Time-resolved fluorescence immunochromatographic assay for the detection of mycoplasma pneumoniae IgM and IgG / 中华核医学与分子影像杂志

Objective:To establish time-resolved fluorescence immunochromatographic assay (TFICA) for rapid and quantitative detection of mycoplasma pneumoniae (MP) immunoglobulin (Ig)M and IgG.Methods:Based on capillary effect and europium nanospheres, rapid TFICA for MP-IgM and IgG detections were developed with the optimized parameters (coupling rates of antigens or antibodies to microspheres, dilution of labeled nanospheres, fixture concentrations on test line and serum dilutions). The methodological performances were estimated such as sensitivity, specificity, stability. By testing 55 healthy control samples, the reference values of TFICA were obtained. The reliability was evaluated by Kappa test from detecting sera of 88 cases (33 patients and 55 healthy controls) using TFICA and commercial kits by chemiluminescence immunoassays (CLA). Results:After screening the assay conditions, the mass ratios of mouse anti-human IgG and MP antigen with nanospheres were 1∶20 and 1∶100 respectively; the work dilutions of nanobeads conjugated with anti-human IgG and MP antigen were 1∶200 and 1∶100 respectively; the spraying concentrations of MP antigen and goat anti-human IgM were 0.5 and 1.0 g/L on the test line respectively, and the working dilutions of serum sample were both 1∶300. In the MP-IgM and IgG detections, the linear working ranges were (0.78-70.00)×10 3 relative unit (RU)/L and (0.17-200.00)×10 3 RU/L, while the sensitivities of the assays were 0.78×10 3 and 0.17×10 3 RU/L, respectively. No cross reactions were found with antithyroid peroxidase antibody, anticardiolipin antibody or thyroglobulin antibody. In these MP-IgM and IgG assays, the relative standard deviations were 3.7%-14.8% and 2.9%-14.0%, the average reduction rates of fluorescence were 13.7% and 14.2% respectively after incubation at 37 ℃ for 5 d. The reference values of MP-IgM and IgG were 3.33×10 3 and 2.61×10 3 RU/L, while the Kappa values between TFICA and CLA were 0.79 and 0.76, respectively. Conclusion:TFICA is a simple, sensitive, specific and quantitative method for detecting MP-IgM and IgG antibodies, and may show great promise for future clinical use.

Comparison of Rapid Influenza Diagnostic Tests With Digital Readout Systems and Conventional Rapid Influenza Diagnostic Test for Influenza Virus Detection

@#Introduction: Rapid diagnosis for influenza virus infection is essential for proper patient management, delivering prompt treatment and reducing unnecessary antiviral therapy. Early diagnosis helps in disease prevention and control. Real-time reverse transcription-polymerase chain reaction (RT-PCR) assay yields high sensitivity and specificity in detecting influenza virus infection. However, it is relatively expensive and requires trained personnel and special equipment. In this study, we compared two rapid influenza diagnostic tests (RIDTs): digital readout systems (STANDARD™ F Influenza A/B FIA, fluorescence immunoassay) and conventional visual confirmation (QuickNavi™-Flu2, chromatography immunoassay) with the real-time RT-PCR assay. Methods: Two hundred ninety-eight respiratory samples were obtained from patients suspected of influenza infection at Siriraj Hospital from December 2018 to December 2019. Results: Real-time RT-PCR results showed the detection of influenza A virus in 99 samples (60%), influenza B virus in 61 samples (37%) and co-infection by both viruses in 5 samples (3%) by the real-time RT-PCR assay. The QuickNavi™-Flu2 sensitivity for detecting influenza A and B viruses were 81.73% and 84.85%, and the specificity was 100%. The STANDARD™ F Influenza A/B FIA sensitivity for detecting influenza A and B viruses were 84.62% and 83.33%, respectively. The specificity for influenza A virus detection was 99.25% and 94.74% for influenza B virus. Conclusion: The STANDARD™ F Influenza A/B FIA and the QuickNavi™-Flu2 showed acceptable and comparable sensitivity and specificity. Both RIDTs are potential alternative methods of real-time RT-PCR for rapid screening of influenza virus infection.

