Latex Nanoparticle-enhanced Turbidimetric Immunoassays Based On 3 Monoclonal Antibodies And Its Application Research For GP73 Detection

ObjectHepatocellular Carcinoma(HCC)is the fifth largest mortality rate of malignant tumors in the world.Due to the lack of effective early diagnosis and pre-clinical screening for HCC in high risk populations,the majority of patients can be treated only with loco regional therapies,resulting in limited survival benefits and high recurrence rates.Therefore,early diagnosis and early treatment of HCC patients is particularly important.At present,Alpha Fetoprotein(AFP)is the most important and most routine serological marker for the diagnosis of HCC.But the sensitivity of AFP is 39-64%,the specificity is 76-91%,the diagnostic accuracy for detecting early HCC has been questioned as its low sensitivity and specificity.Therefore,a serum biomarker with superior diagnostic accuracy is required in diagnosing HCC.In recent years,some experts found that the sensitivity and specificity of Golgi Protein 73(GP73)were higher than AFP in diagnosis of HCC.To evaluate the value of GP73 as an early diagnostic serum marker in HCC,the Latex Nanoparticle-Enhanced Turbidimetric Immunoassay(LTIA)was established for the quantitative detection of serum GP73.First,we evaluated the characterization of the two latex nanoparticles.Then the rapid and sensitive LTIA using latex nanoparticles immobilized anti-GP73 polyclonal and monoclonal antibodies were performed and the clinical application was assessment.Methods(1)Characterization of the Latex Nanoparticles.The particle size and morphology of the two kinds of latex nanoparticles were determined by Transmission Electronic Microscopy(TEM).The size distribution(hydrodynamic diameter)and zeta potential of latex nanoparticles were evaluated by Dynamic Light Scattering(DLS).The hydrodynamic diameter and concentration of nanoparticles were measured with the Nanoparticle Tracking Analysis(NTA).The carboxylation content was measured with the Ftir-850 Fourier Transform Infrared(FTIR)spectrometer and conductometric titration.(2)The LTIA assay based on polyclonal antibodies.An optimization of the assay conditions was performed on the BECKMAN analyzer.The major variables included the particle size of latex particles,the concentration of 1-ethyl-3-[3-dimethylaminopropyl]Carbodiimide Hydrochloride(EDC)and N-hydroxysulfosuccinimide(NHS),the number of antibodies,the amount of latex-immobilized antibody reagent,and the concentration of Polyethylene Glycol(PEG-6000).The clinical relevance was tested on 80 HCC patients,and the reference range was determined using 80 healthy individuals.(3)The LTIA assay based on 3 monoclonal antibodies.An optimization of the assay conditions was performed on the BECKMAN analyzer.The major variables included the concentration of EDC and NHS,the number of antibodies,the amount of latex-immobilized antibody reagent,and the concentration of PEG-6000.The clinical relevance was tested on 80 HCC patients,and the reference range was determined using 80 healthy individuals.Results(1)The 86 nm and 107 nm latex nanoparticles demonstrated excellent monodispesity;the hydrodynamic diameter and concentration of the two nanoparticles are similar;the values of zeta potential were-39.2 and-41.9 mV,indicating that the surface charge of latex nanoparticles was strong enough to ensure the long-term stability.The carboxylation content was measured by conductometric titration was 0.6 mmol/g.(2)The LTIA based on polyclonal antibodies for quantitative measurement serum GP73 was successfully established.On the basis of optimal experimental conditions,the linear range of this method is 20-320 ng/mL;Within-run Coefficient of Variation(CV)obtained in this study was<8%,total CV<10%;the lower detection limit is 2.11 ng/mL;and the sensitivity and specificity was 94.6%and 72.4%,respectively.(3)The LTIA based on 3 monoclonal antibodies for quantitative measurement serum GP73 was successfully established.On the basis of optimal experimental conditions,the linear range of this method is 10-350 ng/mL;Within-run CV obtained in this study was<8%,total CV<10%;the lower detection limit is 1.82 ng/mL;and the sensitivity and specificity was 96.7%and 93.3%,respectively.ConclusionsThe LTIA based on polyclonal antibodies and 3 monoclonal antibodies were successfully established,which provided a new method for clinical detection of GP73.The study showed that the application of LTIA has good methodological performance,and the LTIA based on 3 monoclonal antibodies was more superior than polyclonal antibodies in linear relationship,lower detection limit,sensibility and specificity.