Distinct T-cell receptor profiles associated with hepatitis B e antigen seroconversion during entecavir treatment.

BACKGROUND & AIMS: T-cell receptor (TCR) repertoire is ambiguously changed in chronic hepatitis B (CHB) patients during antivirus therapy. We tried to assess TCR repertoire dynamics and its clinical significance upon HBeAg seroconversion in CHB patients. METHODS: Twenty CHB patients undergoing 1-year entecavir (ETV) treatment were enrolled, including 10 complete response (CR) vs 10 non-complete response (NCR) patients based on HBeAg seroconversion at week 48. The TCRß complementarity-determining region 3 (CDR3) of peripheral CD4+ and CD8+ T cells at weeks 0, 12 and 48 was analyzed by unbiased high-throughput sequencing. The TCR repertoire profiles and their correlations with serological parameters were analyzed. RESULTS: The diversity of TCRß repertoires was decreasing in CR patients but increasing in NCR patients. The distribution pattern of TCR repertoires stratified according to clonotype frequencies changed in the opposite direction between CR and NCR patients. Narrow amounts of newly appearing clonotypes in CR patients experienced a more intensive and robust expansion and this phenomenon could occur as early as week 12 for the CD4+ subset but later at week 48 for the CD8+ subset. There existed some CR-exclusive clonotypes with a relatively low but increasing frequency at week 48. The number of unique TCRß clonotypes was positively correlated with the ALT or HBV DNA level in CR patients but showed no or negative correlation in NCR patients. CONCLUSION: Distinct TCR profiles contribute to predicting HBeAg seroconversion in CHB patients during ETV treatment and certain TCRß CDR3 motif may be utilized for CHB immunotherapy in the future.

Antibodies Specific to Membrane Proteins Are Effective in Complement-Mediated Killing of Mycoplasma bovis.

The metabolic inhibition (MI) test is a classic test for the identification of mycoplasmas, used for measuring the growth-inhibiting antibodies directed against acid-producing mycoplasmas, although their mechanism still remains obscure. To determine the major antigens involved in the immune killing of Mycoplasma bovis, we used a pulldown assay with anti-M. bovis antibodies as bait and identified nine major antigens. Among these antigens, we performed the MI test and determined that the growth of M. bovis could be inhibited effectively in the presence of complement by antibodies against specifically membrane protein P81 or UgpB in the presence of complement. Using a complement killing assay, we demonstrated that M. bovis can be killed directly by complement and that antibody-dependent complement-mediated killing is more effective than that by complement alone. Complement lysis and scanning electron microscopy results revealed M. bovis rupture in the presence of complement. Together, these results suggest that the metabolic inhibition of M. bovis is antibody-dependent complement-mediated killing. This study provides new insights into mycoplasma killing by the complement system and may guide future vaccine development studies for the treatment of mycoplasma infection. Furthermore, our findings also indicate that mycoplasmas may be an appropriate new model for studying the lytic activity of membrane attack complex (MAC).

Determination of sulfonamides in meat with dummy-template molecularly imprinted polymer-based chemiluminescence sensor.

In this study, a molecularly imprinted polymer capable of recognizing 15 sulfonamides was first synthesized with sulfabenz as the dummy template. The calculation results from computation simulation showed that the specific 3D conformation of the template had an important influence on the polymer's recognition ability. Then, the polymer was used as recognition reagent to prepare a chemiluminescence sensor on a conventional 96-well microplate for the determination of the residues of 15 sulfonamides in meat (chicken and pork). Due to the 4-(imidazol-1-yl)phenol-enhanced luminol-H2O2 system, the limits of detection for the 15 analytes were in the range of 1.0-12 pg/mL. The recoveries from the standard fortified blank samples were in the range of 72.7-99%. Furthermore, one assay could be finished within 30 min, and the sensor could be reused 4 times. Therefore, this sensor could be used as a very useful tool for routine screening of residues of sulfonamides in meat samples. Graphical abstract Assay procedures of the molecularly imprinted polymer-based chemiluminescence sensor for determination of sulfonamides.

The development of a fluorescence polarization immunoassay for aflatoxin detection.