Value and clinical application of multiplex bead-based flow fluorescent immunoassay in the detection of autoantibodies in primary biliary cholangitis / 临床肝胆病杂志

Objective To investigate the value of multiplex bead-based flow fluorescent immunoassay (MBFFI) in the diagnosis of primary biliary cholangitis (PBC) by analyzing its results in detecting antimitochondrial antibody M2 subtype (AMA-M2), gp210 antibody, and sp100 antibody. Methods A total of 340 patients who attended The Affiliated Hospital of Qingdao University from August 2018 to June 2020 and were diagnosed with PBC were enrolled as PBC group, and 143 patients with other diseases and 117 individuals undergoing physical examination were also enrolled. MBFFI and immunoblotting test (IBT) were used to detect AMA-M2, gp210, and sp100 autoantibodies in serum, and the Kappa-test was used to compare the consistency of the two methods in detecting the same autoantibody; the receiver operating characteristic (ROC) curve was used to evaluate the value of MBFFI detection of three antibodies in the diagnosis of PBC; the chi-square test was used to compare positive. Results In the PBC group, the two methods showed the best consistency in detecting AMA-M2(Kappa=0.874) and showed relatively good consistency in detecting gp210 and sp100 antibodies (Kappa=0.713 and 0.749). MBFFI had the highest sensitivity of 72.06% in detecting AMA-M2; it had a sensitivity of 44.71% in the combined detection of gp210 and sp100 antibodies, which was higher than its sensitivity in detecting each antibody alone; it had a sensitivity of 82.65% in the combined detection of AMA-M2, gp210, and sp100 antibodies, which was higher than its sensitivity in detecting each antibody alone. Combined detection of the three antibodies had the largest area under the ROC curve of 0.907 0 in the diagnosis of PBC. Conclusion MBFFI has good consistency with IBT in detecting autoantibodies associated with PBC, and the combined detection of AMA-M2, gp210, and sp100 antibodies by MBFFI has higher sensitivity and value in the diagnosis of PBC.

Value and clinical application of multiplex bead-based flow fluorescent immunoassay in the detection of autoantibodies in primary biliary cholangitis / 临床肝胆病杂志

Objective To investigate the value of multiplex bead-based flow fluorescent immunoassay (MBFFI) in the diagnosis of primary biliary cholangitis (PBC) by analyzing its results in detecting antimitochondrial antibody M2 subtype (AMA-M2), gp210 antibody, and sp100 antibody. Methods A total of 340 patients who attended The Affiliated Hospital of Qingdao University from August 2018 to June 2020 and were diagnosed with PBC were enrolled as PBC group, and 143 patients with other diseases and 117 individuals undergoing physical examination were also enrolled. MBFFI and immunoblotting test (IBT) were used to detect AMA-M2, gp210, and sp100 autoantibodies in serum, and the Kappa-test was used to compare the consistency of the two methods in detecting the same autoantibody; the receiver operating characteristic (ROC) curve was used to evaluate the value of MBFFI detection of three antibodies in the diagnosis of PBC; the chi-square test was used to compare positive. Results In the PBC group, the two methods showed the best consistency in detecting AMA-M2(Kappa=0.874) and showed relatively good consistency in detecting gp210 and sp100 antibodies (Kappa=0.713 and 0.749). MBFFI had the highest sensitivity of 72.06% in detecting AMA-M2; it had a sensitivity of 44.71% in the combined detection of gp210 and sp100 antibodies, which was higher than its sensitivity in detecting each antibody alone; it had a sensitivity of 82.65% in the combined detection of AMA-M2, gp210, and sp100 antibodies, which was higher than its sensitivity in detecting each antibody alone. Combined detection of the three antibodies had the largest area under the ROC curve of 0.907 0 in the diagnosis of PBC. Conclusion MBFFI has good consistency with IBT in detecting autoantibodies associated with PBC, and the combined detection of AMA-M2, gp210, and sp100 antibodies by MBFFI has higher sensitivity and value in the diagnosis of PBC.

