Development of a Time-Resolved Fluorescence Immunoassay for the Diagnosis of Hepatocellular Carcinoma Based on the Detection of Glypican-3.

Glypican-3(GPC3), an oncofetal protein, is a potential novel marker for hepatocellular carcinoma (HCC). In this study, we attempted to establish a new method to detect serum GPC3 using the antibodies identified in our previous research, and then evaluated its clinical application for the diagnosis of HCC. Herein, a sandwich time-resolved fluorescence immunoassay (TRFIA) for detecting serum GPC3 was developed. The detection limit, analytical recovery, specificity and precision of the proposed TRFIA assay were satisfactory. A total of 415 patients were collected and divided into seven groups: hepatocellular carcinoma (101), colorectal cancer (67), gastric cancer (44), esophageal cancer (15), cirrhosis (55), hepatitis (61), normal liver (72). Using this proposed method, the concentration of serum GPC3 in these clinical samples was detected. The results demonstrated that the levels of GPC3 in serum from HCC patients were significantly higher than that in others. Compared with the results of chemiluminescence immunoassay (CLIA), a high consistency (Kappa =0.84) was observed. Thus, an effective, sensitive and reliable TRFIA-GPC3 kit for diagnosing HCC was successfully developed. It offers a suitable alternative to existed methods of determining GPC3 and is expected to be used in clinic in the future.

Rapid and Sensitive Lateral Flow Immunoassay Method for Procalcitonin (PCT) Based on Time-Resolved Immunochromatography.

Procalcitonin (PCT) is a current, frequently-used marker for severe bacterial infection. The aim of this study was to develop a cost-effective detection kit for rapid quantitative and on-site detection of PCT. To develop the new PCT quantitative detecting kit, a double-antibody sandwich immunofluorescent assay was employed based on time-resolved immunofluorescent assay (TRFIA) combined with lateral flow immunoassay (LFIA). The performance of the new developed kit was evaluated in the aspects of linearity, precision, accuracy, and specificity. Two-hundred thirty-four serum samples were enrolled to carry out the comparison test. The new PCT quantitative detecting kit exhibited a higher sensitivity (0.08 ng/mL). The inter-assay coefficient of variation (CV) and the intra-assay CV were 5.4%-7.7% and 5.7%-13.4%, respectively. The recovery rates ranged from 93% to 105%. Furthermore, a high correlation (n = 234, r = 0.977, p < 0.0001) and consistency (Kappa = 0.875) were obtained when compared with the PCT kit from Roche Elecsys BRAHMS. Thus, the new quantitative method for detecting PCT has been successfully established. The results indicated that the newly-developed system based on TRFIA combined with LFIA was suitable for rapid and on-site detection for PCT, which might be a useful platform for other biomarkers in point-of-care tests.

The Establishment of an HE4-CLIA Method and the Combined Analysis of HE4 and CA125 in Ovarian Cancer.

BACKGROUND: The human epididymal secretory protein 4 (HE4) is a novel, verified biomarker for the early diagnosis of ovarian cancer. METHODS: Magnetic beads were coated with capture antibodies and were used with acridinium ester labeled detection antibodies in a sandwich-type immunoassay. The patient's HE4 serum levels were measured simultaneously with the chemiluminescence immunoassay (CLIA) kit we developed and electrochemiluminescence immunoassay (ECLIA) kit from Roche (Mannheim, Germany). CA125 was also detected by time-resolved fluoroimmunoassay. The diagnostic value was analyzed. RESULTS: The assay demonstrated a linear range from 2.5 to 2,000 pmol/l, with an analytical sensitivity of 2.5 pmol/l. The reproducibility, recovery, and specificity of the immunoassay were demonstrated to be acceptable. Compared with the ECLIA kit from Roche in 124 serum samples (40 patients with ovarian cancer, 35 patients with benign gynecological diseases, and 49 health controls), there is a satisfied correlation coefficient of 0.875. The area under the receiver-operating curve (ROC-AUC) was 0.903 (95% CI was 0.839-0.966, P < 0.001) for HE4, 0.787 (95% CI was 0.694-0.879, P < 0.001) for CA125, and 0.914 (95% CI was 0.866-0.962, P < 0.001) for combined analysis of HE4 and CA125. CONCLUSIONS: A quantitative method (HE4-CLIA) for detecting HE4 in serum was successfully established. Preliminary clinical sample analysis showed HE4-CLIA has a certain clinical value in the screening and diagnosis of ovarian cancer. Moreover, in distinguishing benign from malignant ovarian lesions, HE4 has higher demonstrated accuracy than CA125.

