Double Antibody Sandwich Research Articles

Expression of sB7-H4 in Serum and Lymphoma Tissues of Patients with Malignant Lymphoma and Its Value for Diagnosis and Re-Examine of Lymphoma

To study the soluble B7-H4 (sB7-H4) expression in serum and lymphoma tissues of patients with malignant lymphoma (ML) and its value for diagnosis and re-examination lymphoma. The serum samples from 83 cases of ML were collected, among them 69 cases of newly diagnosed ML were enrolled in group A including 11 cases of Hodgkin's lymphoma (NHL group) and 58 cases of non-Hodgkin's lymphoma (NHL group), the serum samples from 14 cases of relapsed ML were enrolled in group B; at the same time the serum samples of 50 healthy persons conformed by physical examination were collected and enrolled in control group. The double antibody sandwich ELISA was used to detect the serum level of sB7-H4 in each group, and immunohistochemistry method was used to detect the expression of sB7-H4 in malignant lymphoma and reactive lymphoid hyperplasia tissues. The serum level of sB7-H4 in the group A was significantly increased in comparison with the group B and control group, and the level of group B was significantly higher than that in the control group (P<0.05); the serum level of sB7-H4 in the NHL group was significantly increased in comparison with HL group and control group, and the level of HL group was higher than that of control group (P<0.05). The expression of sB7-H4 in reactive lymphoid hyperplasia tissues was negative, but the positive expression rate in malignant lymphoma tissues was 47.50%, suggesting the positive rate of sB7-H4 in malignant lymphoma tissues was significantly higher than that of reactive lymphoid hyperplasia tissues (P<0.05). The high expression of sB7-H4 in serum and lymphoma tissues of patients with malignant lymphoma has a certain value for the diagnosis and re-examination of patients with malignant lymphoma.

Prevalence of severe Citrus tristeza virus infection in Khasi mandarin throughout the north-eastern states of India: an evidence study based on coat protein gene analysis

Citrus tristeza virus (CTV) infected plant samples from the North Eastern states of India namely Arunachal Pradesh (AR1), Assam (AS1), Nagaland (NL1), Meghalaya (M1), Sikkim (SK1) and Tripura (TR1) were examined through double antibody sandwich enzyme linked immunosorbent assay and reverse transcriptase polymerase chain reaction using coat protein (CP) gene specific primers, yielding amplicon of 672 base pair for all the isolates. The sequence analysis revealed nucleotide (nt) and amino acid (aa) sequence similarity with other exotic CTV isolates. AR1 and AS1 shared similarity with T3 isolate (Florida) at 98–99% and 95–99% for nt-aa respectively, whereas, M1 showed 99% similarity with stem pitting isolate B165 (Bangalore). SK1 and NL1 were 95–97% and 97–99% similar with Hawaiian isolate HA at nt-aa level. TR1 showed 97% homology with seedling yellowing Israeli isolate VT. The CP gene sequence based phylogenetic analysis grouped present isolates into four clades with reference severe isolates (i.e. VT, B165, HA and T3), indicating their probable evolution. TR1 and NL1 are the putative recombinant isolates and ASI and SK1 were the major and minor parents respectively. This might be due to the conserved nature of CP gene. Moreover, the CP gene sequences of present isolates showing nucleotide identity to the VT, T3 and B165 isolates also suggest the prevalence of severe CTV isolates in Khasi mandarin growing areas comes under North East (NE) region of India.

Expression of human epididymal protein 4 in patients with epithelial ovarian cancer and its effect on prognosis

Objective To investigate the expression of human epididymal protein 4 (HE4) and its effect on prognosis in patients with epithelial ovarian cancer (EOC). Methods From 2009 to 2014, ninety-eight cases of epithelial ovarian cancer (EOC group) and 29 cases of benign ovarian tumor (benign group) and 100 healthy subjects (control group) were selected as research subjects.The serum HE4 was detected by double antibody sandwich enzyme-linked immunosorbent assay (ELISA), and the positive expression was determined by HE4>150 pmol/L in serum.The concentration and positive rate of serum HE4 were compared among the three groups.The median serum HE4 level of EOC patients before treatment was divided into high level HE4 group and low level HE4 group.The median survival time of the two groups of EOC patients was compared. Results The median value of serum HE4 concentration (four percentile) in EOC group was 421.8 (68.7 ~ 2173.9) pmol/L, 47.8 (35.24 ~ 68.5) pmol/L in the benign group and 43.8 (32.91 ~ 70.4) pmol/ L in the control group.The serum concentration of HE4 in the EOC group was significantly higher than that in the benign group and the control group (P=0.000). The serum HE4 concentration in group EOC was significantly higher than that in benign group and control group (P 0.05). The positive rate of serum HE4 in the EOC group was 74.5 (73/98), the positive rate of the benign group and the control group were 0, the difference between the three groups was statistically significant (P=0.000). The positive rate of serum HE4 in the EOC group was significantly higher than that in the benign group and the control group (P 0.05). The median survival time of EOC patients in low level HE4 group was 66.2 months, while that in high level HE4 group was 36.9.The difference between two groups was statistically significant (P=0.001). Conclusion The serum HE4 level of EOC patients was significantly higher than that of patients with benign ovarian tumors and healthy controls.The higher serum HE4 level, the worse the prognosis, and the shorter the survival time of patients.Therefore, the expression level of serum HE4 can be used for the diagnosis and prognosis of ovarian cancer. Key words: Epithelial ovarian cancer; human epididymis protein 4; carbohydrate antigen 125; survival time

