Establishment Andevaluation Of Solid Phase Time-resolved Fluorescence Immunochromato Graphy For Luteinizing Hormone Detection

At present,the detection of luteinizing hormone(LH)is mainly divided into qualitative detection and quantitative detection.Qualitative detection mostly adopts colloidal gold immunochromatography,namely in vitro ovulation diagnostic paper.The detection of ovulation diagnostic paper does not need instrument,can be interpreted with the naked eye.It is simple to operate and can be used for home self-test and hospital preliminary test.However,due to its low sensitivity,it is susceptible to various external factors and is only suitable for the initial screening.The main quantitative detection methods include radioimmunoassay,enzyme-linked immunosorbent assay,chemiluminescence immunoassay and time-resolved fluorescence immunoassay.Among them,the radioimmunoassay method has some problems such as radionuclide contamination,short shelf life of markers and so on.Enzyme-linked immunosorbent assay is time-consuming and complex to operate,and the enzymaticallabeling tend to affect the spatial structure of the marker,which limits the sensitivity of detection.Chemiluminescence is one of the most widely used detection methods in clinical practice,with high sensitivity and specificity.Time-resolved fluorescence immunoassay technology overcomes the radionuclide pollution problem of immunoradiometric assay,time consuming,complicated process and low sensitivity problems of the enzyme immunoassay method,and high equipment requirement problem of chemiluminescence method.Conventional time-resolved fluorescence immunoassay technologyis carried out in miro-plate,it is necessary to wash and separate combined marker from free marker,with a complex operating steps,long cycle,large sample consumption,hardly for automation,difficultly to be widely used in biomedical research and clinical test.Inthis study,we combine the advantages of time-resolved fluorescence immunoassay and chromatography,use the double-antibody sandwich methodto solidify the antibody conjugated time-resolved fluorescence microspheres on the glass fiber,established a new solid-phase time-resolved immunochromatography method,for the quantitative detection of luteinizing hormone(LH).Compared with traditional liquid phase time-resolved immune chromatography(that is,the antibody conjugated time-resolved fluorescence microspheres is stored in sample diluent),the solide-phase method has higher sensitivity and accuracy,shorterreading time and lowercross reactionwith high concentration of homologous sex hormone in clinical samples,longer shelf life,lower cost,moreover,it more easy to use.After several rounds of practical trials,it has been proved that it is suitable for large-scale clinical application.MethodLH monoclonal antibody(detecting antibody)and sheep anti-chicken IgY(quality control antibody)were labeled on Europium(?)chelated microparticle(eu-cm)and then processed on the fluorescent binding pad.Another strain of LH monoclonal antibody(coating antibody)was coated at the T line(detection line)of cellulose nitrate membrane.Chicken IgY antibody was coated at C line(quality control line)of the cellulose nitrate membrane.The coated cellulose nitrate membrane,fluorescent binding pad and sample pad were assembled as immunochromatographic reagent strip,to establish a time-resolved fluorescence immunochromatographic method.LH in serum samplewas captured by LH detection antibody labled with Eu-CM,form the antigen-antibody reaction compounds,the compound move forwardalong the nitrocellulose membrane,when reachs T position in nitrocellulose membrane,was captured with coating antibody,to form coating antibody-LH-detecting antobody-Eu-CM compound and be fixed on the T line,the remained liquid keep moving to C line,formed chicken IgY-goat anti chicken IgY-Eu-CM compound,and be fixed in C line.Immune fluorescence detector were used to detect the fluorescence signal and calculate the ratio of T/C position(T/C),LH concentrations in the serum samples were calculated by standard curve..The main tasks of this study including screen of LH antibodies,optimize the formulation of fluoresce binding pad processing solution and process,optimize the formulation of sample pad and the formulation of antibody coating.Under the best experimental conditions,the performance of the proposed method was evaluated in terms of the minimum detection limit,linear range,precision,accuracy,specificity and stability.The specimens with high reliability assayed by CLIAwere collected,and the established method was used for repeated detection to verify the reliability of the method.ResultThis study established a LHdetecting method based on time-resolved fluorescence immunoassay and chromatography technique,and related testing kits is also made,the minimum detection limit is 0.165 mIU/mL,and detection range is 0.5-200 mIU/mL for LH.The method has,no cross reaction with human chorionic gonadotropin(HCG),follicle stimulating hormone(FSH),thyroid stimulating hormone(TSH),and has good stability,can be stored at room temperature for two years.Compared with Abbott automatic chemiluminescence analysis system,the linear correlation coefficient between the two detection methods is 0.9874(n=120),and the two methods are highly correlated.The detection time of this method is only 15 minutes(chemiluminescence method takes 30 minutes),the uesd fluorescence immunoanalyzer is smaller in size,the testing cost is lower,the testing process is more convenient.Clinical trial results have proved that it has greater advantages in sensitivity,reading time,specificity,storage period,and other aspects,and low cost,accurate results,convenient detection,commercial trial production.To sum up,this study provides a testing kits with a rapid and accurate methodfor clinical detection of serum LH,the indicators(sensitivity,specificity,precision,stability and accuracy)all meet the requirements of clinical testing,it also provides a new detection method for clinical testing of other hormones,with a broad application prospects.