Time-resolved fluoroimmunoassay of zearalenone in cereals with a europium chelate as label

A competitive indirect time-resolved fluoroimmunoassay(TRFIA) was developed for detection of zearalenone(ZEN) in cereals,in which ZEN conjugated to bovine serum albumin(BSA) is used as solid-phase antigen.A competitive indirect TRFIA was conducted by simultaneously incubating ZEN in standard or extracted samples with anti-ZEN monoclonal antibody over ZEN-BSA coated plates,and then determining the bound ZEN monoclonal antibody with goat anti-mouse europium conjugate.Samples were extracted with methanol/water...

Eu-Chelate Construct Ultrasensitive Time-Resolved Fluoroimmunoassay-Assay of Pepsinogen I

Eu-chelate were used to construct a two-site sandwich-type assay for pepsinogen Ⅰ （PGI） with time-resolved fluoroimmunoassay （TRFIA） as a detection technique. On the noncompetitive assay, captured monoclonal antibodies （McAbs） coated on wells were directed against a specific antigenic site on the PGI. Another McAbs, called as labeling McAbs, were prepared with the Eu-chelate of N-（p-isothiocyanatobenzyl）-diethylenetriamine-N, N, N, N-tetraacetic acid and directed against a different antigenic site on the PGI. The fluorescence counts of bound Eu^3＋ -McAbs were measured with the auto DELFIA1235 system. The PGI in sera from healthy volunteers were determined by PGI-TRFIA. The within-run and between-run CVs of the PGI-TRFIA were 1.9% and 4.7%, respectively, and the recovery rate was 102.65%. The assay had a detection limit of 0.05 μg· L^-1. The PGI-TRFIA provided a linear response from 3.5 to 328 μg· L^-1. The cross-reacting rate with pepsinogen Ⅱ was negligible. The linear correlation of PGI-TRFIA and radioimmunassay measurements resulted in a correlation coefficient of 0.977. The means of healthy volunteers were 154 ±43 μg·L^-1 for serum PGI. The availability of a highly sensitive, reliable, and convenient method for quantifying PGI will allow investigations into the possible diagnostic value of this analyte in various clinical conditions, including gastric carcinoma, duodenal ulcer, gastritis and severe atrophic gastritis.

Determination of Hydrocortisone in Cosmetics by CdSe/CdS Quantum Dots-Based Fluoroimmunoassay Using Magnetic Fe304/Au Nanoparticles as Solid Carriers

This work demonstrated the feasibility of detecting hydrocortisone in cosmetics using a novel CdSe/CdS quan- tum dots-based competitive fluoroimmunoassay with magnetic core/shell Fe3Oa/Au nanoparticles （MCFN） as solid carriers. Hydrocortisone antigen was labeled with the synthesized core/shell CdSe/CdS quantum dots （QDs） to form the antigen-QDs conjugate. Meanwhile, hydrocortisone antibody was incubated with MCFN and the immobilized antibody was obtained. The immobilized antibody was then mixed sequentially with hydrocortisone and a slightly excess amount of the QDs-labeled hydrocortisone antigen, allowing their competition for binding with the antibody immobilized on MCFN. The bound hydrocortisone and the antigen-QDs conjugates on MCFN were removed subsequently after the mixture was applied to a magnetic force. The analyte concentration was obtained by measuring the fluorescence intensity of the unbound hydrocortisone antigen-QDs conjugates. The proposed method was characterized by simplicity, rapidity, and high sensitivity with a wide linear working range of 0.5 to 15000 pg·mL^-1 and a low detection limit of 0.5 pg.mL^- 1. The proposed method was successfully applied to the determination of hydrocortisone in cosmetics with satisfactory results.

Simultaneous detection of different serum pepsinogens and its primary application

AIM: To develop the simple, rapid and sensitive dual-label time-resolved fluoroimmunoassay for pepsinogens in human serum.METHODS: Based on two-site sandwich protocol, mono-clonal antibodies (McAbs) against pepsinogen Ⅰ (PG Ⅰ) and PG Ⅱ were co-coated in 96 microtitration wells, and tracer McAbs against PG Ⅰ and PG Ⅱ were labeled with europium (Eu) and samarium (Sm) chelate, respectively. Diluted serum samples of Eu3+- and Sm3+-McAbs were added into microtitration wells simultaneously. After washing, fluorescence of bound Sm3+ and Eu3+ tracers was detected. RESULTS: The detection limit was 0.2 μg/L for PG Ⅰ and 0.05 μg/L for PG Ⅱ. The assay range was 5.0-320.0 μg/Lfor PG Ⅰ and 1.0-55.0 μg/L for PG Ⅱ. The average re-covery rate was 102.7% for PG Ⅰ and 98.8% for PG Ⅱ. Sera from healthy controls and patients with gastric dis-ease were analyzed. The PG detected by dual-label as-say was in good agreement with that detected by single-label assay or by enzyme-linked immunosorbent assay. CONCLUSION: Dual-label assay can provide high-throughput serological screening for gastric diseases.