Analysis of NAT non-reactive results implicated in HBsAg ELISA reactive blood donors by multi-assays / 中国输血杂志

【Objective】 To investigate NAT non-reactive results implicated in HBsAg ELISA reactive voluntary blood donors in Shenzhen. 【Methods】 HBsAg ELISA+ but NAT-blood samples were collected, and HBsAg was further retested by TRFIA, Roche ECLIA and neutralization test. HBV DNA of individual donation was detected by commercial Roche MPX and Uultrio Elite, and virus nucleic acid was extracted via 2.5 mL. Molecular characterizations of HBsAg+ /NAT-samples were determined by quantitative polymerase chain reaction(qPCR) and nested PCR amplifification of the precore and core promoter regions and HBsAg(S) region. HBV serological markers were detected, and the samples with suspicious results were followed up and detected by multi-assay. 【Results】 Among 67 602 samples, 73(0.11%) HBsAg ELISA+ and NAT-blood samples were enrolled in the study. 15(20.5%, 15/73) were confirmed HBsAg+ by TRFIA, ECLI and five alternative DNA assays, and the other 2(2.7%, 2/73) were further identified as HBsAg+ by follow-up study. In 17 confirmed HBsAg+ samples, the viral loads ranged undetectable to 378 IU/mL, with the median of 10.1 IU/mL. Weak correlation was found between HBsAg and HBV DNA load(R2=0.394 4). 【Conclusion】 Some Hepatitis B virus infected blood samples may miss even with different HBsAg assays. Multi-assays with high sensitivity should be combined for blood screening to ensure blood safety..The inconsistent results should be followed up and further tested for hepatitis B serological markers to assist the confirmation.

Newborn Screening and Diagnosis of Infants with Congenital Adrenal Hyperplasia

Congenital adrenal hyperplasia (CAH) is an autosomal recessive endocrine disorder which can manifest after birth with ambiguousgenitalia and salt-wasting crisis. However, genital ambiguity is not seen in male babies and may be mild in female babies, leading to amissed diagnosis of classical CAH at birth. In this review, we provide a standard operating protocol for routine newborn screening forCAH in Indian settings. A standardization of first tier screening tests with a single consistent set of cut-off values stratified by gestationalage is also suggested. The protocol also recommends a two-tier protocol of initial immunoassay/time resolved fluoroimmunoassayfollowed by liquid chromatography tandem mass spectrometry for confirmation of screen positive babies, wherever feasible. Routinemolecular and genetic testing is not essential for establishing the diagnosis in all screen positive babies, but has significant utility inprenatal diagnosis and genetic counseling for future pregnancy.

Dendritic cell and M2 macrophages are associated with the severity of disease in IgA nephropathy patients / 中华肾脏病杂志

Objective@#To investigate the number and distribution of dendritic cells (DCs), macrophages and M2 macrophages in renal tissues of patients with IgA nephropathy (IgAN) and their correlation with clinicopathological parameters, and explore its role in the progression of IgAN.@*Methods@#Renal tissue samples from 42 patients aged ≥18 years with IgAN were collected by kidney biopsy in Guangdong Provincial People's Hospital from January 2018 to June 2018. The patients were divided into different groups according to Oxford classification and Lee grade classification criteria. The distribution and number of DCs (CD209), macrophages (CD68) and M2 macrophages (CD68 and CD206) were detected by immunohistochemistry. Spearman correlation test was used to analyze the correlation between the number of DCs and macrophages in renal tissues and clinical pathological parameters.@*Results@#The number of DCs in the glomeruli of the M1, T0 and C1 groups increased significantly compared with the M0, T1 and C0 groups, and the number of DCs in the renal interstitium of the T1 group increased significantly compared with the T0 group (all P<0.05). The number of glomerular macrophages in group C1 was significantly higher than that in group C0. The number of macrophages in S1, T1 and Lee IV-V tubulointerstitial groups was significantly higher than that in S0, T0 and Lee II-III groups (P<0.05). The number of M2 macrophages in the S1, T1 and Lee IV-V groups was significantly higher in the tubulointerstitial group than in the S0, T0 and Lee II-III groups (all P<0.05). The blood urea nitrogen, serum creatinine and 24 h urine protein levels in the T1 and Lee IV-V groups were significantly higher than those in the T0 and Lee II-III groups, and the serum albumin levels in the Lee IV-V group were significantly lower (all P<0.05). Spearman correlation analysis showed that the number of DCs in renal interstitium was positively correlated with the proportion of tubulointerstitial fibrosis. There were a positive correlation between the proportion of glomerular segmental sclerosis and tubulointerstitial fibrosis and renal interstitial macrophages and M2 macrophages. The number of M2 macrophages was positively correlated with serum creatinine and 24 h urine protein (both P<0.05).@*Conclusions@#The number of DCs and M2 macrophages in kidneys are positively correlated with the clinicopathological features of IgAN patients, which indicates that they may be associated with disease progression.