A fluorescence polarization immunoassay (FPIA) was developed for the analysis ofaflatoxins (AFs) using an anti-aflatoxin B1 (AFB1) monoclonal antibody and a novel fluorescein-labeled AFB1 tracer. The FPIA showed an IC50 value of 23.33 ng/mL with a limit of detection of 13.12 ng/mL for AFB1. The cross-reactivities of AFB1, AFB2, AFG1, AFG2, AFM1, and AFM2 with the antibody were 100%, 65.7%, 143%, 23.5%, 111.4%, and 2%, respectively. The group-specificity of anti-AFB1mAb indicated that the FPIA could potentially be used in a screening method for the detection of total AFs, albeit not AFG2 and AFM2. The total time required for analyzing 96 samples in one microplate was less than 5 min. This study demonstrates the potential usefulness of the FPIA as a rapid and simple technique for monitoring AFs.

[Relationship between hepatitis B virus polymerase gene mutation patterns of rtM204I/V and pre-core/basal core promoter mutations].

OBJECTIVE: To investigate the relationship between mutations of rtM204V/I (methionine to valine or isoleucine at position rt204 of reverse transcriptase domain) in the hepatitis B virus (HBV) polymerase gene and the G1896A and G1899A single mutations in the pre-eore (PC) region and the A1762T and G1764A double-mutations in the basal core promoter (BCP) region. METHODS: A total of 2,849 hepatitis B complete genome sequences were retrieved from the GenBank/EMBL/DDBJ. The amino acid sequence of the of reverse transcriptase domain and genome sequences of the PC region and the BCP region were aligned using MEGA4 software. Data were calculated using Microsoft Excel and evaluated using SPSS 13.0 statistical software. RESULTS: Among the 2, 849 HBV complete genome sequences, 217 (8%) strains were identified with Y(I/V) DD and 120 of those had the YIDD mutation and 97 had the YVDD mutation. Of the 1543 strains (54.2%) with PC-BCP mutations, seven mutation patterns of G 1896A-G 1899A-G 1896A-G 1899A-A 1762T/G 1764A, A 1762T/G 1764AG 1896A, A 1762T/G 1764A-G 1899A, and A 1762T/G 1764A-G 1896A-G 1899A were identified. of YMDD and PC-BCP had a higher incidence than the single YMDD mutation (76% vs 24.0%, x2=45.283, P=0.000). The double-mutations of YIDD and PC-BCP had a higher incidence than the double-mutation of YVDD and PC-BCP (85% vs 64.9%, x2=11.836, P=0.000). The double-mutation for lamivudine resistance of YMDD and PC-BCP had a higher incidence than the double pre-existent YMDD and PC-BCP mutations (89.3% vs 58.9%, x2=27.084, P=0.000). The three mutation patterns of G1896A-G1899A (P=0.000, OR=7.573), A1762T/G1764A-G1899A (P=0.000, OR=6.539) and A1762T/G1764A-G1896A-G1899A (P=0.000, OR=6.596) were associated with a greater risk of developing the YIDD mutation, according to binary logistic analysis. CONCLUSION: There is a relationship between the HBV YI/VDD mutation and PC-BCP mutations. Different PC-BCP mutation patterns have different effects on the YI/VDD mutation.

Development of a rapid multi-residue assay for detecting ß-lactams using penicillin binding protein 2x*.

OBJECTIVE: To develop a rapid multi-residue assay for detecting 16 demanded by the European Union (EU). METHODS: A recombinant penicillin-binding protein (PBP) 2x* from Streptococcus pneumoniae R6 was expressed in vitro and six ß-lactams were conjugated to HRP by four methods. A rapid multi-residue assay for ß-lactams was established with PBP2x* and HRP-conjugate. RESULTS: PBP2x* was expressed and purified successfully and the ideal HRP-conjugate was identified. The multi-residue assay was developed. After optimization, penicillin G, ampicillin, amoxicillin, cloxacillin, dicloxacillin, oxacillin, nafcillin, cephalexin, ceftiofur, cefalonium, cefquinome, cefazolin, cefoperazone, cephacetrile, and cephapirin can be detected at levels below MRL in milk with simple pretreatment. CONCLUSION: This assay developed can detect all 16 ß-lactams demanded by the European Union (EU). The whole procedure takes only 45 min and can detect 42 samples and the standards with duplicate analysis.

