Comparative study on two serological detection methods of mycoplasma pneumoniae in children with mycoplasmal pneumonia / 中国医师进修杂志

Objective To compare the sensitivity and specificity of dot immunogold method (DIM) and particle agglutination (PA) for the diagnosis of mycoplasma pneumoniae (MP) infection. Methods The 190 serum specimens of 113 children with mycoplasmal pneumonia (infection group) and 50 serum specimens of 50 health children (health group) were tested for MP by PA and DIM- A and B. Results In infection group, the positive rates of DIM- A and B were 82.63% (157/190) and 84.74%(161/190), and there was no statistical difference (χ2 = 0.31, P>0.05); the positive rate of PA (titer ≥1:160) was 70.00%(133/190), the positive rate of PA was significantly lower than that in DIM-A and B, and there were statistical differences (P0.05); the positive of PA was 8.00% (4/50), the positive rate of PA was significantly lower than that in DIM- A and B, and there were statistical differences (P<0.05 or<0.01). Conclusions Compared with the PA, DIM has low sensitivity and poor specificity for clinical diagnosis. DIM is not suitable for clinical diagnosis of MP infection.

Comparative research of multiple antigens dot immunogold filtration assay and imaging diagnosis for two kinds of echinococcosises / 重庆医学

Objective To compare and study the value of multiple antigens dot immunogold filtration assay (DIGFA ) and ima-ging diagnosis for rapid diagnosis of two kinds of echinococcosises .Methods 167 cases of hydatid patients diagnosied by pathologi-cal examination were divided into the DIGFA group for diagnosis of DIGFA and the control group for imaging diagnosis .Results The diagnosis rate of cystic echinococcosis (CE) in the DIGFA group was 74 .60% and control group was 90 .48% (P<0 .01);the diagnosis of alveolar echinococcosis(AE) in the DIGFA group was 92 .68% and the control group was 73 .17% (P<0 .05);when the cystica<5 cm ,the diagnosis rate of AE and CE in the DIGFA group was 91 .67% and 61 .11% (P<0 .05) ,when the cystica 5- <10 cm ,the detection rate of AE and CE in the DIGFA group was 94 .12% and 71 .43% (P<0 .05) .When the cystica≥10 cm ,<5 cm or between 5 - < 10 cm ,the detection rate of CE in DIGFA group was 94 .12% ,61 .11% ,71 .43 ,respectively (P<0 .05);The totle detection rates of the AE and CE in DIGFA group were 92 .68% and 74 .60% (P<0 .05) .Conclusion Imaging di-agnosis for the CE was higher and the DIGFA diagnosis for the AE was higher and the DIGFA also had clinical significance espe-cially applicated to the early diagnosis of AE .With the help of the imaging diagnosis ,the DIGFA could diagnose two kinds of echi-nococcosises correctly and it provided the benefits of specificity and sensitivity and performed easily .

A comparison of two methods for anti-human immunodeficiency virus antibody of double-antigen sandwich assay / 中国基层医药

Objective To investigate the sensitivities and specificities of enzyme-linked inununosorbent assay(ELISA)and immunogold labeling technique in the detection of anti-human immunodeficiency virus antibody.Methods(1)Sensitivity test:15 sera from patients with acquired immunodeficiency syndrome(AIDS)diagnosed by western blotting were diluted proportionately,we detected the anti-human immunodeficiency virus antibody in all diluted sera using ELISA and immunogold labeling technique,and recorded the maximum dilutions of the two methods that the anti-human immunodeficiency virus antibody was positive.(2)Specificity test:200 sera from patients with nonAIDS were detected by ELISA and immunogold labeling technique,and we recorded the negative rates of the two methods.Results(1)Sensitivity test:The maximum dilution of ELISA was significantly higher than that of immunogold labeling technique(P ＜ 0.05).(2)Specificity test:The negative rates of the two methods were 100％,and there was no significant difference between the two methods.Conclusion The sensitivity of ELISA in the detection of antihuman immunodeficiency virus antibody is better than that of immunogold labeling technique,but the sensitivities and specificities of the two methods are consistent when the serum is detected,so the immunogold labeling technique is able to replace ELISA as a screening test for AIDS.

