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Objective To compare the effect of different sutures and suture method on corneal neovascularization ( CNV) in rabbit models. Methods NV was induced by placing sutures at the corneal periphery of rabbits (n = 45). To observe the NV status, 45 rabbits were randomly divided into 5 equal groups. Group A applied 8-0 absorbable suture (A1 single loop parallel suture, A2 single loop vertical suture). In group B, 10-0 nylon suture was used (B1 double loop parallel suture, B2 double loop vertical suture, B3 three loop radial suture). The development of CNV was observed with slit lamp microscope and photographed. Therefore the effective model for neovascularization induction was selected. Histological examination, immunofluorescent staining and ELISA analysis for the vascular endothelial growth factor( VEGF) were performed before suture, 7 and 14 days after suture. Results Sutures fell off and CNV gradually atrophied in group Al and A2; At the 14th day after suture, Sparse or short cluster CNV grew into the corneal margin in group B1 and B2, while CNV was vigorous and grew in bundles in group B3. The expression of VEGF in aqueous humor increased in B3 group after suturing, and increased in 14 days as compared with 7 days after suture. Corneal edema, neovascularization and little immunofluorescence staining for VEGF were detected in group B3 after 7 days suture. More neovascularization and immunofluorescence staining for VEGF were detected in group B3 after 14 days suture. Conclusion Corneal NV can be induced successfully in rabbit model by suturing. The method of 10-0 thread with three sets of circular seams (B3) is stable and effective.
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To produce specific antibodies against malachite green ( MG) , one special hapten was synthesized and characterized, and conjugated to carrier protein as immunogen. The immunogen showed excellent reactogenicity and immunogenicity. One specific monoclonal antibody (mAb, named MG-DA4-C7) with high sensitivity and specificity for MG in indirect competitive enzyme-linked immunoassay ( icELISA ) was screened. The isotype was IgG1 and the light chain was κ type. After optimization of ELISA conditions, the proposed icELISA showed a 50% inhibition value ( IC50 ) of 0. 96 μg/L, a linear range ( IC20-IC80 ) of 0. 1-8. 1 μg/L and a limit of detection ( LOD, IC10 ) of 0. 05 μg/L for determination of MG. The assay showed cross-reactivity of 18. 1%, 26. 5% with crystal violet and brilliant green, respectively, and negligible cross-reactivity with other metabolites of MG (<0 . 1%) . The average recoveries of MG from spiked fish samples were from 87. 3% to 107. 3%. Good correlation (R2=0. 999) was obtained between the results of icELISA and those of liquid chromatography-tandem mass spectrometry analysis. The proposed icELISA is suitable for the determination of MG in fish samples in a simple and sensitive manner.
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2-Amino-3-hydroxylpyridinE-H2O2-horseradish peroxidase (HRP) voltammetric enzymE-linked immunoassay based on N-heterocyclic substrate has been successfully applied for the detection of carcionembryonic antigen(CEA) in human serum. 2-Amino-3-hydroxylpyridine is oxidized with H2O2 catalyzed by HRP, and the resulting electroactive product produces a sensitive voltammetric peak at the potential of 0.36 V(vs. SCE) in Britton-Robinson (B-R) buffer solution. By this voltammetric peak, free HRP can be measured and immunoassay of HRP label can be developed. The enzymE-catalyzed reaction conditions and voltammetric detection conditions have been optimized, and the electrode procedure of the enzymatic product was investigated. The selected optimum reaction conditions were that the reaction medium was pH 6.0 B-R buffer solution for 10 mL reaction solution containing 1.0 mL of 0.2 mol/L B-R buffer solution, 3.0 mL of 8.0 mmol/L 2-amino-3-hydroxylpyridine solution and 1.5 mL of 0.5 mmol/L H2O2 solution, and the reaction time was 30 min at 37 ℃. The optimum detection conditions were that the supporting electrolyte was pH 7.0 B-R buffer solution for 10 mL of the overall detection solution containing 5 mL of reaction solution and 1.0 mL of 0.2 mol/L B-R buffer solution. The optimum instrumental conditions for the detection were chosen as follows: the initial potential, 0.00 V; the final potential, -0.80 V; the potential scanning rate, 400 mV/s; the mercury drop standing time, 7 s. Under the optimum conditions, the linear range for detection of free HRP was 4.0×10-4-1.0 μg/L with a detection limit of 0.12 μg/L. Based on the new immunoassay system, the linear range of the detection to CEA was 0.50-80 μg/L and the detection limit was 0.50 μg/L, which is 10 times lower than that of traditional spectrophotometric enzymE-linked immunosorbent assay method. The 2-Amino-3-hydroxylpyridinE-H2O2-HRP voltammetric enzymE-linked immunoassay new system exhibits excellent performance having wider linear range and lower detection limit. The new method is inexpensive and simple. It has a great potential in clinical diagnosis.