Correlation of fluoroquinolone resistance with expression of qnrA gene in Kluyvera spp.

The objective of the present study was to examine whether the expression of qnrA may contribute to a high level of resistance among parent and induced strains of Kluyvera spp. Two clinical isolates of ciprofloxacin-resistant Kluyvera spp. were obtained from livers of diseased chickens, and upon induction with ciprofloxacin, six strains with increased resistance were produced. Point mutations in qnrA, aac(6')-Ib-cr, gyrA, gyrB, parC, and parE were investigated by polymerase chain reaction (PCR) amplification and DNA sequencing, and expression levels of acrAB and qnrA in all strains were investigated by quantitative real-time PCR (qRT-PCR). The induced strains contained the same mutations in quinolone resistance-determining region as those of the parent strains. qRT-PCR showed that the expression of the acrA gene was not detected in any strain and acrB gene expression was unchanged between induced and parental strains. However, difference in expression of qnrA was observed, which correlated well with the level of quinolone resistance in the parent and induced strains. The induced high resistance was not affected by mutations in qnrA and aac(6')-Ib-cr, by new mutations in the quinolone resistance-determining region of gyrA, gyrB, parC, and parE, or by the expression level of acrAB. These data suggest that the expression of qnrA may be a factor contributing to the high level of resistance among parent and induced strains of Kluyvera spp.

[Identification the relationship between mutation patterns of rtM204I/V in the polymerase gene and genotypes of hepatitis B virus].

OBJECTIVE: To investigate the relationship between the mutation patterns of rtM204V/I (methionine to valine or isoleucine at position rt204 of reverse transcriptase domain) in hepatitis B virus (HBV) polymerase gene and HBV genotypes. METHODS: A total of 2849 HBV complete genome sequences were retrieved from the GenBank/EMBL/DDBJ. HBV genotypes were determined by using MEGA4 software. The amino acid sequences of the reverse transcriptase (RT) domain were aligned. Data were analyzed using SPSS 13.0. RESULTS Among the 2849 HBV complete genome sequences, 217 strains with Y (I/V) DD were identified. Of them, 120 had YIDD mutation and the genotype/subgenotype distribution was as follows: A (2), B(B2 19), C(C1 1, C2 78, C5 1), D(17), E(1), G(1); 97 had YVDD mutation and the genotype/subgenotype distribution was as follows: A(17), B(B2 22), C(C1 3, C2 48), D(3), G(3), H(1). There is a significant difference in the mutation patterns of Y (I/V) DD among genotypes of A-D, A-C, and between genotype A and B, P < 0.01.There is a difference in the mutation pattern of Y (I/V) DD among genotypes of B-D, between genotype C and D, P < 0.05. Genotype A has a higher tendency to develop YVDD mutation, whereas genotype D has a higher frequency to develop YIDD mutation. The rtM204V-rtL180M mutations were more frequently found in subgenotype B2 than in subgenotype C2 while the rtM204V-rtL180M-rtV173L mutations were more associated with subgenotype C2 (P < 0.01). CONCLUSION: Different HBV genotype/subgenotype may select different mutation pattern in the YMDD domain. Subgenotype C2 is more diversity and complexity than other HBV genotypes/subgenotypes.

Phage M13KO7 detection with biosensor based on imaging ellipsometry and AFM microscopic confirmation.

A rapid detection and identification of pathogens is important for minimizing transfer and spread of disease. A label-free and multiplex biosensor based on imaging ellipsometry (BIE) had been developed for the detection of phage M13KO7. The surface of silicon wafer is modified with aldehyde, and proteins can be patterned homogeneously and simultaneously on the surface of silicon wafer in an array format by a microfluidic system. Avidin is immobilized on the surface for biotin-anti-M13 immobilization by means of interaction between avidin and biotin, which will serve as ligand against phage M13KO7. Phages M13KO7 are specifically captured by the ligand when phage M13KO7 solution passes over the surface, resulting in a significant increase of mass surface concentration of the anti-M13 binding phage M13KO7 layer, which could be detected by imaging ellipsometry with a sensitivity of 10(9)pfu/ml. Moreover, atomic force microscopy is also used to confirm the fact that phage M13KO7 has been directly captured by ligands on the surface. It indicates that BIE is competent for direct detection of phage M13KO7 and has potential in the field of virus detection.