Multiplex measurement of twelve tumor markers using a GMR multi-biomarker immunoassay biosensor

Tumor markers play an important role in the early diagnosis and therapeutic effect monitoring of tumors. Combined detection of multiple tumor markers is a realistic way of improving the sensitivity and specificity of cancer diagnosis. To achieve this, we studied and designed a giant magneto resistance (GMR) multi-biomarker immunoassay biosensor that can simultaneously detect twelve kinds of tumor markers by integrating a GMR sensor chip, a microfluidic device, a magnetic nano-beads label, and a double antibody sandwich immunoassay method. As a proof of concept, the proposed immunosensor was utilized to detect 12 tumor markers (AFP, CEA, CYFRA21-1, NSE, SCC, PG I, PG II, CA19-9, total PSA, free PSA, free-β-hCG, Tg) and to screen patients with lung cancer, liver cancer, digestive tract cancer, prostatic cancer, etc. The immunosensor showed excellent sensitivity, accuracy, precision and stability. Designed as a POCT device, the immunosensor also allows for portability, able to perform rapid detection wherever necessary. As a multi-analyte assay, it provides significant advantages over single-analyte tests in terms of cost per test, labor and convenience. The system's ability to simultaneously measure the concentration of multiple markers in serum samples with excellent sensitivity and accuracy allows the immunosensor to be used for early tumor diagnosis.

Development of an immunochromatographic assay for the specific detection of Bacillus thuringiensis (Bt) Cry1Ab toxin

Cry1Ab has been widely used in genetically modified (GM) crops and its amino acid sequence had high identity to Cry1Ac toxin. Existing nanogold immunochromatographic strips cannot distinguish Cry1Ab from Cry1Ac toxin. In this study, a rapid (5–6 min), qualitative nanogold immunochromatographic strip was successfully developed for the specific detection of Cry1Ab toxin. The assay was based on double antibody sandwich format with the visual detection limit (vLOD) of 0.1 μg mL−1. The results of immunochromatographic assay were all positive validated against the DAS-ELISA (recoveries between 109.6 and 111.8%). In addition, 10%, 5% and 0% error probability results were found in 20 times repeated tests for Cry1Ab concentration of 0.1, 0.2, 0.5 and 1 μg mL−1, respectively, demonstrating the reproducibility of the test strip. Furthermore, the test strip could be stored for 3 months under dry conditions without significant loss of sensitivity. Furthermore, the practical sample analysis results showed that the test strip was able to detect the presence of Cry1Ab in GM materials containing as low as 0.5% MON 810 Bt maize which indicated the practical value of the test strip. To our knowledge, this is the first report on the detection of Cry1Ab by immunochromatographic assay without interference from Cry1Ac toxin.

Survey for the identification and geographical distribution of viruses and virus diseases of maize (Zea mays L.) in Ethiopia

A survey to identify the distribution of virus diseases of maize was made in major maize growing regions of Ethiopia. Four surveys were conducted between 2015 and 2017. A total of 284 maize fields have been surveyed, of which 846 leaf samples with virus like disease symptoms were collected from 191 fields. There were no symptoms indicating for virus presence in maize grown in 93 fields which were exempted from sampling, while in contrast up to 100% disease incidence was recorded in maize grown in the Benishangul-Gumuz, Oromia and South Nations, Nationalities and People (SNNP) regions. Symptoms varied in maize ranging from the most common mosaic and streak to severe leaf mottling and necrosis as well as stunting and pre-mature plant death. Double or triple antibody sandwich (DAS or TAS) enzyme-linked immuno-sorbent assay (ELISA) to test for eight common maize viruses such as Maize chlorotic mottle virus (MCMV), Sugarcane mosaic virus (SCMV), Maize streak virus (MSV), Maize dwarf mosaic virus (MDMV), Maize mosaic virus (MMV), Maize stripe virus (MSpV), Wheat streak mosaic virus (WSMV) and Johnsongrass mosaic virus (JGMV) confirmed the presence of MCMV, SCMV and MSV in the leaf samples. Single or mixed infections with MCMV and SCMV frequently occurred and triple infections of MCMV, SCMV and MSV were found in 1% of samples from SNNP region. Sequence analysis of the coat protein genes of randomly selected seropositive samples of the three viruses showed little variability within the studied isolates and those retrieved from the GenBank. Our results indicated maize lethal necrosis disease (MLND) caused by MCMV and SCMV co-infection was the most important disease in SNNP and Oromia while maize streak disease was the predominant virus infecting maize in Benishangul-Gumuz.

