Biomedical subjects

Weiling Fu

Publications and source records attributed to Weiling Fu.

11 recordsLinked to original sources

PubMed2006

Rapid and simultaneous quantification of 4 urinary proteins by piezoelectric quartz crystal microbalance immunosensor array.

BACKGROUND: Urinary proteins are predictive and prognostic markers for diabetes nephropathy. Conventional methods for the quantification of urinary proteins, however, are time-consuming, and most require radioactive labeling. We designed a label-free piezoelectric quartz crystal microbalance (QCM) immunosensor array to simultaneously quantify 4 urinary proteins. METHODS: We constructed a 2 x 5 model piezoelectric immunosensor array fabricated with disposable quartz crystals for quantification of microalbumin, alpha1-microglobulin, beta2-microglobulin, and IgG in urine. We made calibration curves after immobilization of antibodies at an optimal concentration and then evaluated the performance characteristics of the immunosensor with a series of tests. In addition, we measured 124 urine samples with both QCM immunosensor array and immunonephelometry to assess the correlation between the 2 methods. RESULTS: With the QCM immunosensor array, we were able to quantify 4 urinary proteins within 15 min. This method had an analytical interval of 0.01-60 mg/L. The intraassay and interassay imprecisions (CVs) were <10%, and the relative recovery rates were 90.3%-109.1%. Nonspecificity of the immunosensor was insignificant (frequency shifts <20 Hz). ROC analyses indicated sensitivities were > or =95.8% and, specificities were > or =76.3%. Bland-Altman difference plots showed the immunosensor array to be highly comparable to immunonephelometry. CONCLUSIONS: The QCM system we designed has the advantages of being rapid, label free, and highly sensitive and thus can be a useful supplement to commercial assay methods in clinical chemistry.

Alpha-Globulins↗

PubMed2006

Expression and purification of a soluble B lymphocyte stimulator mutant modified with the T-helper cell epitope.

The DNA encoding soluble B lymphocyte stimulator (134-285 amino acids, sBLyS) mutant with residues 217-224 replaced by two glycines (named msBLyS) was constructed. The sequence encoding a foreign immunodominant T-helper epitope from ovalbumin (OVA) was then coupled to the 5'-end of msBLyS cDNA. After being sequenced, the recombinant DNA was ligated into the prokaryotic expression vector pQE-80L. The recombinant protein was produced in E. coli DH5alpha after induction with IPTG with the yield of more than 40% of total bacterial protein. The recombinant protein was purified with Ni-NTA chromatography and Sepharcryl S200 chromatography to a purity of more than 98%. The BALB/c mice, immunized with the recombinant protein, produced anti-BLyS antibodies at a high level, which indicated that the recombinant BLyS mutant modified with T-helper epitope elicited polyclonal antibodies with cross-reactivity with BLyS in vivo. This recombinant protein may therefore be used as immune inhibitor of BLyS for treating BLyS -associated autoimmune diseases.

Animals↗

PubMed2006

[Experimental studies of carcino-embryonic antigen immunosensor based on piezoelectric resonate technology of quartz crystal].

In order to construct a new type of piezoelectric quartz immunosensor for the determination of carcino-embryonic antigen (CEA), the sensor detection pools were consisted of plastic loops and crystals that were 10 MHz quartz AT-cut with gold coated electrodes and the immobilization of the monoclonal antibody against CEA onto gold electrode surface of the quartz crystal was accomplished via Thiol method. Then the immunosensors were used in clinical laboratory. The experimental results showed that the piezoelectric immunosensor had good response to CEA, the frequency shifts were linearly dependent on CEA concentration in the range of 1.56 to approximately 50.00 ng/ml, other antigens such as alpha fetoprotein (AFP), prostate specific antigen (PSA), human chorionic gonadotropin (hCG) did not interfere with the sensor's response. The results obtained from this method were in satisfactory accordance with those obtained by radio immunoassay(P>0.05), The correlation coefficient was 0.90. Piezoelectric immunosensor for determination of CEA has the advantage of high sensitivity, high specificity, unnecessary labeling, simple performing, rapid analysis, low cost, real-time detection and repeated use, etc. It can be used for detecting serum CEA in clinical laboratory.

Antibodies, Monoclonal↗

PubMed2004

A novel piezoelectric quartz micro-array immunosensor based on self-assembled monolayer for determination of human chorionic gonadotropin.