Development of dot immunogold filtration assay kit for rapid detection of antibody to schistosome in human sera / 中国血吸虫病防治杂志

Objective To develop a rapid kit applied to the field for detection of antibody to schistosome in human sera. Methods A new kit for rapid detection of antibody to schistosome was developed through improving the dot immunogold filtration assay (DIGFA). A total of 100 cases of sera from chronic schistosomiasis patients and 140 from healthy people, HBV patients and the people infected with other parasites were detected by the kit. The sensitivity, specificity, Youden's index and Kappa value were utilized as the evaluation standard. Results The sensitivity of detecting antibody to schistosome, specificity, Youden's index and Kappa value were 92% , 95.08% , 0.87 and 0.87, respectively. The cross reaction to patients with clonorchiasis was 5%. Conclusion DICFA kit is practical for antibody to schistosome detection in the field because of its advantages such as smaller serum needed and faster in reaction.

Using recombined SSB antigen expressed in Pichia pastoris to detect anti-SSB antibody by dot immunogold filtration assay / 临床检验杂志

Objective To establish a new,rapid,simple and reliable assay for detecting autoantibody SSB.Methods A new dot immunogold filtration assay(DIGFA) was developed,in which the recombinant SSB protein expressed in Pichia pastoris was bound to nitrocellulose(NC) membrane and colloidal gold-labeled staphylococus protein A(SPA) was used as an indicator.Results The sensitivity and specificity of DIGFA were 100% and 98.75%,respectively.The agreement between DIGFA and ENA dot assay was 99.01%.Conclusion DIGFA for detecting autoantibody SSB is a good,rapid,simple and accurate assay for clinical diagnosis.

Detection of anti-histoplasma antibody in sera of immunized mice by dot immunogold filtration assay / 第三军医大学学报

Objective To study a new and rapid method for detection of anti-histoplasma antibody by dot immunogold filtration assay (DIGFA). Methods DIGFA was developed by coating purified protein derivative of histoplasmin (P-HTPM) on nitrocellulose membrane as membrane antigen and labeling SPA with colloidal gold. Anti-histoplasma antibodies in sera from mice immunized with Histoplasma were detected by DIGFA. Results All immunizedsera with Histoplasma were positive by DIGFA as well as the results detected by ELISA. The sera immunized respectively with Monilia and Penicillium marneffei were negtive by DIGFA while 1 of 8 immunizedsera with Blastomyces dermatitidis and 2 of 9 immunizedsera with Paracoccidioides brasiliensis were found to be positive. Conclusion DIGFA was a valuable and rapid method to detect histoplasmaantibody in serum.

THE APPLICATION OF PRE-EMBEDDING DOUBLE-LABELED IMMUNO-ELECTRON MICROSCOPIC METHOD IN THE NEUROANATOMICAL STUDIES / 解剖学报

Objective The co existence of two kinds of neurotransmitters in the cell bodies or axon terminals and the relationship between a neurotransmitter and its relevant receptor were observed at the ultrastructural level. Methods The pre embedding double labeled immuno electron microscopic techniques were used by peroxidase immunohistochemistry combined with immunogold silver labeled method. Results In the sections of the striatum,a large number of the SP like immunoreactive(\|LI) axon terminals(immunoperoxidase reaction products)and SP receptor(SPR)\|LI neuronal cell bodies and dendrites(immunoglold silver grains)were seen in the present study.Some SP\|LI axon terminals were observed to form symmetric axon somatic or axon dendritic synaptic contacts with SPR\|LI neuronal cell bodies and dendritic profiles.On the other hand,in the sections of caudal spinal trigeminal nucleus,a substantial number of axon terminals of the two kinds of the vesicular glutamate transporters,the differentiation associated Na + dependent inorganic phosphate cotransporter(DNPI)and vesicular glutamate transporter of type1(VGluT1),were labeled by peroxidase immunohistochemistry and immunogold silver grains.Some axon terminals were labeled by both DNPI and VGLuT1 immunoreaction formed asymmetric synaptic contact with non labeled dendrite profile.Conclusion\ The pre embedding double labeled immuno electron microscopic technique has the advantage of higher sensitivity and greater preservation of the antigen in the many tissues.Thus,the present study favors the application of this technique to study the co existence of two kinds of the neurotransmitters in the cell bodies or axon terminals and analyse the relationship between a neurotransmitter and its relevant receptor in the field of the neuroanatomy.