Heatstroke induces liver injury via IL-1ß and HMGB1-induced pyroptosis.

BACKGROUND & AIMS: Liver injury is a common complication of heat stroke (HS), and often constitutes a direct cause for patient death. The cellular and molecular mechanism underlying HS-induced liver injury remains unclear. Recent evidence indicates that inflammasome plays an important role in mediating sterile inflammation triggered by tissue damage. Using a rat HS model, we identified a novel mechanism by which inflammasome-dependent interleukin-1ß (IL-1ß) activation and hepatocyte pyroptosis mediate HS-induced liver injury. METHODS: To induce HS, rats were subjected to heat exposure. Inhibition of inflammasomes was achieved by RNA silencing and pharmacologic inhibitor prior to heat exposure. Inflammasome assembly, caspase-1 activation, histological changes, as well as serum levels of liver enzymes were measured. RESULTS: We demonstrated that the onset of HS activated inflammasome in the liver as evidenced by increased capase-1 activity and the association of inflammasome components NOD-like receptor family pyrin domain containing 3 (Nlrp3) and apoptosis speck-like protein containing a caspase-recruitment domain (ASC); and the activated inflammasome, in turn, induced IL-1ß activation and hepatocyte pyroptosis, and subsequent augmented liver injury. HS-induced hepatocyte inflammasome activation seems to be high-mobility group box 1 (HMGB1) dependent. Inhibition of Nlrp3, caspase-1, or HMGB1 prevented HS-induced liver inflammation and ameliorated liver injury. CONCLUSIONS: These findings demonstrate an important role of HMGB1 in mediating inflammasome activation in the development of liver injury following HS, and suggest that targeting inflammasome may represent a novel therapeutic strategy to limit cell death and prevent liver failure after HS.

Development of a clinical chemiluminescent immunoassay for serum GPC3 and simultaneous measurements alone with AFP and CK19 in diagnosis of hepatocellular carcinoma.

BACKGROUND: Glypican-3 (GPC3) is an oncofetal antigen that shows great promise as a biomarker for diagnosis of hepatocellular carcinoma (HCC), but there is no reliable kit that can be used to detect it in clinics. The aim of this study is to develop a stable performance kit for GPC3 detection in clinics. DESIGN AND METHODS: The paired antibodies were identified through cycle-screening methods based on our previous research. Then, a double antibodies sandwich chemiluminescent immunoassay for detecting serum GPC3 was developed. The performance of the developed GPC3 diagnostic kit was evaluated by detecting the concentration of serum GPC3 and assessing its single or combined use with alpha fetoprotein (AFP) and cytokeratin 19 fragment (CK19) for HCC diagnosis. RESULTS: The assay demonstrated a linear range of 10-800 ng/ml, the cross-reactivity rate at 0.018% (AFP), 0.020% (carcino-embryonic antigen), and 0.021% (CK19), respectively. The minimum detectable concentration was 0.05 ng/ml; the intraassay coefficient of variation (CV) and interassay CV were both less than 10%, with good stability and reproducibility. GPC3 has a high sensitivity (54.2%) and specificity (99.4%) in diagnosing HCC. The level of GPC3 in HCC was robust higher than that in healthy or other liver diseases' sera (108.67 ± 230.04 ng/ml vs. 3.99 ± 7.68 ng/ml). The diagnostic sensitivity of GPC3 single or combined with CK19 and AFP for HCC was evaluated, and the rates were 54.2 and 90.6%, respectively. CONCLUSIONS: An applicable chemiluminescent immunoassay with stable performance against GPC3 in diagnosing HCC has been established and the combination of GPC3 with CK19 and AFP could improve the diagnostic sensitivity for HCC.