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PURPOSE: This study aimed to investigate the positive rate of 3 serologic methods and polymerase chain reaction (PCR) and the changes of IgG and IgG subclasses in children with Mycoplasma pneumoniae pneumonia (MP). METHODS: Fifty children with pneumonia admitted to Daejeon St. Mary's Hospital, Korea, during MP outbreaks were evaluated for the diagnostic antibody status using 3 serologic methods: indirect micro-particle agglutinin assay (MAA, Serodia-Myco II, Fujirebio, Tokyo, Japan), cold agglutinins and enzyme-linked immunoassay (EIA, Platelia M. pneumoniae IgM & IgG BIO-RAD, Marnes-la-Coquette, France) and PCR. The levels of antibody for MP in each method were measured 2 times during hospitalization: at presentation and at discharge (mean interval, 6.5 days). The levels of IgG and IgG subclasses (IgG1, IgG2, IgG3 and IgG4) were also analyzed 2 times (at presentation and at discharge) using stored sera. RESULTS: At presentation, the positive rates of the diagnostic methods were 52%, 38%, 30% and 12% for MAA, cold agglutinins, EIA and PCR assay, respectively. Following analysis of the repetitive measurement of the antibody, the positive rates of the diagnostic methods were 76%, 60% and 56% for MAA, cold agglutinins and EIA, respectively. The mean IgG level of MP patients increased during hospitalization (973+/-184 vs. 1,040+/-205 mg/dL; P=0.008). Among the IgG subclasses, the levels of IgG1 and IgG3 showed a significant increase during hospitalization (553+/-129 vs. 611+/-151 mg/dL, P=0.003 for IgG1; 43+/-27 vs. 47+/-30 mg/dL, P=0.005 for IgG3). CONCLUSION: For the accurate and relatively rapid diagnosis of MP, a paired sample examination is mandatory, especially within a short-time period. The sensitivity of serologic tests for the diagnosis of MP may differ among commercially available kits. IgG1 and IgG3 appear to be the main IgG subclasses that show an increase after MP infection.
Subject(s)
Child , Humans , Agglutinins , Cold Temperature , Cryoglobulins , Disease Outbreaks , Hospitalization , Immunoassay , Immunoglobulin G , Immunoglobulin M , Korea , Mycoplasma , Mycoplasma pneumoniae , Pneumonia , Pneumonia, Mycoplasma , Polymerase Chain Reaction , Serologic Tests , TokyoABSTRACT
The objective of the present study was to correlate ophthalmic and haematological findings, compared with the serological data obtained by indirect fluorescent antibody test (IFATT) and by dot-blot linked immunoassay (DBELIA) in 51 dogs with uveitis due to possible ehrlichiosis infection. Thirty-four positive IFAT and forty-four positive DBELIA results were obtained in serum samples.The high correlation between uveitis and a positive serology for Ehrlichia canis was established. The DBELIA test was moresensitive for the diagnosis of ehrlichiosis than IFAT.
O objetivo do presente trabalho foi correlacionar os achados oftálmicos e hematológicos, comparados aos dados sorológicos obtidos por meio da reação de imunofluorescência indireta (RIFI) e dot-blot ELISA (DBELIA) de 51 cães com uveíte, possivelmente devido a erliquiose. Trinta e quatro soros sangüíneos foram positivos pela RIFAT e quarenta e quatro pelo DBELIA. Foi estabelecida uma alta correlação entre uveíte e sorologia positiva para a Ehrlichia canis. O DBELIA foi mais sensível para o diagnóstico da erliquiose comparativamente a RIFI.
Subject(s)
Animals , Dogs , Ehrlichia canis , Enzyme-Linked Immunosorbent Assay , Fluorescent Antibody Technique/veterinary , Infections/veterinary , Serology , Uveitis/veterinaryABSTRACT
Objective To express TmpA recombinant antigen of Treponema pallidum in E.coli and to develop an Enzyme linked Immunoassay (EIA) based on the recombinant antigen for diagnosing syphilis. Methods The target TmpA gene was amplified by polymerase chain reaction (PCR). The PCR products of the gene were inserted into pBluescript T vector, and then expressed in E.coli, using pQE 30 system. Then the recombinant antigen was purified by an affinity chromatography and used for the development of EIA. Results The antigenicity of the recombinant antigen was identified by western blotting (WB) and EIA. The sensitivity and specificity of EIA were 100%(10/10) and 100% (20/20), respectively. The positive rates of anti TmpA antibodies were 91.67% (11/12) for the patients with Ⅰ phase of syphilis and 100% for the patients of Ⅱ and late stage of the disease. Conclusions The TmpA recombinant protein can be used to diagnose syphilis since 97.2% (35/36) of patients with syphilis were positive for the anti TmpA antibody by EIA.
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Objective:To improve the method for isolating platelet-rich plasma (PRP). Methods:Whole blood was collected from 8 healthy donors and then PRP was separated by both the tube method and the syringe method respectively. Samples were activated to get serum rich-in growth factors (SRGF).Platelets in the SRGF were counted and the level of TGF-?1 was assayed by enzyme-linked immunosorbent assay (ELISA).Results:The end product of syringe method has both a higher platelet count in PRP (P=0.003) and a higher level of TGF-?1 in SRGF(P=0.041) than that of tube method.Conclusion:The syringe method is an effective method in preparation of PRP.