Hepatitis B virus genotypes, subgenotypes, precore, and basal core promoter mutations in the two largest provinces of Pakistan.

BACKGROUND AND AIM: Hepatitis B virus (HBV) genotyping has been done in most countries, but unfortunately, in Pakistan, HBV genotypic distribution is still unclear. The aim of the present study was to determine the prevalent genotype and subgenotype in the two most populated provinces in Pakistan: Punjab and Sind. METHODS: In total, 236 HBV DNA-positive samples were selected for genotyping by polymerase chain reaction-restriction fragment length polymorphism (RFLP). The RFLP results were further confirmed with whole genome and partial genome sequencing. RESULTS: Genotype D was detected as the most prevalent (93.22%) genotype in all eight cities of both provinces; genotype C was present in 5.93% and genotype A was present in 0.85% of the samples. The D1 subtype was present in 84%, and D2 was present in 8% of 25 whole genome-sequenced samples. The C2 subtype was detected in 58.33% of S gene-sequenced samples, while D1 was detected in the remaining 41.67% of 24 samples sequenced for the S gene. Subtype D1 is the most dominant in D, while C2 is dominant in genotype C. Eight- and 15-bp deletion mutations were also detected in genotype D samples. Other precore and basal core promoter (BCP) mutations included T1915 (100%), A1679 (86.96%), T1762 (39.13%), and A1764 (30.43%), which were also detected in the genotype D samples. CONCLUSION: Genotype D subtype D1 is the most prevalent HBV strain in Pakistan with 8-bp deletion mutants the most common in HBV carriers.

Soluble angiopoietin receptor Tie-2 in patients with acute myocardial infarction and its detection by optical protein-chip.

The Tie-2 receptor has been shown to play a role in angiogenesis in atherosclerosis. The conventional method assaying the level of soluble Tie-2 (sTie-2) was ELISA. However, this method has some disadvantages. The aims of this research are to establish a more simple detection method, the optical protein-chip based on imaging ellipsomtry (OPC-IE) applying to Tie-2 assay. The sTie-2 biosensor surface on silicon wafer was prepared first, and then serum levels of sTie-2 in 38 patients with AMI were measured on admission (day 1), day 2, day 3 and day 7 after onset of chest pain and 41 healthy controls by ELISA and OPC-IE in parallel. Median level of sTie-2 increased significantly in the AMI patients when compared with the controls. Statistics showed there was a significant correlation in sTie-2 results between the two methods (r=0.923, P<0.01). The result of this study showed that the level of sTie-2 increased in AMI, and OPC-IE assay was a fast, reliable, and convenient technique to measure sTie-2 in serum.

[Distribution of hepatitis B virus genotypes and subgenotypes among chronically infected patients in Xinjiang Uighur.].

OBJECTIVE: To investigate the distribution of Hepatitis B virus genotypes and subgenotypes among patients with chronic hepatitis B in Xinjiang Uighur. METHODS: The HBV genotypes and subgenotypes were analyzed by PCR-restriction fragment length polymorphism in 109 patients with chronic hepatitis B. RESULTS: Two HBV genotypes, genotype C (45.9%) and genotype C/D (29.4%) were prevalent, genotype B (8.3%) and genotype D (16.5%) were also found in Xinjiang Uighur. Genotype C had two subgenotypes, C1 (54%) and C2 (46%). Genotype B had only one subgenotype, i.e. Ba. The subgenotype C2 was associated with cirrhosis and hepatocellular carcinoma. CONCLUSION: In Uygurs, the most common HBV genotypes were C and C/D, and the subgenotype C2 was associated with cirrhosis and hepatocellular carcinoma.

[Characteristics of HBcAg(18-27) CTL epitopes of the main epidemic HBV strains in China].