Aberrant CpG island hypermethylation and down-regulation of Oct-6 mRNA expression in human hepatocellular carcinoma.

BACKGROUND: Aberrant CpG island hypermethylation is a major epigenetic mechanism that can inactivate the transcription of cancer-related genes. PURPOSE: This study aimed to investigate whether Oct-6 transcription was regulated by CpG island methylation in hepatocellular carcinoma (HCC). METHODS: Quantitative real-time PCR and the MassARRAY platform (Sequenom) were employed in 38 HCC tissues samples and four cell lines. RESULTS: The levels of Oct-6 mRNA were decreased by more than twofold in 31 of 38 tumor tissues compared to that of adjacent non-cancerous tissues. Among the 31 tumor tissues with lower levels of Oct-6 mRNA, 17 tumor tissues also had higher methylation levels in Oct-6 CpG island. Based on these results, we hypothesized that CpG island hypermethylation may down-regulate Oct-6 mRNA expression in HCC. To confirm this hypothesis, we also analyzed the changes in Oct-6 mRNA expression and CpG island methylation in four HCC cell lines (Huh7, Bel-7402, HepG2 and SMMC-7721) after treatment with 0.1, 0.5 and 2.5 µM 5-Aza-2-deoxycytidine (5-Aza-CdR), a demethylating agent. The results demonstrated that the CpG island methylation levels decreased and Oct-6 mRNA levels increased in a dose-dependent manner in both Huh7 and Bel7402 cells, but there were only slight changes in HepG2 cell. Interestingly, there were no significant alterations of Oct-6 mRNA levels observed in SMMC7721 cell; although lower levels of CpG island methylation were detected after treatment with 5-Aza-CdR. CONCLUSIONS: Our study shows that CpG island hypermethylation contributes to down-regulation of Oct-6 mRNA expression in HCC.

Determination of median levels of the free beta subunit of human chorionic gonadotropin in women from Mainland China using a new time-resolved fluoroimmunoassay.

BACKGROUND: The free beta subunit of human chorionic gonadotropin (free beta-hCG) is an important serum marker for biochemical screening. Its weekly median value varies with ethnicity. Most of the fluorometers for lanthanide chelates are designed for the detection of signals from europium (Eu(3+)) chelates only. METHODS: We developed a two-site, one-step assay using two monoclonal antibodies (MAbs) against free beta subunit and beta subunit with Eu(3+) chelates as labels. Using the present assay, we evaluated 24,634 normal serum samples in Chinese pregnant women during 8-20 weeks of gestation. RESULTS: The detection limit using this assay was <0.05 ng/mL. The within-run and between-run imprecision was <6.0% and 7.0% using control material. Free beta-hCG concentrations measured using the current assay in 999 maternal serum samples correlated well with those obtained by samarium (Sm(3+))-labeled DELFIA free hCGbeta assay (r=0.987). The medians for 8-20 weeks for maternal serum free beta-hCG were higher in the women from mainland China compared to reports from other countries. CONCLUSIONS: The present assay is suitable for use in biochemical screening of women in mainland China. Our study on the median concentrations of free beta-hCG will help establish reference values that are specific for ethnic populations from the Chinese mainland. These will be useful for studying the importance of ethnic factors in biochemical screening.

Dexmedetomidine Attenuates Acute Lung Injury Induced by Heatstroke and Improve Outcome.