Microfluidic Chip for Methamphetamine Detection Based on Time-resolved Fluoroimmunoassay / 分析化学

A novel polydimethylsiloxane ( PDMS)-poly-L-lysine ( PLL) microfluidic chip, comprised of 24 reaction channels with 2. 5 μL volume of each reaction channel only, was proposed for quantitative analysis of methamphetamine ( MET) based on time-resolved immunoassay techniques. The chip utilized the adsorption characteristics of PDMS and PLL towards proteins to immobilize MET complete antigens ( MET-BSA) on the surface of reaction channel. Then the competition reaction could happen between MET-Ag in the sample solution and MET-BSA on the inner surfaces of the reaction channels with MET-Ab in the reagent. The surface of latex microspheres was labeled by lanthanide, which could emit red fluorescence under the exposure of ultraviolet ( UV ) . Based on the principle of competitive immunoassay, the more MET-Ag, the less the fluorescence intensity in the reaction channel. The detection results of this chip were acquired using UV-irradiation fluorescence imaging method. With this method, 24 samples could be detected and analyzed simultaneously on a chip by just taking the fluorescence image of the chip. The method allows the detection of MET antigens ranging from 100 ng/mL to 3000 ng/mL, with less sample consumption and high-throughput. This chip is suitable for the police preliminary screening work and has a good application prospects.

Evaluation of Double Antigen Sandwich Time-resolved Fluoroimmunoassay for Specific Total Antibodies to Treponema Pallidum / 现代检验医学杂志

Objective To evaluation the performance of double antigen sandwich time-resolved fluoroimmunoassay(TRFIA)for specific total antibody to Treponema pallidum (TP).Methods Specific total antibody to TP was detected by a double antigen TRFIA based on recombinant multi epitope chimeric antigen.The methodological precision,low limit of detection,accuracy,linearity,reference standard coincidence rate and other analytical performance indicators were evaluated,and clinical comparison research trials were completed.The x2 test was used for the difference between two methods results,the P ＜0.05 which represents the difference was statistically significant.Results The intra-assay and inter-assay coefficients of var iation (CV) were both less than 10％ respectively.The low limit of detection was 0.05 mIU/ml.The relative deviation of de tecting the national standard was not exceed 10％.The linear range was 1.50～ 155.00 mIU/ml and the linear correlation co efficient could be reached 0.999 9.The performance of detection national reference could meet the national accreditation requirements.The consistent rate was 100 ％ when the TRFIA methodology detected the standardized serum plate.The parallel test of TRFIA and treponema pallidum gelatin agglutination test (TPPA) were completed,the total coincidence rate was 99.56％,and the Kappa index was 0.990 6.Conclusion Their result showed that the TRFIA methodology is high sensitivity,accuracy,wide linear range,and highly clinical coincidence rate,which is valuable for clinical application.

Myelin-associated glycoprotein associated neuropathies: clinical features and antibody detection / 中华神经科杂志