OBJECTIVE: To study the characteristics of the virology background of HLA-A2 restricted HBcAg(18-27) epitope mutations in HBV infected patients in China. METHOD: 30 HBV sequences with different genotypes from Genbank were analyzed by bioinformatics and the mismatched primers were designed for constructing a PCR-RFLP method to screen HBcAg(18-27)V/I in China. The distributions of HBcAg(18-27)V/I of 160 samples with HBV genotype B/C infection from 8 areas in China were screened and analyzed by PCR-RFLP and sequencing. The affinity of HBcAg(18-27)V/I to HLA-A0201 was analyzed through referencing the bioinformatics websites. RESULTS: We successfully constructed a PCR-RFLP method for screening HBcAg(18-27)V/I from genotype B/C, and only 3 samples with HBcAg(18-27)V sequence were found in the 160 samples (3/160, 1.88%). The affinity of HBcAg(18-27)I to HLA-A 0201 was lower than the one of HBcAg(18-27)V through bioinformatic analysis (HLA ligand score was 123 vs 156, and the SYFPEITHI score was 22 vs 24). CONCLUSION: The last amino acid of most HBcAg(18-27) sequences of epidemic HBV strains in China is isoleucine, and not valine. Therefore HBcAg(18-27) sequence background in different HBV genotypes should be thoroughly considered when using it as a reference or control in immunological research about HBV.

[Kinetics of HBV mutants conferring adefovir resistance (rtn236t) and a method to detect them rapidly].

OBJECTIVE: The aim was to build a PCR-RFLP method for detecting rtN236T mutants and to observe their kinetics in chronic hepatitis B (CHB) patients. METHODS: Seven CHB patients who had suboptimal viral response or viral breakthrough under adefovir mono-therapy were studied. Part of the HBV reverse transcriptional gene from serial sera samples was sequenced with PCR products or cloned HBV DNA; mutations at rt236 were simultaneously analyzed by a PCR-RFLP assay. Genetic diversity of HBV was observed by calculating Hamming distance within domains B, C and D of RT. RESULTS: Three patients had viral breakthrough and one with suboptimal viral response had adefovir-resistance mutants, one had rtA181V mutation and three had rtN236T mutation. A novel PCR-RFLP assay based on restriction enzyme HpaI or DraI for on the detection of rtN236T mutant was established, which detected 10% minor strains with 100% specificity. Mutants (rtA181V or rtN236T) appeared 0-8 months earlier than the viral breakthrough, then afterwards became the dominant ones. In one patient after stopping the adefovir therapy, 3 months later a wild type virus re-took again the mutant one (rtN236T); in one patient who developed a rt236T mutant after 132 weeks of adefovir treatment, a novel mutant (rtN236V) appeared and then became the dominant one while adefovir treatment continued. CONCLUSIONS: A rapid and easy method was established to detect rtN236T mutants. Mutants for adefovir-resistance accumulated rapidly then became dominant, but they could be taken over again by a wild type or novel mutant HBV.

[Analysis of clinical features and virological characteristics in patients infected with three different HBV subgenotypes].

OBJECTIVE: To investigate the clinical characteristics and the pattern of precore and core promoter mutations of hepatitis B virus (HBV) subgenotypes Ba, C1 and C2. METHODS: A cohort of 151 patients with chronic HBV infection in Guangdong province of China was enrolled in this study. HBV subgenotypes were determined by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP). Precore and core promoter mutations were analysed using nucleotide sequencing. RESULTS: Of the 151 patients, 80, 51 and 20 were infected with subgenotypes Ba, C1 and C2 respectively. No significant differences were found in HBeAg positivity and liver functional indexes among these three subgenotypes when age and sex were matched. Virologically, HBV/Ba showed the highest frequency of A1896 mutation but the lowest frequency of T1762/A1764 mutation. HBV/C1 was associated with the highest tendency to develop T1762/A1764 mutation, but the lowest prevalence of A1896 mutation. HBV/C2 was associated with an intermediate tendency to develop A1896 and T1762/A1764 mutations. CONCLUSION: Different mutation patterns in precore and core promoter regions are responsible for HBeAg-negative HBV infections among different subgenotypes.

[Changes of serum HBsAg in HBeAg positive chronic hepatitis patients with sustained viral response to long-term lamivudine treatment].