Ultrastructural Analysis of GABA- and Glycine-Immunoreactive Nerve Terminals on Jaw-Closing and Jaw-Opening Motoneurons in the Rat / 대한해부학회지

Previous studies have shown that inhibitory synaptic inputs are different between in spinal and trigeminal motor systems and activities of jaw closing and opening alpha motoneurons are different during a chewing cycle. This study examined the distribution of inhibitory synapses made on masseter and digastric motoneurons by using retrograde tracing of wheat germ agglutinin conjugated to horseradish peroxides (WGA-HRP) combined with postembedding immunogold labeling on serial ultrathin sections.Many boutons IR (immunoreactive) to GABA and/or glycine were found to appose on two kinds of motoneurons, which were containing pleomorphic vesicles (a mixture of round, oval and flattened vesicles) and exhibited symmetrical synaptic contacts on the somata. Packing density and synaptic covering % were higher in digastric than in masseter motoneurons. Of 703 boutons apposing on 12 masseter motoneurons, 6.08+/-3.51, 29.67+/-8.89 and 17.78+/-5.22% were IR to GABA only, glycine only, and both GABA and glycine, respectively. Of 637 boutons apposing on 11 digastric motoneurons, 6.37+/-4.64, 19.74+/-8.25 and 12.01+/-5.38% were IR to GABA only, glycine only, and both GABA and glycine, respectively. Proportions of glycine IR boutons were higher than that of GABA IR boutons in both masseter and digastric motoneurons. Packing density and proportion of boutons IR to GABA and/or glycine were higher in jaw closing than in jaw opening motoneurons (packing density, 12.03+/-1.58 vs 10.28+/-2.99; proportion of IR boutons, 53.54+/-8.94% vs 38.12+/-9.38% in jaw closing and opening motoneurons, respectively). These results provide ultrastructural evidence that GABA and glycine act as important neurotransmitters for modulation of jaw movement and that proportion of inhibitory synapses is higher in jaw closing than in jaw opening motoneurons.

Study and application of immunogold filtration assay for detecting serum antibodies against helicobacter pylori / 中国实用内科杂志

Objectives To establish a simple,quick,accurate serodiagnostic assay for detecting serum antibodies against Helicobacter pylori(H.pylori).Methods We established quick immunocolloidal gold filtration assay(DIGFA) by combining colloidal gold labelling technique with filtration method.145 sera from cases with H.pylori infection were detected by DIGFA,and the results were compared with that of ELISA and pathological examinations.In addition,blocking test and human IgG protein detection were conducted.The sensitivity,specificity of DIGFA were analysed.Besides,DIGFA was applied to investigate the H.pylori infection situation among persons of different ages.Results The coincidence rate reached 95 1% compared with the ELISA,and it was 88 8% compared with pathological the examination.The detectable minimum amount of IgG was 10?g/mL.The antibodies detected by DIGFA proved to be specific antibodies by blocking test.The human H.pylori infection rate among 216 persons reached 42 5% by DIGFA.Conclusions DIGFA can be applied effectively in clinical diagnosis and epidemiological survey.

Expression and Localization of Laminin on Rectus Femoris Muscle in the Developing Rats / 대한해부학회지

Laminin is an extracellular matrix-associated protein, which is largely present in the basement membranes of the human placenta, striated muscle and Schwann cell. Laminin has been proposed to promote attachment, spread, motility, growth and development of tissues or cells. In this study, we investigated the formation, localization and migration of the laminin in developing rectus femoris muscle of fetal and newborn rats. Experimental animals, fetal or newborn rats (Sprague-Dawley strain), were divided into 8 groups (14 day-old, 16 day-old, 18 day-old, 20 day-old and 22 day-old fetal rats, 1 day-old, 3 day-old, 5 day-old and 7 day-old newborn rats). The specimens of each group were prepared for detection of the laminin by immuno- histological and immunogold electron microscopic methods. 1. The primitive rectus femoris muscle of 14 day-old fetal rats consisted of mesenchymal cells and myoblasts. Laminin stained with a few gold particles were observed in the cytoplasm of myoblasts and rough endoplasmic reticulum of mesenchymal cells. 2. In the rectus femoris muscle of 16 day-old fetal rats, laminin was strongly expressed in myoblasts and mesenchymal cells. Gold particles were distributed in the cytoplasm of myoblasts and mesenchymal cells. 3. In the rectus femoris muscle of 18 and 20 day-old fetal rats, strong laminin immune reactions were observed in the basement membrane of muscle cells. A few gold particles distributed on the basal lamina of muscle cells and in the RER of fibroblasts. 4. In the rectus femoris muscle of 22 day-old fetal rats and 1 day-old newborn rats, strong laminin immune reactions were seen in the basement membrane of muscle cells. Numerous gold particles were located on the basal lamina of muscle cells and the RER of fibroblasts. 5. In the rectus femoris muscle of 3 day, 5 day and 7 day-old newborn rats, moderate laminin immune reactions were detected in the basement membrane of the muscle cell. A few gold particles were located on the basal laminae of muscle cells and RER of fibroblasts. These results suggest that the distribution of laminin in the rectus femoris muscle is related to myogenicity. Laminin seemed to be secreted by myoblasts of rectus femoris muscle at the fetal stage, but by fibroblasts in the muscle at neonatal and adult stages.