INTRODUCTION: Dexmedetomidine (DEX) has been demonstrated to inhibit inflammatory response and protect against multiorgan injury in various scenarios. The objectives of the present study were to ascertain whether DEX is able to attenuate acute lung injury (ALI) under heatstroke (HS), and to explore the underlying mechanism. METHODS: Male C57BL/6 mice were exposed to ambient temperature of 39.5 ±â€Š0.2°C until core temperature reach 43°C. DEX or 0.9% saline was injected i.p. immediately. At the end of the experiment, bronchoalveolar lavage fluid (BALF) and lung tissue were harvested. RESULTS: HS induce ALI and pulmonary dysfunction, while DEX treatment could significantly inhibit lung injury and improve respiratory dysfunction under HS. The overall effect was beneficial and improved the 72 h cumulative survival rate of mice with HS. Furthermore, HS significantly elevated the levels of cytokines in BALF, as well as increased the activity of toll-like receptor 4 (TLR4)/MyD88/nuclear factor-κB (NFκB) signaling pathway in lung tissue, while DEX treatment could inhibit such effects. Finally, DEX could upregulate the expression of caveolin 1 downregulated by HS, which may contribute to the inhibition of TLR4/MyD88/NFκB signaling pathway. DISCUSSION: In conclusion, the present results indicated that DEX may protect against lung inflammatory response and injury under HS via TLR4/MyD88/NFκB signaling pathway, and caveolin-1 may participate in the effects.

Noninvasive Prenatal Testing of Rare Autosomal Aneuploidies by Semiconductor Sequencing.

Rare autosomal aneuploidies (RAAs) can cause miscarriage or other pregnancy complications and lead to inconsistent results of noninvasive prenatal testing (NIPT), but many NIPT providers have not yet started to provide related services. Our aim was to develop a semiconductor sequencing platform (SSP)-based method for detecting RAAs when pregnant women performed NIPT. Fifty-three aneuploidy samples with verified karyotyping or array comparative genomic hybridization (aCGH) results were collected and subjected to RAAs detection using an SSP to develop a method by genomic sequencing. Various trisomies on all chromosomes other than chromosomes 17 and 19, four multiple aneusomies, one monosomy and five sex chromosome abnormalities were got by our method which can directly identify RAAs via a z-score. Then, artificial mixtures of 10% and 5% DNA were created by adding fragmented fifty-three tissue samples and used in an NIPT simulation to develop a bioinformatics analysis method which can use in NIPT. And the results were in accordance with those of karyotyping and aCGH. Therefore, our method has potential for use in NIPT. Finally, 23,823 clinical plasma samples were tested to verify the performance of our approach. Karyotyping or aCGH was performed on the positive clinical samples. In total, 188 of 23,823 clinical samples were positive (T2, n = 1; T7, n = 1; T13, n = 15; T18, n = 45; T21, n = 125; and multiple aneusomies, n = 1) and verified by karyotyping or aCGH; no sample was a false negative. Several false positives were detected, one of which showed maternal copy number variation (CNV). One case of multiple aneusomies was caused by a maternal tumor. The method developed enables detection of RAAs without increasing costs.

[Preparation and evaluation of a time-resolved fluoroimmunoassay kit for CA50 determination].

OBJECTIVE: To prepare a rapid and sensitive diagnostic kit for detection of CA50 based on time-resolved fluoroimmunoassay. METHODS: A sandwich-TRFIA diagmostic kit was developed using anti-CA50 monoclonal antibody and all parameters of the kit were evaluated. RESULTS: The linear measurement range of the kit was (5 - 300) U/ml. The sensitivity was 0.2 U/ml. The intra- and inter-assay coefficients of variation were 4.3% - 8.2% and 7.7% - 11.2%, respectively. There was no cross-reaction with CEA, CA12-5, CA15-3 and AFP. The cross reactivity with CA19-9 was 0.7 U/ml. The correlation coefficient of detection results of 107 blood samples between this newly developed kit and commercially available CA50 RIA kit was 0.901. CONCLUSION: This newly developed CA50-TRFIA kit is a valuable test tool for clinical application with even better sensitivity, specificity and accuracy.

[Preparation of time-resolved fluoroimmunoassay kit for human alpha-fetoprotein detection].

