;good linearity in the detection range of CRP from 0 to 200 mg/L;higher than 20-fold Interfering substances(haemoglobin,cholesterol,triglycerides,γ-globulin,serum albumin)at CRP concentrations had no crossover effect on the results of the method.Conclusion:In this thesis,specific photoreceptor microspheres were prepared and successfully applied to the analysis of serum CRP,thus developing a homogeneous photoexcitation chemiluminescence assay for CRP with its own intellectual property.The breakthrough in the core technology of specific photoreceptor microspheres is expected to surpass the imported products and accelerate the domestic replacement. |
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