Establishment and performance evaluation of a domestic chemiluminescence immunoassay of serum HE4

Objective To establish and evaluate a domestic Chemiluminescence immunoassay(CLIA) for detecting serum human epididymis protein4(HE4). Methods To establish a double antibody sandwich CLIA for the determination of serum HE4. Fluorescein isothiocyanate (FITC) and alkaline phosphatase (ALP) were used to label two different monoclonal antibodies of HE4. The separation could be realized by the magnetic particles coated with anti-FITC antibodies. Measure the relative light unit (RLU) after adding substrate solution, the RLU is proportional to the concentration of HE4. After evaluating the analytical performance including sensitivity, precision, accuracy, linearity, specificity under the optimized condition, this study compared the method with the commerical HE4 kits as well. Results The precision of with-in lot , and with-out lot are less than 4.0% and 5.0% respectively; Recovery is within 90.0%-110.0%; LoB is 5.0 pmol/L; functional sensitivity is 15.0 pmol/L; measure range is 15.0-1 500.0 pmol/L; the Correlation coefficient is bigger than 0.99 comparing with commercial kits. The AUC(area under curve) of three methods is 0.895, 0.900 and 0.896 respectively. Conclusions This study established a sensitive CLIA method with high repeatability and wide measure range; it has good correlation with commercial HE4 test kits.(Chin J Lab Med, 2018, 41: 615-620) Key words: Proteins; Luminescent measurements; Immunoassay; Ovarian neoplasms; Evaluation studies

First Report of Cocksfoot Mottle Virus Infecting Dactylis glomerata in Forage Production Fields in California.

Orchardgrass (Dactylis glomerata L.) is a cool season perennial grass commonly used in pastures and also grown for hay and silage. A Sobemovirus infecting orchardgrass, cocksfoot mottle virus (CfMV), was originally described in 1963 in England (Serjeant 1964). The symptoms of CfMV are visible in spring with yellow streaking in mild cases, but these symptoms can progress into whitening of the leaves, followed by necrosis and plant death in severe cases (Serjeant 1964). Although beetles (Oulemma spp.) are thought to introduce this virus into pastures, mechanical transmission is believed to be responsible for the spread of this virus once it has been introduced into an area (Serjeant 1964). This virus is associated with stand decline in orchardgrass fields in Europe, Japan, New Zealand (Mahy and Van Regenmortel 2010), Canada (Bittman et al. 2006), and recently in U.S. seed production fields in Oregon, where 79% of the orchardgrass fields surveyed in 2016 were infected with CfMV (Gilmore et al. 2017). Subsequently, in early April of 2017, several forage growers in northern California that were experiencing extensive die-off in their fields requested testing for CfMV and other pathogens. Orchardgrass samples were tested for CfMV using double antibody sandwich ELISA, with antiserum developed to an isolate of CfMV from British Columbia, Canada (R. R. Martin). Ten samples, collected (into individual plastic bags) from areas of the field with die-off, were ELISA positive for CfMV, and one was also positive for cereal yellow dwarf virus (CYDV). Five healthy samples collected from this same field tested negative for CYDV and CfMV. Total RNA was extracted from four of the CfMV-ELISA-positive samples following the nucleic acid extraction protocol of Tzanetakis et al. (2007) and used for cDNA synthesis using the SuperScriptIII First-Strand Synthesis SuperMix kit (Invitrogen by Life Technologies, CA). PrimeSTAR HS DNA Polymerase (Takara Clonetech, Japan) was used for polymerase chain reaction with CfMV-specific primers CfCP-F1 (GATGGAGCCAGTCTCTCG) and CfCP-R2 (ATCCGTCAATCTTCAAGC) (Gilmore et al. 2017) using a BioRad thermal cycler with the following program: 98°C for 2 min; 40 cycles of 98°C for 15 s, 55°C for 5 s, and 72°C for 45 s; and 72°C for 7 min. The predominant band at ≈750 bp was reamplified and sequenced using CfCP-F1 and CfCP-R2 primers. A BLAST search indicated one sample with 99% nucleotide sequence identity to CfMV reported in the Willamette Valley, OR, and three samples that were most closely related (≈95 to 96% identity) to CfMV reported in Ohio. Sequences have been submitted to GenBank (accession nos. MG765457 to MG765460). To our knowledge, this is the first report of CfMV in orchardgrass forage production fields in California. Although infection with CfMV can be lethal (Serjeant 1964), it is also possible that the presence of CfMV in combination with the drier and hotter summers experienced in the last few years may have weakened the plants and increased susceptibility to other pathogens, leading to the devastation observed in orchardgrass fields in northern California. Growers are encouraged to look for symptoms of CfMV in early spring and prevent mechanical transmission of CfMV by harvesting clean fields first, or by cleaning machinery thoroughly between fields. Additionally, growers are encouraged to grow CfMV-resistant cultivars, if possible. Cultivars Cheam-VR, Haida-VR, and Chilliwack-VR are listed as resistant to CfMV.