Prokaryotic expression and purification of human Smith D1 antigen / 中华检验医学杂志

Objective To obtain recombinant human Smith D1 (Sm D1) antigen and establish detecting assay.Methods Human Smith D1 antigen was synthesized by PCR using human Leukemic cDNA. The prokaryotic expression vector pGEX-ST-Sm D1 was constructed and transformed into E.coli.BL21 cell.Protein expressed under the induction of IPTG.We established DIGFA for detecting anti-Sm D1 antibodies with purified Sm D1 antigens.Results Sequence and restriction analysis revealed Sm D1 gene was cloned in frame into pGEX-5T,SDS-PAGE profile showed a clear protein band with a relative molecular weight of 39 000 and western blotting indicated that the expressed product specifically reacted to polyclonal anti-human Sm D1 genes.There was no significant difference between DIGFA and IB.The agreement between DIGFA and IB was 91.7% as calculated by Kappa statistical method.The sensitivity and specificity of DIGFA were 100% and 83.3% repectively.Conclusions Human Sm D1 gene is successfully cloned、 expressed and purification.The DIGFA,using purified Sm D1 antigens,is as good as IB,rather simpler, more rapid and reliable assay.

Immunohistological and Immunogold Electronmicroscopic Studies on Fibronectin in Lung and Liver of CS gas Exposed Rats / 체질인류학회지

Fibronectin (FN) is a major extracellular matrix glycoprotein, highly expressed in developing rat lungs. Several observations suggest that it play an important role in many developmental processes of animals. In vitro, FN can affect the adhesion, migration, proliferation, differentiation, and even apoptosis of various cell types. This study was undertaken to describe the distribution and localizations of fibronectin in the alveolar septum of lungs and liver lobules after CS gas exposure to experimental rats. The experimental rats (Sprague-Dawley strain), weighing 150~200 gm were sacrificed at 6 hours, 12 hours, 24 hours, 48 hours and 72 hours after CS gas exposure. The specimens of lung and liver were prepared for fibronectin immunoreactions of alveolar septa and liver lobules on experimental group, and for fibronectin reactions on the cytoplasm of type I alveolar cells, type II alveolar cells, fibroblasts, alveolar macrophages and interstitium in alveolar septa of lungs. All of specimens for immune reactions were observed with light and electron microscopes. The results obtained were as follows. 1. The liver lobules showed mild fibronectin reactions at the 6 hours and 12 hours after CS gas exposure. 2. The alveolar macrophages revealed strong fibronectin reactions. But alveolar septa showed weak FN reactions at 6 hours after CS gas exposure. 3. At 12 hours and 24 hours after CS gas exposure, the interstitial pneumonitis were seen in the lung alveoli. The gold particles were increased in the cells of alveolar septa, weak fibronectin reactions were revealed in the alveolar septum. 4. At 48 hours and 72 hours after CS gas exposure, FN reactions of alveolar septa were moderate, and the gold particles in the alveolar cells were markedly decreased. These results suggest that CS gas exposure to rats induces the increase of the fibronectin in lung and liver.

Expression of Surfactant-D Protein and TNF-alpha in the Interaction of Pneumocystis Carinii and Alveolar Macrophages in Pneumocystis Carinii Pneumonia

Alveolar macrophages participate in the host defense against P. carinii, but the mechanisms in degradation and clearance of the organism from lung has not been well established. We observed the transmission and scanning electron microscopic features and evaluated the expression of TNF-alpha and Surfactant-D in the interaction of P. carinii with alveolar macrophages. Expression of TNF-alpha and Surfactant-D in the experimentally induced P. carinii pneumonia in rat was examined by immunohistochemistry and immunoelectron microscopy. Electron microscopically, the alveolar macrophages phagocytized trophozoites and cysts of P. carinii micro-organisms. Immunohistochemically TNF-alpha was strongly expressed in the cytoplasms of alveolar macrophages. Postembedding immunogold labeling for Surfactant-D protein was expressed on the pellicles of trophozoites and cysts, P. carinii micro-organisms in the cytoplasms of macrophages, free floating surfactant materials and multilamellar bodies of type II epithelial cells. We conclude that alveolar macrophages interacted with P. carinii micro-organisms respond with increased expression of TNF-alpha. TNF-alpha may bind to P. carinii and exert a direct toxic effect upon the micro-organisms. Surfactant-D protein may augment binding of P. carinii to the alveolar macrophages and enhance the clearance of the micro-organisms.