Progress on Study of Luminescence of Rare Earth Organic Chelates

Based on the investigation of the luminescence of a series of rare earth organic chelates, some relationships between luminescence and the structure of the chelates were proposed: the intensity of sensitized luminescence of central lanthanide ions(Ln 3+ ) in a rare earth organic chelate depends on (1)the suitability of the energy gap between the excited triplet energy level of the ligands and the lowest excited energy level of Ln 3+ ions; (2)the rigidity and planarity of the structure of the chelate molecule; (3)the existence of a suitable secondary ligand which may increase rigidity and the stability of the chelate molecule; and (4) the existence of a suitable π conjugated system in the chelate molecule. According to the above relationships, 25 novel organic ligands were designed and synthesized, and their lanthanide chelates were prepared. Investigation of the photoluminescence for the new chelates shows that some of the chelates are strongly luminescent, and are applied to fluoroimmunoassay for determination of human immunoglobulin(IgG), to preparation of fluorescent plastics, and to determination of growth hormone for plants. Two novel spectroscopy probe techniques for structure of coordination compounds and biological molecules were proposed and developed based on vibronic spectroscopy of Tb 3+ complexes and fluorescence of Ce 3+ .

Caspase-3 mRNA expression in rats with apoptosis of retinal photoreceptor cells

BACKGROUND： Apoptosis of retinal photoreceptor cells is a commonly pathological characteristic of various eye diseases, while caspase-3 is an important regulating gene and plays a key role in apoptosis. OBJECTIVE： To measure the expression of caspase-3 mRNA in rats with apoptosis of retinal photoreceptor cells by using real-time fluorescent quantitative polymerase chain reaction and compare with those of the normal rats. DESIGN： Randomized controlled animal study. SETTING： Zhongshan Ophthalmological Center of Sun Yat-sen University. MATERIALS： A total of 36 female SD rats of 50 days old and clean grade and weighing （150±10） g were provided by Experimental Animal Center of Northern Area of Sun Yat-sen University. All rats were randomly divided into normal control group （n =6） and N-methyl-N-nitrosourea （MNU） group （n =30）, and they were observed at 12 hours, 1, 2, 3 and 5 days after model establishment, with 6 rats at each time point. METHODS： The experiment was carried out at Zhongshan Ophthalmological Center, Key Laboratory of Ophthalmology by State Ministry of Education from March to December 2004. Rats in the normal control group were intraperitoneally injected with saline and rats in the MNU group were intraperitoneally injected with 40 mg/kg MNU. And then, retinal photoreceptor injured models were established. At 12 hours, 1, 2, 3 and 5 days after model establishment, the rats were sacrificed for enucleating right eyeballs, isolating retina immediately and extracting total RNA. The expression of caspase-3 mRNA in retina was measured with real-time fluorescent quantitative polymerase chain reaction. MAIN OUTCOME MEASURES： Expression of caspase-3 mRNA in retina of rats in the two groups. RESULTS： A total of 36 SD rats were involved in the final analysis. The expressions of caspase-3 mRNA in the rat retina of both groups at the five time points （12 hours, 1, 2, 3 and 5 days） after model establishment were 1.52×105, 18.35×105, 25.14×105, 29.25×105, 13.72×105 and 12.24×105, respectively. The expression of caspase-3 mRNA in the MNU group increased after 12 hours of intraperitoneal injection, and rose to the top on the 2nd day, which was 19 times as many as that of the normal control group. Then, it decreased gradually and was still 8 times as many as that of the normal control group on the 5th day. CONCLUSION： The expression of caspase-3 mRNA is related to apoptosis of retinal photoreceptor cells, while caspase-3 plays an important role in occurrence and development of apoptosis of retinal photoreceptor cells.