Development of a double-antibody sandwich ELISA for rapid detection to C-peptide in human urine

C-peptide level is recognized as an important indicator of diabetes diagnosis. A sensitive and specific double-antibody sandwich enzyme-linked immunosorbent assay for the detection of C-peptide based on double antibody sandwich method was studied in this paper. The rabbit and hen were innunized with PLL-C-peptide and BSA-C-peptide respectively to obtain specific Yolk antibody (IgY) and polyclonal antibody used to construct the sandwich ELISA for the measurement of C-peptide. The limit of detection was 0.51 μg/mL and the half maximal inhibitory concentration (IC50) was 3.26 μg/mL. The method developed in the study showed no evident cross-reactivity with other similar analogs. The detection standard curve of C-peptide exhibited a good linearity (R2 = 0.9896, n = 15). 17 types of the urine of diabetes patients on c-peptide levels compared with the hospital type of diabetes information, with a conclusion of a high consistent rate. Therefore, the methods could be selectively used for rapid screening of C-peptide in human urine, and the type of diabetes has some referential significance.

First Report of Moroccan Watermelon Mosaic Virus in Zucchini in Iran.

Cucurbits are among the most important vegetable crops in Iran, growing both in open fields and in greenhouses. They are ranked first in economic value, second in yield, and third in acreage and are susceptible to a number of viruses (Bananej and Vahdat 2008). Whitefly-transmitted viruses, namely, cucurbit chlorotic yellows virus (CCYV) and cucumber yellows stunting disorder virus (CYSDV), and the aphid-borne watermelon mosaic virus (WMV), zucchini yellow mosaic virus (ZYMV), papaya ringspot virus (PRSV), and cucurbit aphid-borne yellows virus (CABYV), have all previously been reported from Iran (Bananej and Vahdat 2008; Bananej et al. 2013). During November 2017, 12 samples were collected from cucumber (Cucumis sativus) and 14 from zucchini (Cucurbita pepo) plants grown in open fields that showed virus-like symptoms such as leaf mosaic, mottling, deformation, and yellowing symptoms in Kerman province in the south of Iran. The majority of both plant species were heavily infested by Bemisia tabaci, whereas no aphid colonies were observed. Leaf samples collected from symptomatic plants were subjected to total RNA extraction (Chatzinasiou et al. 2010). Reverse transcription PCR assays used for the detection of CCYV, CYSDV, and CABYV (Orfanidou et al. 2014) showed that all samples were infected with CABYV (26/26), whereas three cucumber samples were infected with CCYV and four with CYSDV. Moreover, double antibody sandwich ELISA was used for detection of WMV, ZYMV, PRSV, and Moroccan watermelon mosaic virus (MWMV) using polyclonal antisera kindly provided by H. Lecoq (INRA, France). None of the samples (0/26) was found to be infected with WMV, ZYMV, or PRSV; however, 9 out of 14 zucchini samples were positive for MWMV infection, whereas none of the 12 cucumber plants were positive for MWMV infection. For further confirmation, the specific primers MoDetF/MoDetR (5′-AARGAYGTTGATGTTGGRAC-3′, 5′-CCATARTCAYTCTTGACYCCATC-3′) were used to amplify a 211-bp product of the capsid protein gene of MWMV. These results confirmed the presence of the virus in the nine zucchini plants. Two isolates were further characterized by sequencing the amplicon, and the BLASTn analysis of the obtained sequences (accession nos. LT985986 and LT985987) showed 100% nucleotide identity with MWMV isolates from Tunisia and Greece (accession nos. EF579955 and LN810061). To our knowledge, this is the first report of MWMV infecting zucchini in Iran. After the detection of the virus in several countries of the Mediterranean region, this report further extends the information on its geographical distribution in the Middle East. The high sequence similarity of the Iranian MWMV isolate with those from the other Mediterranean countries, but not sub-Saharan Africa, indicates their recent rapid spread in the Mediterranean region (Yakoubi et al. 2008). Further surveys should concentrate on the spread, incidence, and epidemiology of this virus in Iran.

