Research Of Magnetic Particle Chemiluminescence Based On CTnI Detection

Cardiac troponin I(cTnI)is the gold standard for clinical diagnosis of acute myocardial infarction.Because of its low concentration in human blood,clinical diagnosis requires high detection sensitivity of it.Chemiluminescence immunoassay(CLIA)has been widely used in the detection of myocardial markers due to its high sensitivity,fast reaction speed and high degree of automation.In this paper,a chemiluminescence immune reaction system based on the structure of "antibody A-antigen-antibody B" on the surface of magnetic particles was constructed with acridine sulfonamide as chemiluminescence agent.The detection sensitivity of the system was improved by optimizing reaction conditions,constructing the detection technology of antibody orientation controlled by p H,introducing streptavidin biotin system and exploring the blocking effect of PEG.The main work is as follows:1)Construction and optimization of magnetic particle chemiluminescence method based on cTnI detection.Firstly,magnetic particles modified with antibody A and antibody B coupled with acridine sulfonamide were prepared.By using acridine sulfonamide as chemiluminescence agent,a double antibody sandwich chemiluminescence immune reaction system was constructed by combining reaction substrates.Then the system was optimized in many aspects.For example,optimizing the preparation method of magnetic particle antibody complex,optimizing carboxyl activation conditions,adjusting the dosage of acridine sulfonamide,improving the feed ratio of reactants,improving the formula of buffer,improving environmental conditions and operation methods,etc.The sensitivity of the final system was significantly improved.2)PH regulates the orientation coupling of antibody on the surface of magnetic particles and enhances the sensitivity of cTnI chemiluminescence detection.The chemical coupling method,which is often used to combine proteins with nanomaterials,almost occupies the active groups of antibodies without selection.It is easy to make the antibody binding direction random,so that the antigen binding site can not be fully exposed and lose its activity.Because the charge distribution and hydrophilicity of antibody have different performance at different p H conditions,this chapter developed a simple and efficient p H controlled antibody orientation coupling method by adjusting the optimal p H and antibody dosage when antibody binded to the surface of magnetic particles,and applied it to cTnI detection.The results showed that the binding efficiency,antibody orientation and detection sensitivity of cTnI antigen on the surface of magnetic particles were significantly improved after p H control.At the same time,PEG was modified on the surface of the magnetic particles to improve the hydrophilicity of the magnetic particles and the stability of the detection system.The results showed that when 1 mg magnetic particle corresponded to 80 μg antibody,when the coupling p H was reduced from 7.8 to 5.8,the antibody binding amount increased by 60%,the exposure degree of Fc site decreased by 75%,and the detection signal intensity increased by 8 times.The detection limit of the system was 0.046ng/m L,the linear range was 0.05 ～ 100 ng/m L,and the repeatability was less than 8%,which met the national requirements(the C.V% of manual operation can not exceed 15%).3)Objective to study the effect of streptavidin(SA)-biotin(SB)system on enhancing the sensitivity of cTnI chemiluminescence detection.On the basis of the above work,SA-SB system was introduced to improve the chemiluminescence signal.Firstly,acridine sulfonamide was labeled on SA surface to construct ‘acridine sulfonamide-SA’ complex,and the coupling number of acridine sulfonamide on SA surface was calculated.Then SB was modified on the surface of antibody B to prepare ‘biotin-antibody B’ complex.After that,acridine sulfonamide SA was coupled with biotin antibody by using the strong biological binding between SA and SB,which significantly increased the coupling amount of chemiluminescence agent.Finally,the ‘magnetic particle-antibody A-antigen-biotinantibody B-acridine sulfonamide-SA’ complex was constructed by the specific binding between antigen and antibody,and the cTnI antigen of different concentrations was detected.Finally,the minimum detection limit of the system was 0.037 ng/m L,the linear range was 0.04 ～ 100 ng/m L,and the repeatability of the system was good,which met the national standard(the C.V% of manual operation can not more than 15%).4)The study of PEG blocking to reduce the non-specific adsorption on the surface of magnetoresistance.SA-SB amplification system significantly increased the coupling amount of acridine sulfonamide,and the chemiluminescence signal was improved,but the blank value was also high,and the minimum detection limit had a large optimization space.In this chapter,PEG was introduced to block the surface of magnetic particles,which reduced the non-specific adsorption of chemiluminescence agent on magnetic particles,thus reducing the blank value and improving the stability.Through the design of different PEG feeding time and quantity,combined with the coupling principle of antibody on the surface of magnetic particles,the blocking principle of PEG on the surface of magnetic particles was explored.Finally,the system was used to detect cTnI antigen of different concentrations.The minimum detection limit of the system was 0.021 ng/m L,and the linear range was 0.03 ～ 100 ng/m L.the repeatability was good,which met the national standard(the C.V value of manual operation can not more than 15%).