OBJECTIVE: To prepare time-resolved fluoroimmunoassay (TRFIA) kit for detecting human alpha-fetoprotein (hAFP). METHODS: Sandwich TRFIA kit for hAFP detection was developed using monoclonal antibody of anti-AFP. RESULTS: AFP-TRFIA kit was capable of detecting AFP within the range of 1-1 000 U/ml with sensitivity of 0.17 U/ml and without cross-reactivity with CEA, CA12-5, CA15-3, or CA19-9. The intra- and inter-assay coefficients of variation were (3.3-5.9)% and (3.7-6.5)%, respectively. The prepared AFP-TRFIA reagent could withstand preservation at 4 degrees celsius; for 1 year and at 37 degrees celsius; for 7 days with the cutoff value of 12 U/ml in healthy subjects (n=426). The correlation coefficient of the detection results between this kit and commercially available AFP kit (Wallac OY, Finland) for 60 blood samples was 0.995. CONCLUSION: The prepared TRFIA kit for hAFP detection meets the demand of clinical application with good sensitivity, precision, specificity, stability and accuracy.

[Screening and preliminary identification of mimetic peptides of Plasmodium falciparum-infected erythrocyte membrane surface protein 1].

OBJECTIVE: To screen and identify mimetic peptides of Plasmodium falciparun-infected erythrocyte membrane surface protein 1 in order to explore anti-adhesive agent against cerebral malaria. METHODS: Phage-borne peptide KLYLIAEGSVAA was used as panning targets to select target binders in a disulfide-constrained heptapeptides library. Three rounds of biopanning were carried out and then ELISA and competitive ELISA were used to evaluate the binding character between phage-borne peptides and ICAM-1. The insert DNA sequences of positive clones were determined and their amino acid sequences were deduced. RESULTS: After three-round panning, 22 clones were randomly chosen from the third panning and analyzed. Three clones showed positive interaction with ICAM-1, and two of them possessed the amino acid sequence C-ITAVPVR-C, the other one was C-DIMGGYN-C. These peptides specifically inhibited the binding of 15.2 antibody to ICAM-1 detected by competitive ELISA. CONCLUSION: Two kinds of mimetic peptides of PfEMP-1 have been obtained, which can bind with ICAM-1 specifically.

[Identification of the binding site on ICAM-1 for red blood cells infected by Plasmodium falciparum].

OBJECTIVE: To identify the binding site on ICAM-1 to PRBCs in order to explore anti-adhesive agent against cerebral malaria. METHODS: Monoclonal antibody 15.2 against ICAM-1 domain 1 was chosen as target molecule to screen mimetic peptides of ICAM-1 from a 12-mer random peptide library. Three rounds of biopanning were carried out and then ELISA, competitive ELISA, dot-ELISA and Western blotting were used to evaluate the binding character between phage-borne peptides and McAb 15.2. The insert DNA sequences of positive clones were determined and their amino acid sequences were deduced. RESULTS: Thirty clones from the third round were randomly selected, and 26 of them were found positive by sandwich ELISA. The competitive ELISA test proved that most phage-borne peptides could competitively inhibit the binding of antibody (15.2 McAb) with ICAM-1. Analysis of DNA and amino acid sequences indicated that over a half positive phage clones expressed 12-mer peptide KLYLIAEGSVAA. Comparison of peptide K(XX) L(XXX) GSV with the 64-73 aa of primary sequence of ICAM-1 showed a 50% homogeneity. CONCLUSION: These peptides displayed by phage may be analogs of ICAM-1, K..L...GSV probably plays a significant role on the binding reaction of ICAM-1 and PRBCs.

Development of an amplified luminescent proximity homogeneous assay for quantitative determination of hepatitis B surface antigen in human serum.