OBJECTIVE: HBsAg loss is rare in chronic hepatitis B patients, even in the patients with long-term nucleos(t)ide analogue therapy; therefore information about serum HBsAg kinetics will be of value in understanding this unusual occurrence. METHODS: Forty-five consecutive patients were studied, which were all HBeAg positive and never had antiviral therapy prior to lamivudine treatment; they then achieved rapid and good viral responses (defined as undetectable HBV DNA [Roche Lightcycler, less than 1000 copies/ml] at treatment week 24 and they remained so until week 156). Abbott Architect HBsAg assay was used to quantify serum HBsAg and HBV genotypes were determined by direct sequencing. RESULTS: Twenty-six (57.8%) patients had HBeAg loss during the observation and one patient had HBsAg loss following his HBeAg seroconversion. Serum HBsAg levels decreased to 39.5% (median) of their baseline values at week 12, but no further significant reductions of serum HBsAg were found afterwards. Changes of serum HBsAg were comparable between patients with or without HBeAg loss. Serum HBsAg levels at their baselines were higher in HBV genotype B (HBV/B, n = 21) patients than in genotype C (HBV/C, n = 24) patients. HBV/B patients achieved many more HBsAg reductions than HBV/C ones (75.5 vs. 26.0%, median, P less than 0.05) in the first 12 treatment weeks, however HBsAg levels at week 156 were comparable between these two subgroups. HBsAg changes mainly showed two distinct patterns: a biphasic pattern (HBsAg levels were less than 60% of baseline ones at week 12 and 24, n = 25) and a maintaining pattern (HBsAg levels were greater than 80% of the baseline ones at week 12 and 24, n = 14). Logistic regression analysis showed that low serum HBsAg at baseline (odds ratio 0.020, 95% confidence interval 0.002-0.743, P less than 0.05) and HBV/C infection (odds ratio 8.206, 95% confidence interval 1.070-62.948, P less than 0.05) were the determinants of the occurrences of the maintaining pattern. CONCLUSION: In patients we examined, their HBsAg changes were mainly presented as either a biphasic pattern or a maintaining pattern, which were associated with HBV genotypes (B/C) but not with HBeAg loss. This might explain that why HBsAg loss is a rare occurrence even with long-term lamivudine therapy.

[Development of a fluorescence polarization immunoassay for sulfamerazine].

A fluorescence polarization immunoassay (FPIA) based on a monoclonal antibody for the determination of sulfamera (SMR) was developed. The fluorescein-labeled SMR and sulfamethazine (SMZ) were synthesized and purified by thin layer chromatography (TLC). The optimized SMR FPIA had a dynamic range from 5.4 to 218.8 ng x mL(-1) with IC50 value of 23.4 ng x mL(-1) and a detection limit of 2.3 ng x mL(-1). The specificity of the FPIA for SMR was investigated using other 16 sulfonamides and the cross-reactivity for SMR, SMZ and sulfadiazine (SDZ) was 100%, 25% and 8.6%, respectively.

[Development of fluorescence polarization immunoassay for determination of pesticides and veterinary drugs].

Fluorescence polarization immunoassay (FPIA) is a fast screening technique based on immune competition and fluorescence polarization principle and is most used in the determination of small molecular substance (antigen). FPIA is based on the increase in polarization of the fluorescence of small fluorescent-labeled antigen when bound by specific antibody. FPIA is a homogeneous technique and not affected by solution color and the sensitivity of instrument. No separation step is required for FPIA. Simplifying the assay and minimizing the analysis time are the most notable advantages of FPIA over other immunoassays and FPIA is suitable to screening a large number of samples. The technique has been applied to the determination of pesticides and veterinary drugs in environment and food samples, while no studies have been reported in the correlative field in China. The present paper presents the principle and history of FPIA and its application in the screening determination of pesticides and veterinary drugs.

Immunosensor interface based on physical and chemical immunoglobulin G adsorption onto mixed self-assembled monolayers.