Objective To investigate the clinical and electrophysiological characteristics of anti-myelin-associated glycoprotein (MAG)-associated peripheral neuropathy, as well as its antibody detection methods, treatment and prognosis. Methods Six cases of IgM paraproteinemia and anti-MAG antibody-associated peripheral neuropathy were summarized. All of the patients came from Peking Union Medical College Hospital and Qilu Hospital since April 2014 to February 2018. The clinical features, electrophysiological characteristics, and auxiliary examinations including anti-MAG antibody results were analyzed, and the treatment and prognosis were followed. Results Of the six patients, five were male and one was female. The age of onset was 50-77 years and the duration was three months to six years. All the six cases suffered from numbness of distal limbs and gradually progressed to the proximal limbs, of which three cases had muscle weakness. Walking instability occurred in five cases. One patient had electricity-like pain in the lower extremities. Reflexes in the four limbs decreased in five cases, and gloves and socks-like deep sensation decreased in six cases. Five patients underwent lumbar puncture and the cerebral spinal fluid protein ranged from 0.75 to 1.33 g/L. Serum monoclonal proteins were found in six patients, of which four were IgM kappa and two were IgG kappa and IgM kappa biclonal. Four cases were diagnosed with monoclonal gammopathy of undetermined significance and two cases were diagnosed with Waldenstr?m's macroglobulinaemia. Abnormal electromyography was detected in all the six cases, suggesting demyelinating peripheral nerve damage with secondary axonal damage, which was sensory predominant and more severe in lower limbs than upper limbs. In all the six cases, anti-MAG-IgM antibodies were all positive by indirect immunofluorescence assay based on transfected cells and peripheral nerve tissues. After the diagnosis, three patients underwent RCD (rituxima + dexamethasone + cyclophosphamide) chemotherapy. One patient improved (the modified Rankin scale (mRS) score was 3 before treatment and 2 three years after treatment), one patient was stable, and one patient was still in follow-up. One patient was treated with rituxima, and the condition improved (the mRS score was 3 before treatment and 1 one year after treatment). Conclusions MAG-associated peripheral neuropathy can present a slow progressed distal symmetric sensomotor peripheral neuropathy with predominant sensory symptoms. Electromyography shows demyelinating change. Monoclonal protein is usually IgM kappa. MAG-IgM antibodies detection by indirect immunofluorescence assay based on transfected cells and peripheral nerve tissues can support the diagnosis. RCD chemotherapy may be effective.

In vitro effect of diammonium glycyrrhizinate on the growth of human hair follicles and Wnt/β-catenin signaling pathway / 中华皮肤科杂志

Objective To evaluate the effect of diammonium glycyrrhizinate extracted from the Chinese traditional medicine licorice root on the growth of human hair follicles cultured in vitro,and to detect the expression of wnt/β-catenin signaling pathway-related molecules.Methods Isolated hair follicles were cultured with diammonium glycyrrhizinate at different concentrations of 0.1,0.01,0.001 and 0.000 1 μmol/L for 10 days,and the hair follicles cultured in Williams' E medium without diammonium glycyrrhizinate served as a control group.The length of hair follicles was measured under a microscope every day,the morphologic changes of hair follicles were observed,and photos were taken.Immunofluorescence assay was performed to assess the proliferation of hair matrix cells,as well as to determine the expression of β-catenin,glycogen synthase kinase 3β (GSK3β),phosphorylated GSK3β (p-GSK3β) and lymphoid enhancer factor-1 (Lef1) in the Wnt/β-catenin signaling pathway.Statistical analysis was carried out by repeated-measures analysis of variance and one-way analysis of variance.Results As repeated-measures analysis of variance showed,only 0.01 μmol/L diammonium glycyrrhetate showed significantly promotive effect on the growth of hair follicles compared with the medium alone (P ＜ 0.05),and there were no significant differences in the length of hair follicles between the other concentration groups and the control group.Compared with the control group,the transition to the catagen phase of human hair cycle was delayed in the 0.01-μmol/L diammonium glycyrrhetate group,while it did not change in the other diammonium glycyrrhetate groups and control group.Immunofluorescence assay showed that the number of ki67-positive hair matrix cells was obviously increased in the 0.1-,0.01-,0.001-μmol/L diammonium glycyrrhizinate groups compared with the control group,while there was no difference between the 0.000 1-μmol/L diammonium glycyrrhizinate group and the control group.One-way analysis of variance revealed that the expression of β-catenin,p-GSK3β and Lef1 significantly differed among all the groups (F =12.604,16.65,15.266 respectively,P ＜ 0.05),while no significant difference in the expression of GSK3β was found among these groups (F =1.472,P ＞ 0.05).Least significant difference (LSD)-t test revealed that the expre-ssion of β-catenin,p-GSK3β and Lef1 in the hair matrix cells was significantly higher in the 0.1-,0.01-,0.001-μmol/L diammonium glycyrrhizinate groups than in the control group (all P ＜ 0.05),but there was no significant difference between the 0.000 1-μmol/L diammonium glycyrrhizinate group and the control group (P ＞ 0.05).Conclusion Diammonium glycyrrhetate at the concentration of 0.01 μmol/L shows markedly promotive effect on the in vitro growth of hair follicles,and can increase the proliferative activity of hair matrix cells and delay the transition to the catagen phase,which may be associated with the activation of Wnt/β-catenin signaling pathway.