Impact of donor-specific antibodies on long-term graft survival with pediatric liver transplantation

BACKGROUND In a previous paper,we reported a high prevalence of donor-specific antibody(DSA)in pediatric patients with chronic rejection and expressed the need for confirmation of these findings in a larger cohort.AIM To clarify the importance of DSAs on long-term graft survival in a larger cohort of pediatric patients.METHODS We performed a retrospective analysis of 123 pediatric liver transplantation(LT)recipients who participated in yearly follow-ups including Luminex testing for DSA at our center.The cohort was split into two groups according to the DSA status(DSA-positive n=54,DSA-negative n=69).Groups were compared with regard to liver function,biopsy findings,graft survival,need for re-LT and immunosuppressive medication.RESULTS DSA-positive pediatric patients showed a higher prevalence of chronic rejection(P=0.01),fibrosis(P<0.001)and re-transplantation(P=0.018)than DSA-negative patients.Class II DSAs particularly influenced graft survival.Alleles DQ2,DQ7,DQ8 and DQ9 might serve as indicators for the risk of chronic rejection and/or allograft fibrosis.Mean fluorescence intensity levels and DSA number did not impact graft survival.Previous episodes of chronic rejection might lead to DSA development.CONCLUSION DSA prevalence significantly affected long-term liver allograft performance and liver allograft survival in our cohort of pediatric LT.Screening for class II DSAs in combination with assessment of protocol liver biopsies for chronic antibodymediated rejection improved early identification of patients at risk of graft loss.

Establishment and application of immunofluorescence using monoclonal antibody in diagnosis of gastric cancer

Monoclonal antibodies against gastric cancer(MG)were purified by ion-exchangechromatography on DEAE-cellulose(DE-52)column and coupled with CNBr-activated Sepharose4B.The Sepharose 4B linked with MG monoclonal antibodies was then incubated with serum andascitic fluid of patients with gastric cancer and benign diseases for 2h.After being blocked by nor-mal mouse serum,the MG-Sepharose was mack to react with the MG-labeled with fluoresceinisothiocyanate for 30min.According to immunoaffinity chromatography principle,the MG-labeledwith fluorescein isothiocyanate was separated from the MG-Sepharose with 0.01 mol/L of PBS-3mol/L of KCNS solution and then detected with a fluorospectrometer.The amount of fluorescencein the solution of separation represented the amount of the antigens defined by MG monoclonalantibodies.The mean value plus 2 standard deviations of the benign disease group was arbitrarilyset as their positive threshold.The positivity rates were 61.1%(22/36)in serum and 75%(18/24)inascitic fluid of patients with gastric cancer.It suggests that the determination of MG-Ags in the se-rum and ascitic fluid of patients may be helpful for the diagnosis of gastric cancer.

High-Sensitivity Detection of Fruit Tree Viruses UsingBacterial Magnetic Particles

Prunus necrotic ring spot virus （PNRSV） and grapevine fanleaf virus （GFLV） were detected by fluoroimmunoassay using bacterial magnetic particles （BMPs）, and a double antibody sandwich enzyme linked immunosorbent assay （DAS-ELISA）. For the fluoroimmunoassay, fluorescein isothiocyanate labeled anti-PNRSV antibody or anti-GFLV antibody was conjugated onto BMPs of Magnetospirillum gryphiswaldense MSR-1. With this method, a very low minimum antigen concentration （1×10^6 dilution of the original sample concentration） could be detected. Using DAS-ELISA, the minimum antigen detection concentration was the original sample concentration. Thus, comparing these two methods, a BMP-based method could increase the sensitivity up to six orders of magnitude （10^6） higher than an ELISA-based method of detection PNRSV and GFLV.

Use of fluorescent europium chelates as labels for detection of microcystin-LR in Taihu Lake, China

A method using the time-resolved fluorescence technology to establish a highly sensitive microcystin-LR （MC-LR） indirect com- petitive immunoassay was proposed in this work. This method was used to monitor the MC-LR level in source water and treated drinking water from Taihu Lake. Algae in the water samples were removed by eentrifugation, and the MC-LR level was quantified using this method. Testing results showed that the sensitivity of this method was 0.01 μg/L, and the dynamic measuring range was from 0.05 to 2 μg/L. The av- erage recovery was 115%, and the variation （CV） within and between different batches were 7.3% and 9.7%, respectively. Testing results also indicated that this time-resolved fluoroimmunoassay was sensitive and accurate in measuring MC-LR level, especially for quantitative analy- sis MC-LR level in bulk water.

Determination of angiotensin Ⅱ by using europium-labelled streptavidin

There has recently been a great deal of interest in time-resolved fluoroimmunoassay（TRFIA）. Not only the content of proteins, but also the content of haptens canbe detected by TRFIA, e.g. progesterone, digoxin, estrone-3-glucuronide, 17α-hydroxyprogesterone, prostaglandin F2α, etc. We describe here a solid-phase, competitiveTRFIA for angiotensin Ⅱ（AⅡ）. AⅡ is an eight-peptide. It plays an important role