BACKGROUND: Hepatitis B virus (HBV) poses a serious risk to human health and the hepatitis B surface antigen (HBsAg) is a popular biomarker in the diagnosis of HBV infection. A quantitative method with a high degree of accuracy, sensitivity and throughput is needed. METHODS: A novel amplified luminescent proximity homogeneous assay (AlphaLISA) was developed for HBsAg determination. A set of monoclonal antibodies was screened against the main subtypes of HBsAg (adr, ay) to confirm the assay's sensitivity to mutants. Technological processes and reaction conditions were optimized and the assay performance was evaluated. RESULTS: HBsAg concentrations were determined within a linear range of 0.04 to 100 IU ml(-1). The detection sensitivity was established as 0.01 IU ml(-1). Assay sensitivity to mutant HBsAg was achieved through antibody screening. The results demonstrate that the reproducibility, recovery, and specificity of this assay for HBsAg were better than acceptable. Compared with the commercial light-initiated chemiluminescence assay, the correlation coefficient of the novel assay was established as 0.921. CONCLUSIONS: The novel AlphaLISA developed in this study has shorter incubation time and easier protocol than the ones of conventional ELISA. It could be used for the clinical determination of HBsAg in human serum. We established a platform for further development of other biomarkers.

Preparation and characterization of monoclonal antibody against glypican-3.

Glypican-3 (GPC3) has been reported as a novel serum and histochemical marker for hepatocellular carcinoma (HCC) by several groups. As an oncofetal protein, it is expressed abundantly in the fetal liver, inactive in the normal adult liver, and frequently reactivated in HCC. Immunology reagents are urgently needed to proceed with mechanism-related research, clinical validation, and application. In this report, monoclonal antibodies (MAbs) against GPC3 were made from hyperimmune BALB/c mice by injecting 100 µg of purified antigen intraperitoneally. Hybridomas were screened by indirect enzyme-linked immunosorbent assay (ELISA) using purified protein. Finally 13 mouse hybridomas producing MAbs to GPC3 were established. The MAbs obtained were fully characterized using Western blot analysis, immunofluorescence, and immunohistochemistry. The results showed that these antibodies could be used for preliminary application of the next step mechanism-related research and GPC3 expression level analysis.

HLA class II variants in Chinese breast cancer patients.

Alterations of human leukocyte antigen (HLA) class II molecules are relevant to the development of breast cancer and metastatic progression. However, the role of HLA class II polymorphisms in the pathogenesis and progression of breast cancer is unclear. This study aimed to investigate the association between HLA class II variants and breast cancer susceptibility and prognosis in a Chinese population. Sixteen variants in HLA class II were detected with the Sequenom MassArray® iPLEX System in 216 breast cancer patients and 216 healthy controls. An association analysis based on unconditional logistic regression was carried out to determine the odds ratio (OR) and 95% confidence interval (95% CI) for each SNP. Stratified analysis by oestrogen receptor (ER) and progesterone receptor (PR) status was also performed. Among 16 variants, only seven conformed to Hardy-Weinberg proportions in the controls. None of these seven variants showed statistically significant differences between the case and control groups in this Han Chinese population. However, chr6_32737733, a variant in HLA-DQB1, showed significant associations with both ER-negative and PR-negative breast cancer in the best fit to the dominant model. Furthermore, another significant correlation was seen between chr6_32606112, a variant in HLA-DRB5, and PR positivity. These results indicate that although no breast cancer risk variants in HLA class II were found in this Chinese population, HLA-DQB1 chr6_32737733 may be involved in determining a poor prognosis, whereas HLA-DRB5 chr6_32606112 may relate to a good prognosis.

[Preparation and identification of phage-displayed ICAM-1-mimic peptide].

AIM: To obtain bioactive ICAM-1 mimetic peptide. METHODS: Phages displaying P1 and P2 were prepared by phage amplication and PEG precipitation. The binding between phage-displayed peptides and anti-ICAM-1 mAb 15.2 was evaluated by sandwich ELISA and competitive ELISA. Bioactivities of P1 and P2 was detected by immunohistochemical staining. RESULTS: Phage-displayed peptides P1 and P2 could specifically bind to mAb 15.2, and the binding could be competitively inhibited by ICAM-1. Immunohistochemical staining showed that P1 and P2 could mimic the binding of ICAM-1 to its receptor LFA-1. CONCLUSION: Phage-displayed peptides P1 and P2 are bioactive just as native ICAM-1.