An immunosensor interface based on mixed hydrophobic self-assembled monolayers (SAMs) of methyl and carboxylic acid terminated thiols with covalently attached human Immunoglobulin G (hIgG), is investigated. The densely packed and organised SAMs were characterised by contact angle measurements and cyclic voltammetry. The effect of the non-ionic surfactant, Tween 20, in preventing nonspecific adsorption is addressed by ellipsometry during physical and covalent hIgG immobilization on pure and mixed SAMs, respectively. It is clearly demonstrated that nonspecific adsorption due to hydrophobic interactions of hIgG on methyl ended groups is totally inhibited, whereas electrostatic/hydrogen bonding interactions with the exposed carboxylic groups prevail in the presence of surfactant. Results of ellipsometry and Atomic Force Microscopy, reveal that the surface concentration of covalently immobilized hIgG is determined by the ratio of COOH / CH(3)-terminated thiols in SAM forming solution. Moreover, the ellipsometric data demonstrates that the ratio of bound anti-hIgG / hIgG depends on the density of hIgG on the surface and that the highest ratio is close to three. We also report the selectivity and high sensitivity achieved by chronoamperometry in the detection of adsorbed hIgG and the reaction with its antibody.

[Affects of HBV genotypes (B/C) on the levels of serum and intrahepatic HBsAg].

OBJECTIVE: To observe the effects of HBV genotypes on the level of HBsAg in serum and hepatocytes in chronic hepatitis B patients without antiviral therapy. METHODS: Seventy-six chronic hepatitis B inpatients were enrolled into this study, and liver biopsies and histologic diagnosis were performed, and serum samples were collected at the time point of liver biopsy. PCR-RFLP method was adopted to determine the genotype of hepatitis B virus and Abbott Architect HBsAg assay was used to quantify the serum HBsAg. Immunostaining for antigens in liver tissues with monoclonal antibody (for HBsAg) or polyclonal antibodies (for HBcAg) was carried out in consecutive slides. The percentages of hepatocytes for HBsAg stain, hepatocytes for HBcAg nuclear stain and hepatocytes for HBcAg cytoplasm stain were estimated in the ranges of 0 (negative), < or = 1%, 1+; 2% - 5%, 2+; 6% - 25%, 3+; 26% - 50%, 4+; and > 50%, 5+. The distributions of positive cells in slides are described as single or isolated, cluster or widespread. Surface gene was directly sequenced with the serum HBV DNA from 6 patients with genotype B and 8 with genotype C HBV infection, respectively. RESULTS: Four HBV genotypes were detected in 76 patients: 47 patients with B, 21 with C, 3 with D and 5 were infected by genotype B mixed with C HBV infection. Age, gender, serum HBV DNA level, ALT, AST or histological evaluation (grades and stages scores) were not different between the patients infected with genotype B or C HBV. The level of serum HBsAg was not significantly different between the patients infected with genotype B or C HBV, but the proportions of hepatocytes stained with HBsAg was greater in patients with C type HBV infection than B (P < 0.01). In the liver slides from the patients infected HBV genotype B, HBsAg was stained frequently in single or isolated hepatocytes (22/47), and widespread HBsAg-positive hepatocytes were often seen in the patients with C type HBV (8/21), P < 0.01. In the patients with B type HBV, serum HBsAg was positively correlated with serum HBV DNA (r = 0.674, P = 0.000), proportion of hepatocytes with HBcAg in nucleus (r = 0.534, P = 0.000) and in cytoplasm (r = 0.405, P = 0.004). In the patients with C type HBV infection, serum HBsAg had positive correlation only with serum HBV DNA (r = 0.503, P = 0.017). Proportion of HBsAg positive hepatocytes was positively correlated only with the proportion of HBcAg cytoplasm positive hepatocytes in the patients with B type HBV (r = 0.318, P = 0.029) and no correlation with serum HBsAg, HBV DNA, or proportions of hepatocytes with HBcAg in nucleus. Analysis of the first 40 amino acid sequences of surface antigen showed that variations most existed at amino acid 3, 4, 5 and 8. CONCLUSION: Proportion of HBsAg in hepatocytes is significantly greater in the patients with C type HBV than those with B type HBV. Positive correlation between serum HBsAg and viral replication was seems to be more significant in the patients with HBV genotype B infection.