Establishment of Eu3+ / Sm3+ dual?label time?resolved fluoroimmunoassay for measurement of anti?bodies to hepatitis C virus and its clinical application / 中华核医学与分子影像杂志

Objective To establish a time-resolved fluoroimmunoassay (TRFIA) system for simul-taneous measurement of immunoglobulin (Ig)M and IgG antibodies to HCV. Methods Recombinant HCV antigen was fixed on microtiter plates to detect serum HCV antibodies. Eu3+-labeled anti-human IgM and Sm3+-labeled anti-human IgG were prepared. HCV-IgM and HCV-IgG TRFIA were established using indirect assay and further optimized and evaluated. The one-sided 95th-percentile was used to calculate the cut-off (CO) values. Results Defining 1 sample/ CO (S/ CO) as measuring unit, the detection limit was 0.06 S/ CO for HCV-IgM and 0.15 S/ CO for HCV-IgG. When diluted a strong-positive specimen from 1 :12.5 to 1 :51200, there was a good liner range within 1 :12.5 to 1 :12800 for HCV-IgM and 1 :25 to 1 :6400 for HCV-IgG. The average intra-assay CV of HCV-IgM and HCV-IgG were 3.37％ and 3.66％, respectively, and the aver-age inter-assay CV were 6.52％ and 6.75％, respectively. Compared with enzyme-linked immunosorbent as-say (ELISA) kits, the positive conformity rate, the negative conformity rate and total conformity rate were 100.0％(20/ 20), 90.0％(18/ 20), 95.0％(38/ 40) for HCV-IgM TRFIA, and were 100.0％ (20/ 20), 95. 0％(19/ 20), 97.5％(39/ 40) for HCV-IgG TRFIA, respectively. Additionally, the established HCV-IgM and HCV-IgG assay kits presented good stability with a decline in the value of fluorescence of 11.1％and 9.5％ respectively after being stored at 37 ℃ for 7 d. Conclusions The established HCV-IgM and HCV-IgG TRFIA could simultaneously measure HCV-IgM and HCV-IgG antibodies at one step. The method has wider detectable range and may be a more sensitive, stable, and reliable method for diagnosing HCV in-fection.

Dual-antigen sandwich-type time-resolved fluoroimmunoassay for phospholipase A2 receptor antibody, a new marker of membranous nephropathy / 中华核医学与分子影像杂志

Objective To establish a time-resolved fluoroimmunoassay (TRFIA) method for detecting M-type phospholipase A2 receptor (PLA2R) antibody and to investigate the diagnostic value of serum PLA2R antibody for idiopathic membranous nephropathy (IMN).Methods The microplates coated with recombined PLA2R antigen and Eu3+-streptavidin labeled PLA2R antigen were used to establish a dual-antigen sandwich-type TRFIA for PLA2R antibody detection (anti-PLA2R-TRFIA).The serum concentrations of PLA2R antibody in 63 IMN patients (36 males,27 females,age 25-75 years) and 90 healthy volunteers (30 males,60 females,age 22-53 years) were quantitatively analyzed.Kruskal-Wallis H test and MannWhitney u test were used to analyze the data.Results The range of anti-PLA2R-TRFIA was 0-10 mg/L and the sensitivity was 5 μg/L,while ED20,ED50 and ED80 of the standard curve were (0.144±0.012),(0.707±0.029) and (3.466±0.098) mg/L,respectively.The CV of inter-and intra-assay were 4.7％ and 5.1％,respectively.The average concentration of serum PLA2R antibody in healthy volunteers was 0.455(0.320,0.593) mg/L,but in IMN patients it was 2.690(0.717,7.750) mg/L (z=-3.688,P＜0.05).Meanwhile,the serum levels of PLA2R antibody in IMN patients were significantly different between different stages and ages (x2 values:10.328,9.716,both P＜0.05).According to the receiver operating characteristic (ROC) curve,when the diagnostic cut-off was set at 0.80 mg/L for IMN detection,the sensitivity and specificity of anti-PLA2R-TRFIA were 73.0％ (46/63) and 95.6％ (86/90),respectively.Conclusions AntiPLA2R-TRFIA has been well established.This quick and easy-performance method could increase the diagnostic accuracy for IMN.