A novel multi-channel 2 x 5 model of piezoelectric quartz micro-array immunosensor has been developed for quantitative detection of human chorionic gonadotropin (hCG) in serum or urine samples. Every crystal unit of the fabricated piezoelectric hCG micro-array immunosensor can oscillate independently without interfering each other. A 2 x 5 model of micro-array immunosensor as compared with a one-channel immunosensor can provide eight times higher detection speeds for hCG assay. The anti-hCG antibody is deposited on the gold electrode's surface of 10 MHz quartz AT-cut crystal by self-assembled technique using sulfosuccinimidyl 6-[3'-(2-pyridyldithio) propionamido] hexanoate (Sulfo-LC-SPDP), and serves as an antibody recognizing layer. The highly ordered self-assembled monolayers (SAM) ensure well-controlled surface structure and offer many advantages to the performance of the sensor. Compared with conventional antibody immobilization methods, the amount and the reaction activity of antibody monolayer coated by the SAM binding are bigger than those by the SPA method, and less non-specific binding caused by other analytes in sample is found. Under the optimized experimental conditions, the results showed that micro-array immunosensor quantitatively detected serum or urine hCG in the range of 2.5-500 mIU/ml with high precision (CV<5%); other hormones in human serum and urine did not interfere with the determination markedly. Serum and urine samples of 60 patients were detected by the micro-array immunosensor, and the results agreed well with those given by the commercial radioimmunoassay test kit, with correlation coefficient of 0.92. After regeneration with urea solution the coated immunosensor can be reused five times without appreciable loss of activity.

Biosensing Techniques↗

PubMed2004

Two cases of X-linked juvenile retinoschisis with different optical coherence tomography findings and RS1 gene mutations.

The optical coherence tomography (OCT) findings, clinical features, and mutations in the RS1 gene of two unrelated patients with X-linked retinoschisis (XLRS) are reported herein. Two Chinese patients with early onset XLRS were given a comprehensive ophthalmologic examination and OCT investigation. The RS1 gene was screened for sequence alterations in all exons and splice regions. The two patients presented with different phenotypic features and OCT findings. One patient with more severe clinical presentation had a RS1 exon 1 deletion and a P193S mutation was found in the other patient with mild macular involvement. OCT demonstrates the markedly different features of XLRS patients with different RS1 mutations. This study strengthens the role of OCT in the diagnosis and monitoring of XLRS.

Adolescent↗

PubMed2004

[Advances in piezoelectric quartz crystal biosensor and its applications].

Piezoelectric quartz crystal biosensor is a new sensor developed with the comprehensive utilization of the high sensitivity to mass and the surface characteristics of quartz crystal such as density, viscosity, dielectric constant, conductance, and the high specificity of biologic identification molecules. It has the features of high sensitivity, high specificity, simple operation, high analysis speed, low cost, small size, on-line detection, etc. It can be of wide application in the areas of medical laboratory diagnosis, environment monitor, foods sanitary control and industrial production. In this paper, the fundamental principle, structure and its applications are reviewed.

Biosensing Techniques↗

PubMed2002

Loss of heterozygosity and mutations are the major mechanisms of RB1 gene inactivation in Chinese with sporadic retinoblastoma.

We investigated sequence alternation, promoter methylation, and loss of heterozygosity (LOH) of the RB1 gene as possible mechanisms of its inactivation in retinoblastoma. In 42 Chinese patients with sporadic retinoblastoma, the promoter and entire coding region of RB1 were examined for sequence changes. Status of methylation of the CpG-rich island at the 5'end was determined by methylation specific PCR assay. We detected 15 RB1 mutations in 38% (16/42) of the retinoblastoma patients, among them 19% (8/42) were germ-line mutations. A total of nine novel mutations were identified: E54X, S114X, I126S, g73779insG, D718N, IVS2+1G>C, IVS14+1G>C, IVS21+1G>C, and a complex alteration g78177G>T/g78176insTT leading to 543X. Most of them are likely to affect the RB1large pocket domain through the production of truncated gene products. None of the DNA samples showed methylation at the RB1promoter. In 15 cases where both normal and cancerous retinoblastoma tissue specimens were available, allelic loss according to microsatellite markers within or distal to the RB1 locus was analyzed and immunohistological staining for RB1 expression performed. Among them, frequency of LOH at 13q14 was found to be high at 60% (9/15) with no segregation with unilateral tumors. All these nine tumors did not express RB1 protein, showing an association of LOH at the RB1 locus with its loss of expression in retinoblastoma. Our results indicate that the RB1 gene in sporadic retinoblastoma is commonly inactivated because of loss-of-function mutations and loss of heterozygosity but not by the epigenetic phenomenon of promoter hypermethylation.

Child, Preschool↗

PubMed2002

Multichannel piezoelectric genesensor for the detection of human papilloma virus.

OBJECTIVE: To establish a method for rapid detection and sub-typing of human papilloma virus (HPV). METHODS: We utilized the piezoelectric genosensor (PG) technique, which is a combination of the piezoelectric biosensor and gene chips for HPV identification in 22 recurrent biopsy specimens and 22 corresponding original biopsy specimens. The control samples came from normal tissue of healthy persons. A combined reaction took place on the sensor surface between the target genes and probes. The frequency of the piezoelectric sensor will decrease when such reactions occur, and the frequency decrease depends on the concentration of the target gene. Specimens were also analyzed with conventional PCR and dot blot. RESULTS: Of the 22 recurrent specimens, 15 contained HPV6 DNA, 2 HPV11 DNA, and 4 HPV16 DNA. Only one specimen was negative. All the 22 original specimens were positive: 17 harbored HPV6 DNA, 3 sequence homologous HPV11 DNA, and 2 HPV16 DNA. No HPV18 DNA was detected in any specimen. When compared with PCR and dot blot analysis, the results were essentially the same except for one specimen, which was shown to contain other sub-types of HPV. CONCLUSION: Our results show that the piezoelectric genosensor technique is a rapid and specific method to analyze HPV.