B-Fast ELISA: application of a new ELISA technique for the rapid detection of Pepino mosaic virus

In recent decades, many plant diseases have spread worldwide, favored by the steady increase of global trade and transport of plant products and germplasm. Pepino mosaic virus (PepMV) is an emerging virus with a high economic impact on tomato production in Europe, originating geographically from South America. In recent years, a number of new detection methods have been developed, but most are of limited suitability for routine testing. Enzyme-linked immunosorbent assay (ELISA) is still the most widely used method for large-scale testing of plant viruses, despite being limited by the many separate operating steps and long incubation times required. In order to overcome these shortcomings, the B-Fast ELISA technique has been developed, combining the robustness of an ELISA with the speed of PCR. This test allows reliable results to be obtained within only 2 h. Following the simultaneous incubation of a mixture of capture and detection antibody (conjugate) together with the test sample for 1 h, the ELISA wells are washed and substrate is added without any further operating steps. This new ELISA is fully compatible with standard ELISA buffers and equipment. B-Fast ELISA was comprehensively validated and compared with a standard double antibody sandwich (DAS)-ELISA for the detection of PepMV, demonstrating its suitability for sensitive and specific detection of all relevant PepMV strains in tomato. In addition, 15 of 27 randomly selected tomato samples offered in German supermarkets tested positive for the presence of PepMV by both B-Fast and standard DAS-ELISA. Infections were confirmed by RT-PCR, and subsequent sequencing of amplification products demonstrated that Chile 2 was the predominant strain. The successful development of the B-Fast assay shows that it provides a very rapid and convenient screening alternative to standard ELISA.

Effect of estrogen on expression of cysteine-rich secretory protein CRISPLD2 in myocardium of mice with sepsis

To investigate the effect of estrogen on expression of the cysteine-rich secretory protein containing LCCL domain 2 (CRISPLD2) in myocardium of lipopolysaccharide (LPS)-induced mice model of sepsis. Totally 12 female and 12 male Balb/c mice of specific pathogen-free (SPF) level with 7 weeks were served as objectives. The female and male mice were randomly divided into model groups and control groups, respectively, with 6 mice in each group. The model of sepsis was reproduced by intraperitoneal injection of 10% LPS (5 mg/kg), and the mice in control groups were injected with the same volume of normal saline. The general condition of mice during experiment was observed at 24 hours after injection. All the mice were sacrificed and the heart was harvested after collecting the whole blood. The concentration of estrogen in serum was determined by double antibody sandwich enzyme linked immunosorbent assay (ELISA). The myocardial tissue homogenate was prepared at the same time, and the total protein was extracted. The expression level of CRISPLD2 was determined by Western Blot. Pearson correlation analysis was used to analyze the bivariate correlation. All of the experimental mice survived at 24 hours after injection. The mice in the model groups showed disorder and gray signs of body hair, with diarrhea and decreased appetite. No significant abnormality was observed in the control groups. There was no significant difference in the body weight or concentration of estrogen in serum between model and control group of both female and male mice [body weight (g): 24.6±1.8 vs. 24.5±1.3 in male mice, 18.0±0.8 vs. 17.5±1.1 in female mice; estrogen (ng/L): 11.93±2.59 vs. 12.17±3.87 in male mice, 28.20±5.75 vs. 29.82±6.10 in female mice, all P > 0.05]. There was no statistical difference in the expression of CRISPLD2 in myocardium between male control mice and female control mice (gray value: 1.02±0.19 vs. 1.00±0.11, P > 0.05). No significant difference in the expression of CRISPLD2 in myocardium was found between female sepsis mice and female control mice (gray value: 1.05±0.13 vs. 1.00±0.11, P > 0.05). The expression of CRISPLD2 in myocardium of male sepsis mice was significantly lower than that of male control mice (gray value: 0.29±0.08 vs. 1.02±0.19, P < 0.01), and it was significantly lower than that of female sepsis mice (P < 0.01). It was shown by correlation analysis that the expression level of CRISPLD2 in myocardium of sepsis mice was significantly correlated with serum estrogen concentration [R2 = 0.736, 95% confidence interval (95%CI) = 0.560-0.960, P < 0.001]. In female mice with sepsis, the expression of CRISPLD2 is comparable to that of female healthy mice. It is suggested that estrogen can maintain the expression of CRISPLD2 in LPS-induced septic mice at the normal level.

Influences of interaction between DENV-2-infected HUVECs and regulatory T cells on major inflammatory cytokines

Objective To investigate the influences on major inflammatory cytokines after co-culturing regulatory T cells (Treg) with human umbilical vein endothelial cells (HUVECs) that were infected with dengue virus type 2 (DENV-2). Methods Peripheral blood mononuclear cells (PBMC) were extracted from concentrated human leukocytes by density gradient centrifugation.Treg cells were sorted by immunomagnetic beads.Expression of CD4, CD25 and CD127 molecules on the membrane of Treg cells was detected by flow cytometry to identify the purity of Treg cells.HUVECs pretreated with or without sphingosine-1-phosphate S1P type 1 (S1P1)-specific receptor agonist CYM-5442 for 24 h were first infected with DENV-2 and then co-cultured with Treg cells.Expression of IL-6, IL-8, TNF-α, IL-10 and TGF-β at mRNA level was detected by real-time RT-PCR.Levels of IL-6, IL-8, IL-10 and TGF-β in the culture supernatants were detected by a double-antibody sandwich ELISA. Results The purity of Treg cells was (84.3±0.5)%.Expression of NS1 at mRNA level in DENV-2-infected HUVECs first gradually increased and then decreased after reaching the peak at 24 h (3.03±0.26, P<0.01). Enhanced expression of IL-6, IL-8 and TNF-α at mRNA level in HUVECs was observed after DENV-2 infection (P<0.01). Expression of these cytokines at every time point was decreased after co-culturing DENV-2-infected HUVECs with Treg cells (P<0.05), but was still higher than that before infection.CYM-5442 pretreatment decreased the expression of IL-6, IL-8 and TNF-α at mRNA level in DENV-2-infected HUVECs and inhibited the secretion of IL-10 and TGF-β by Treg cells that were co-cultured with DENV-2-infected HUVECs. Conclusion Primary HUVECs infected by DENV-2 can enhance the secretion of IL-10 and TGF-β by Treg cells, and the suppressive cytokines produced by Treg cells can reduce the production of inflammatory cytokines by DENV-2-infected HUVECs. Key words: DENV-2; HUVEC; Treg cell; Cytokine; Immunomodulation