Establishment of a time-resolved fluoroimmunoassay in detection of human epidermal growth factor receptor 2 / 中华核医学与分子影像杂志

Objective To set up a time-resolved fluoroimmunoassay (TRFIA) method for human epidermal growth factor receptor 2 (HER2) detection and to evaluate its performance.Methods Each well of the 96-microwell plate was coated with monoclonal antibody of HER2(H7) and another monoclonal antibody of HER2(E5) was labeled by Eu3+.The sensitivity,stability,specificity,measurement range and reference value of this method were tested.The correlation between chemiluminescence (CLIA) method and TRFIA method was analyzed.Results The sensitivity of HER2-TRFIA method was 0.214 ng/ml.The measurement range was 0.214-1 000 ng/ml.The mean within-run CV and mean between-run CV were 3.48％ and 4.13％,respectively.HER2-TRFIA method had no cross-reaction with HER1 and its reference range was 0-13.20 ng/ml.The correlation coefficient between TRFIA and CLIA was 0.997.The same batch of reagents were found to be stable for more than 6 months at 4 ℃.Conclusions HER2-TRFIA method has high sensitivity,specificity,stability and wide detecting range.It might be suitable for clinical use.

Simultaneous detection of tumor markers in human serum using time-resolved fluorescence microspheres immunochromatographic assay / 中华核医学与分子影像杂志

Objective To develop a time-resolved fluorescent microspheres immunochromatographic assay (TRFMIA) for detection of alphafetoprotein (AFP) and carcinoembryonic antigen (CEA) in human serum and to evaluate its performance.Methods The Eu-time-resolved fluorescent polystyrene particles conjugated with monoclonal antibody AC18# for AFP and AE03# for CEA were used as fluorescent labels.The monoclonal antibody AC17# for AFP,AE05# for CEA and goat anti-rabbit antibody were immobilized on the nitrocellulose membrane as the test lines and control line.Several performances indicators were measured,including linear range,detection limit,and specificity.AFP and CEA were measured by the new method and the results were compared with those obtained by time-resolved fluoroimmunoassay (TRFIA) and electro-chemiluminescence immunoassay (ECLIA) using linear correlation analysis.Results The measurement ranges of AFP were 0.07-1 000.00 kU/L with the intra-and inter-assay CV of 5.93％ and 11.07％,and those of CEA were 0.12-500.O0 μg/L with the intra-and inter-assay CV of 7.53％ and 12.13％ respectively.The average recovery rate of AFP and CEA was 92.77％ and 94.73％,respectively.Measurements obtained by TRFMIA had strong correlation coefficients ranging from 0.93 to 0.97 when compared with those obtained by TRFIA and ECLIA.Conclusion TRFMIA,which can simultaneously detect AFP and CEA,has been successfully established.

Current status and prospects of quantitative immunoassay technologies / 中华检验医学杂志

Quantitative immunoassay technique is the common method of quantitative detection in clinical laboratory. Several important branches of quantitative immunoassay were formed by changing the tracer or the Antigen-antibody complex separation method, including radioimmunoassay, fluorescent immunoassay, enzyme immunoassay, chemiluminescence, colloidal gold, immuno-turbidimetric analysis and homogeneous immunoassay. Different immunoassay techniques have their own characteristics, also apply to different detecting conditions in clinic. This paper reviewed several common kinds of quantitative immunoassay technology, and discussed both their advantages and disadvantages, which provide reference for the application and development of clinical testing technology.