Expression of Neurofilament in Human Retinal Horizontal Cell by Immunogold Labeling

Retinal horizontal cell is second-order neuron that integrates the information from photoreceptors over large retinal areas, mediating the lateral spread of visual signals to distant retina. The Neurofilament proteins, considered as neuronal markers, have been imunolocalized to mammalian retinal horizontal cess. However, the immunolabeling of Neurofilament in human, has been focused on the studies of visual pathway and large ganglion cells The goal of this study is to see whether human retinal horizontal cells are indeed neuronal nature or glial nature by immunogold labeling for electron microscopy. The sections of 65 year-old human retina showed the expression of Neurofilament by horizontal cells, which confirms the evidence of human retinal horizontal cell as neuronal nature.

Expression of Glial Fibrillary Acidic Protein in Human Retinal Macroglia by Immunogold Labeling

There are two types of retinal macroglia: astrocyte and Muller cell. The major component of iintermediate filament in astrocyte is GFAP, recognized as the primary marker of astrocyte. Muller cells do not physiologically express GFAP in normal developing retina. A striking increase in GFAP expression. however, is observed in Muller cells under certain pathological condition. It is also suggested that this kind of changes of Muller cells in human retina is related with aging, and may be an early feature of pathogenesis of Age related macular degeneration. The goal of this study is to investigate the involvement of the human retinal macroglia in normal aging by immunogold labeling for electron. The goal of this study is to investigate the involvement of the human retinal macroglia in normal aging by immunogold labeling for electron microscopy. The sections of aged neurosensory retina showed the evidence of expression of GFAP by Muller cells. The results suggest, therefore, that Muller cells are induced to express GFAP during aging.

Application of Dot-immunogold Filtration Assay in Detection of IgG in Sera of Trichinellosis Patients / 中国寄生虫学与寄生虫病杂志

0.05). Conclusion The dot-immunogold filtration assay is rapid and simple for performing and reading in the detection of IgG against T.s with high sensitivity and specificity.

Immunogold Localization of Type VII Collagen in the Adhesion Complex of Pathologic Cornea

Type VII collagen is one of the major structural components of the corneal epithelial adhesion complex. Using the immunogold technique combined with indirect immunofluorescence analysis, the fine structural distribution of type VII collagen was studied in the corneas obtained from 5 enucleated hyman eyes (age range, 1-77 years) including one pathologic cornea from graft rejection. The findings on normal cornea corroborated the results from previous studies. In pathologic cornea from graft rejection, type VII collagen antibodies generated linear and irregular patchy fluorescence staining along the epithelial-stromal interface and immunogold binding to type VII collagen mainly occurred within the undulating lamina densa, more densealy distributed anchoring plaques and anchoring fibrils. The distribution of type VII collagen in pathologic human cornea from graft rejection is similar to normal human cornea. But, in pathologic cornea, type VII collagen is more densely distributed in superficial stroma and forms more extended anchoring network, which may be derived from the increased secretion of the type VII collagen due to the activated basal epithelial cell during healing process.

STUDIES ON FACTORS INFLUENCING DOT-IGSS FOR ANTIBODIES IN SCHISTOSOMIASIS / 中国血吸虫病防治杂志

By using different blocking and diluting fluids, different developing time and different gold-labeled anti-immunoglobulin (GLA-Ig) which had been preserved in different conditions, the influence on the dot-immunogold-siiver staining (Dot-IGSS) for diagnosis of schis-tosomiasis japonica was studied. The results suggested that using suitable amount of sheep/ bovine serum albumin (BSA) as blocking fluid and calf serum as diluting fluid was a good choice, and 20℃ 20 min was the appropriate time for development. The GLA-Ig without Na3N3 could be preserved in - 20℃ for 2 years if it was not frozen and melted repeatedly.

Evaluation of diagnosis and therapeutic efficacy of dot immunogold filtration assay for schistosomiasis japonica using AWA-TCA as antigen / 中国血吸虫病防治杂志

0.05). Among 36 sera collected at 12 months post-treatment, the antibody negative conversion rates were 80.6% (29/36) and 58.3% (21/36), there was a significant difference between the two assays (P

Application of Dot Immunogold Filtration Assay for Detecting Serum Antibodies in Clonorchiasis Patients / 中国寄生虫学与寄生虫病杂志

0^05). The negative rate of DIGFA in healthy people was 100%(40/40). The cross reaction rate in 20 cysticercosis cases and 25 schistosomiasis cases were 5%(1/20) and 4%(1/25), respectively. Both coincidence rates comparing DIGFA with dot\|ELISA were 90^9%(50/55). Conclusion DIGFA is as sensitive and specific as the dot\|ELISA,and has the advantages of simplicity and without specific equipment.