Biosensing Techniques↗

PubMed2002

[Mutation analysis of retinitis pigmentosa 1 gene in Chinese with retinitis pigmentosa].

OBJECTIVE: To investigate the frequency and pattern of RP1 point mutations in Chinese retinitis pigmentosa (RP) patients and to examine their effects on the development of RP. METHODS: Conformation sensitive gel electrophoresis (CSGE) and direct DNA sequencing were used to determine sequence alterations occurring in the entire coding region of the RP1 gene in 101 Chinese RP patients in Hong Kong. RESULTS: R677X was detected in one RP patient. A nonpathogenic nonsense mutation, R1933X, was identified in three normal individuals and one patient with Stargardt disease. The frequency of RP1 mutations among all RP patients in this study is 1/101. R677X is expected to lead to large disruptions of the encoded protein. Additionally, 10 more missense alterations in the RP1 gene were identified in the subjects of this study. Apart from M479I whose pathogenicity can not be determined currently, other sequence changes are just polymorphisms of the RP1 gene. CONCLUSION: The nonpathogenicity of R1933X indicates that the C-terminal 224 residues of RP1 protein may be not critical for RP1. Recently, a C-termnal truncating mutation, Y1053(1 bp del), was reported to occur in an RP patient. Thus RP can be caused by lack of the region of RP1 protein after codon 1052 but before 1933. To confirm such a proposition, a large genotyping study is necessary and is likely to reveal more RP causative mutations and uncover more sequence alterations different from those of other ethnic groups.

Adolescent↗

PubMed2002

[Study on the mechanism of hepatocytic insulin signal transduction defects in severely scalded rats].

OBJECTIVE: To investigate the mechanism of hepatocytic insulin signal transduction defects in severely scalded rats, so as to clarify the molecular basis of postburn insulin resistance. METHODS: Wistar rats inflicted by 30% III degree scalding on the back were employed as the model. The rat hepatocytic insulin receptor was partially purified by wheat-germ agglutinin (WGA)-sepharose 4B affinity chromatography. The change of receptor tyrosine protein kinase (TPK) activity, the receptor beta-subunit autophosphorylation and the hepatocytic insulin receptor binding behavior of scalded rats during early stage of scalding were observed by means of insulin receptor binding test, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) autoradiography of phosphorylation of insulin receptor and phosphorylation of exogenous substrate. RESULTS: There exhibited no evident changes of hepatocytic insulin receptor maximal binding capacity and affinity at 3 postburn days (PBDs) in scalded rats. The autophosphorylation capacity of the receptor beta-subunit decreased significantly. And the receptor TPK activity decreased obviously and its reaction to insulin stimulation decreased markedly. CONCLUSION: The defects of the insulin receptor signal transduction in hepatocyte leading to the post-receptor defects of insulin biological effects might be molecular mechanism of postburn insulin resistance.

Animals↗

PubMed2002

[Screening for point mutations in rhodopsin gene among one hundred Chinese patients with retinitis pigmentosa].

OBJECTIVE: To test the frequency and pattern of rhodopsin (RHO) mutations in Chinese retinitis pigmentosa (RP) patients and to evaluate their effects in the pathogenesis of RP. METHODS: Genomic DNA was extracted from peripheral blood samples of 100 Hong Kong Chinese RP patients. Sequence variants of the entire coding exons of the RHO gene were tested using PCR, conformation sensitive gel electrophoresis and DNA sequencing. RESULTS: Totally six nucleotide changes were identified, among which three were silent mutations, two missense mutations and one deletion mutation. P347L was found in one RP proband and her three children who also had RP. P327(1 bp del) was novel and detected in a late-onset RP patient of 53 years. Her 26-year-old daughter, also carrying the identified mutation, had no RP phenotypes except for the mottled retinal pigment epithelium (RPE) revealed by fundal examination. Neither of the two mutations was detected in normal controls. CONCLUSION: Two patients had disease-causing mutations in the RHO gene, thus RHO mutations cause about 2.0% (95% confidence interval: 0.2%-7.0%) of all RP among Chinese in Hong Kong. A highly conserved C-terminal sequence QVS(A)PA was altered due to P347L and thereby resulting in an aberrant subcellular localization of rhodopsin. Loss of all six phosphorylatable residues at the C-terminus and the highly conserved C-terminal sequence QVS(A)PA may occur because of P327(1 bp del). To elucidate the predominant biochemical defects in such mutant, transgenic mice and transfected culture cells carrying P327(1 bp del) would be of greatest value.

Adolescent↗