Diagnostic tests for Alport syndrome / 中华检验医学杂志

Alport syndrome, characterized by hematuria, renal failure, sensorineural deafness, and ocular abnormalities, is an inherited glomerular disease caused by mutations in the COL4A5 (X-linked), or COL4A3 and COL4A4 (autosomal recessive) genes.The ultrastructural changes of Alport syndrome can be observed in the electron microscopic analysis of renal biopsies, which are characterized by irregular thinning/thickening, layering and splitting dense layer of the glomerular basement membrane.In the basement membrane of skin and renal biopsy tissue, type IV collagen α chain immunofluorescence test can be used to diagnose Alport syndrome, screen the carriers and determine the mode of inheritance.The analysis and detection of Alport syndrome gene are not only useful for genetic counseling, but also useful for identification of gene carriers, prenatal diagnosis, and identification of suspected patients who can not be confirmed by clinical and pathological examination results.

Detection p16INK4a protein in cervical tissues with time-resolved fluoroimmunoassay / 临床与实验病理学杂志

Purpose To establish a new method for detecting p16INK4a in cervical tissues with time-resolved fluoroimmunoassay (TRFIA).Methods 126 cases of paraffin imbedding tissues of cervix were selected for immunohistochemistry (IHC) of EnVision two-step and TRFIA.Results There were 20 cases of no intraepithelial lesion or malignancy,24 cases of low-grade squamous intraepithelial lesion (LSIL),53 cases of high-grade squamous intraepithelial lesion (HSIL) and 29 cases of squamous cell carcinoma (SCC).In the groups of no intraepithelial lesion or malignancy,LSIL,HSIL and SCC,p161NK4a positive was seen in 1,19,53 and 28,respectively.TRFIA test results displayed p16INK4a positive in 3,17,50 and 27 cases,respectively.Positive of p16 using by TRFIA in no intraepithelial lesion or malignancy,LSIL,above HSIL was 15.00％,70.83％ and 93.90％,respectively (P ＜ 0.01).Conclusion TRFIA is suitable for detecting of p16INK4a protein and demand low detection equipment,p16INK4a expression detected by TRFIA may helpful for large scale detection in various clinical institution.

Expression of indoleamine 2, 3-dioxygenase in condyloma acuminatum lesions / 中华皮肤科杂志

Objective To measure the expression of indoleamine 2, 3-dioxygenase(IDO)in condy-loma acuminatum (CA) lesions, and to evaluate its ability to locally metabolize tryptophan. Methods Immunohistochemical study was performed to observe the protein expression of IDO in skin lesions of patients with CA, and count the number of IDO-positive cells. Immunofluorescence assay was conducted to estimate the relationship between IDO-positive cells and dendritic cells. Epidermal cells and keratinocytes were isolated from warts of 30 patients with CA and prepuces of 11 healthy controls respectively, and both in vitro incubated with tryptophan solution for 4 hours. Then, high-performance liquid chromatography (HPLC)was performed to detect the level of tryptophan metabolite, kynurenine, in the culture supernatant of the above cells, which could reflect the ability of epidermal cells to metabolize tryptophan. Results Rare IDO-positive cells were found in the normal skin, but a lot of IDO-positive cells gathered in the epidermis of the wart tissues. The IDO-positive cell/total cell ratio was significantly higher in the wart tissues than in the normal skin(48.3%± 15.4%vs. 5.2%± 2.4%, P<0.05). The fluorescence signals of IDO-positive cells and CD1a-positive Langerhans cells were not overlapped with each other, suggesting that IDO-positive cells were derived from epidermal cells of the wart tissues. Compared with the keratinocytes from the healthy skin, the epidermal cells from warts had a stronger ability to metabolize tryptophan in vitro. Conclusion A large number of IDO-positive cells exist in CA warts, and may be involved in occurrence of CA.

Effect of lipidemia on time-resolved fluoroimmunoassay for determination of unconjugated estriol / 国际检验医学杂志

Objective To investigate the effect of different degree lipidemia on time-resolved fluoroimmunoassay(TRFIA)for determination of unconjugated estriol(uE3).Methods Mixed serum was prepared by collecting different levels of lipidemia samples which were normal and chylous appearance from male and by mixing with definite value serum of uE3.The levels of uE3 in the sam-ples were measured by TRFIA and the effect of lipidemia on TRFIA for determination of uE3 was evaluated.Results For the ap-pearance of chylous specimens,mild lipidemia increased uE3,mid-or hiper-lipidemia samples reduced uE3 and the effect of both was considerable.Conclusion The chylous lipidemia has variant degree of influence to TRFIA for determination of uE3,then the results effect accuracy of Down′s screening.