Administration of Electroacupuncture and Da-Cheng-Qi Decoction Has a Synergetic Effect in Relieving Injury of Pancreas, Lung and Large Intestine and Inflammatory Reactions in Rats with Acute Pancreatitis

To explore the effectiveness of electroacupuncture (EA) stimulation of Shu-and Mu-points of Lung and Large Intestine Meridians combined with administration of Da-Cheng-Qi Decoction (DCQD) in the treatment of inflammatory injury in rats with acute pancreatitis, so as to reveal their synergetic anti-inflammatory effect. Forty male SD rats were randomly divided into five groups: normal control, model, DCQD, EA, EA+DCQD, with 8 rats in each group. All the rats except those in the normal group received a retrograde biliopancreatic duct injection of sodium Taurocholate (3.0%, 0.1 mL/100 g) to induce an acute pancreatitis model. Twenty-four hours after modeling, EA (4 Hz/50 Hz) was applied to bilateral Shu-points "Feishu" (BL 13) and "Dachangshu" (BL 25), and Mu-points "Zhongfu" (LU 1) and "Tianshu" (ST 25) for 20 min, once every 7 h, 3 times in total. Rats of the DCQD and EA+DCQD groups were given intra-gastric gavage of DCQD (1 mL/100 g) 24 h after modeling, and those of the other 3 groups were given intra-gastric gavage of same dosage of normal saline. The histopathological changes of the pancreas, lung and large intestine tissues were observed after H. E. staining and scored according to Schmidt's and colleagues' methods (the severity of edema, inflammation, hemorrhage, necrosis). The concentrations of interleukin-6 (IL-6) and IL-10 in the serum, and those of myeloperoxidase (MPO) and malonaldehyde (MDA) in the lung and large intestine tissues were detected by using double-antibody sandwich ELISA. After modeling, the histopathological scores of pancreas, lung and large intestine tissues, the concentrations of MPO and MDA in lung and large intestine, and the levels of IL-6 and IL-10 in serum were significantly increased in the model group compared with the normal control group (P<0.05). Following the treatment, the histopathological scores of pancreas, lung and large intestine tissues, the contents of MPO and MDA in the lung and large intestine, and serum IL-6 were considerably reduced in the EA, DCQD and EA+DCQD groups relevant to the model group (P<0.05), while serum IL-10 content was notably increased in the three treatment groups (P<0.05). The therapeutic effect of EA+DCQD was significantly superior to that of simple EA and simple DCQD in down-regulating the histopathological scores of lung and large intestine tissues, the contents of MPO and MDA in the lung and large intestine, and serum IL-6 level, and up-regulating serum IL-10 level (P<0.05). No significant differences were found between the EA and DCQD groups in the above-mentioned 9 indexes (P>0.05). EA and DCQD can relieve the inflammatory injury of the pancreas, lung and large intestine tissues in rats with acute pancreatitis, and EA combined with DCQD has a better synergetic effect in reducing oxidative stress level and inflammatory reaction.

Establishment of time-resolved fluorescence immunochromatographic assay for detection of carbohydrate antigen 19-9

To establish a time-resolved fluorescence immunochromatographic assay for quantitative determination of carbohydrate antigen 19-9 (CA19-9) in serum, we prepared CA19-9 test strips by integrating double-antibody sandwich method and fluorescence immunochromatography technique. Carboxy fluorescent microspheres and nitrocellulose membrane were used as carriers for labeling and coating CA19-9 pairing antibodies. We optimized the process by adjusting the amount of labeling and coating antibody. According to the linear range, lowest detection limit and precision, We evaluated the time-resolved fluorescence immunochromatographic assay of CA19-9. When the amount of labeled antibody was 80 μg for 20 μL fluorescent microspheres, and the concentration of coated antibody on the test line was 1.5 mg/mL, the optimal reaction time was 15 minutes. Assay linear range was 12.5 to 800 U/mL and the minimum detection limit was 6.32 U/mL. The Within-run and between-run coefficient of variation were less than 15%. Average recovery rate was 101%. By detecting 50 clinical samples in parallel with Roche electrochemical luminescence detection kit, correlation coefficient was 0.980 6. The experiment, initially established a fluorescence immunochromatographic detection method to quantitative detection of serum CA19-9, which has a good clinical application prospect.

Assessment of Fasting Glucagon Levels in Subjects with Diabetes and Healthy Controls Using ELISA and RIA

Based on the lack of accurate methods for glucagon analysis, it has been suggested that glucagon levels are elevated in subjects with type 1 diabetes. The availability of more specific methods for the determination of glucagon has indicated that actual glucagon levels are lower than previously assumed. This study aimed to characterize fasting glucagon levels in subjects with type 1 diabetes (T1D) and type 2 diabetes (T2D) compared to their nondiabetic counterparts using two different assays. We recruited 20 healthy, 20 T1D and 20 T2D subjects for the trial. Glucagon samples were obtained using BD P800 blood collection tubes and measured with a double-antibody sandwich ELISA (Mercodia) and a RIA (MP Biomedicals). Using the ELISA, healthy subjects had similar glucagon levels compared to T1D. ELISA based glucagon measurements demonstrated significantly higher fasting glucagon levels in T2D as compared to healthy subjects, while RIA measurements tend to be numerically higher, albeit not statistically different (p=0.201). Median glucagon values obtained by RIA were significantly (p=0.0001) higher than those measured by ELISA in a pooled analysis. In conclusion, T2D show fasting hyperglucagonaemia, whereas T1D present with similar fasting glucagon levels compared to nondiabetic subjects. Furthermore, this study highlights the importance of selective analysis methods in endocrine research. Disclosure M. Brunner: None. M. Haberlander: None. E. Svehlikova: None. K. Eberhard: None. E. Stach: None. R. Lipp: None. B.M. Obermayer-Pietsch: None. T.R. Pieber: Consultant; Self; Arecor, AstraZeneca, Eli Lilly and Company, Novo Nordisk A/S, Sanofi. Employee; Self; CBmed. Research Support; Self; Novo Nordisk A/S, AstraZeneca. H. Sourij: Speaker's Bureau; Self; Boehringer Ingelheim GmbH, Novo Nordisk A/S, Amgen Inc., Sanofi, MSD K.K.. Research Support; Self; AstraZeneca, Boehringer Ingelheim GmbH, MSD K.K., GI Dynamics Inc..

Dynamics of Immune Responses during Experimental Mycobacterium kansasii Infection of Cynomolgus Monkeys (Macaca fascicularis).

To profile the dynamic changes of immune responses for M. kansasii infection, 3 cynomolgus monkeys were experimentally infected with M. kansasii by intratracheal inhalation of 1 × 106 CFU bacteria per monkey. Every 2 to 4 weeks, tuberculin skin testings (TSTs) were performed and blood samples were collected for immunoassay. Multiple cytokines in a single sample were measured by Luminex xMAP technologies. IgM and IgA were detected by double-antibody sandwich ELISA. IgG against PPD and 11 M. tuberculosis proteins were detected by using of indirect ELISA. At week 16, all animals were euthanized for necropsy and histological analysis. Positivities of TSTs emerged from week 2 to 6 postinfection. Leukocyte counts and T lymphocyte subsets experienced moderate increases. Among 44 kinds of cytokines, 36 kinds of them showed increases of different dynamic types and 8 kinds of them showed no specific changes. Total IgM and IgA showed a transient increase at an early infection stage. Positivities of M. tuberculosis specific IgM and IgA emerged as early as week 2 postinfection. All animals showed positive IgG against PPD and negative IgG responses to 38 kDa, MPT64L, TB16.3, 16 kDa, U1, and MTB81 antigens during the infection period. IgG against ESAT-6, CFP10, CFP10-ESAT-6, Ag85b, and 14 kDa antigens reached positive levels. The IgG avidities of PPD, ESAT-6, CFP10-ESAT-6, and Ag85b were all above 50 percent. In conclusion, the data indicate that M. kansasii infection in monkeys can induce positivities of TSTs, increases of multiple cytokines, and cross-reactive antibody responses to M. tuberculosis antigens.

A study of the correlation of insulin resistance and leptin with inflammatory factors and vascular endothelial injury in T2DM patients with CHD

The purpose of this study was to explore the correlation of insulin resistance (IR) and leptin with inflammatory factors and vascular endothelial injury in patients with type 2 diabetes mellitus (T2DM) complicated with coronary heart disease (CHD) was explored. One hundred and fifty normal patients (normal group), 150 patients with pure T2DM (T2DM group) and 150 patients with T2DM complicated with coronary heart disease (T2DM + CHD group) were selected from Xi'an No. 5 Hospital. All the participants met our inclusion criteria. Age, body mass index, waist-to-hip ratio, blood lipid and fasting plasma glucose (FPG), of all the subjects were measured. Chemiluminescent immunoassay was adopted for the detection of FPG and double-antibody sandwich method was used for the determination of fasting plasma leptin, and assay of high-sensitivity C-reactive protein (hs-CRP), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). Insulin resistance index (IRI) was used to evaluate IR and enzyme-linked immunosorbent assay was adopted for the detection of von Willebrand factor (vWF) and endothelin (ET-1). Compared with the control group, patients in the T2DM + CHD group and those in the T2DM group had higher homeostasis model assessment-IR, and higher assay of plasma leptin, hs-CRP, IL-6 and TNF-α (P<0.05), and lower vascular endothelial function (P<0.05). Moreover, compared with the T2DM group, T2DM + CHD group had higher plasma leptin, and higher assay of hs-CRP, IL-6 and TNF-α (P<0.05). IRI was positively correlated with hs-CRP (r=0.521, P=0.001), IL-6 (r=0.359, P=0.001) and TNF-α (r=0.386, P=0.001), leptin was positively correlated with hs-CRP (r=0.305, P=0.001), IL-6 (r=0.259, P=0.002) and TNF-α (r=0.429, P=0.001), and IRI had no correlation with ET-1 (r=0.058, P=0.734) and vWF (r=0.047, P=0.812), that is, it had no direct correlation with vascular endothelial function. Level of leptin was positively correlated with ET-1 (r=0.366, P=0.001) and vWF (r=0.471, P=0.001), that is, it was negatively correlated with vascular endothelial function. Our results showed that leptin, hs-CRP, IL-6 and TNF-α are involved in the occurrence and development of CHD in patients with T2DM. IR has no direct correlation with the occurrence and development of CHD in patients with T2DM.

Effects of parenteral nutrition of ω-3 polyunsaturated fatty acid, arginine and glutamine on cellular immune status of patients following liver cancer surgery

Purpose: To study the effects of parenteral nutrition (TPN), ω-3 polyunsaturated fatty acid (PUFA), Larginine (Arg), and glutamine on cellular immunity of patients who have done the liver cancer (LC) surgery.Methods: Seventy-five (75) LC patients were randomly divided into 5 groups (A - E; 15 cases each), group A, B, C, D and E, in which patients were treated with TPN, TPN + fish oil, TPN + Arg, TPN + glutamine, and TPN + ω-3 PUFA + Arg + glutamine, respectively. Before and after surgery, CD3 +, CD4 + and CD8 + were measured by antibody-sensitized erythrocyte rosette test, and IL-6, IL-10 and TNF-a were assayed with double-antibody sandwich enzyme-linked immunoassay (DAS-ELISA). IgA and IgM were measured nephelometrically.Results: The levels of CD3 +, CD4 + and CD8 + in group A showed no obvious change after surgery (p &gt; 0.05). However, CD3 + and CD4 + increased in groups B, C and D, while CD8 + decreased in group E (p &lt; 0.05). IL-6 in group E was lower than that in any of the other four groups (p &lt; 0.05). IL-10 in group A was lower than that in groups B, C and D, but lower than in group E (p &lt; 0.05). The levels of TNF-a in groups B and C were lower than those in group A, but higher than that in group E (p &lt; 0.05) but lower than in group D. IgA in group E was higher than in the other groups (p &lt; 0.05), while IgM level in group E was lower than in groups A, B and C (p &lt; 0.05).Conclusion: Immunosuppressive status and cellular immunity of patients after liver cancer surgery may be improved by a combination therapy of TPN, ω-3 PUFAs, Arg and glutamine.Keywords: Polyunsaturated fatty acid, Arginine, Glutamine, Parenteral nutrition, Hepatoma, Cellular immunity

Distribution, Incidence and Severity of Maize Lethal Necrosis Disease in Major Maize Growing Agro-ecological Zones of Uganda

The distribution, incidence and severity of maize lethal necrosis (MLN) disease in major maize growing agro-ecological zones (AEZ) of Uganda was determined following field surveys carried out in 16 major maize growing districts from 5 AEZ over three consecutive seasons. A total of 604 maize fields were visited and MLN disease status visually assessed and 3,624 maize leaf samples collected for identification and confirmation of the MLN causal viruses by Double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) and Reverse Transcription Polymerase Chain Reaction (RT-PCR). MLN disease was not widely distributed at an epidemic proportion, with only 36 (5%) of the 604 farms surveyed over three seasons confirmed to have the disease. The MLN incidence and severity was significantly (P &lt; 0.05) higher in the Eastern AEZ during the three seasons. The main MLN-causing viruses detected using DAS-ELISA were Maize chlorotic mottle virus (MCMV) and Sugarcane mosaic virus (SCMV). MCMV was the most prevalent MLN causing virus driving the epidemic in Uganda. The three major districts where MLN disease has been found in all three seasons surveyed are Bulambuli, Tororo and Busia which are hotspots for MLN disease. Strategies to control spread of MLN disease should focus on high risk AEZs and hotspot districts.