Sandwich Method Research Articles

Development of a fluorescence immunochromatography for quantitative detection of cartilage oligomeric matrix protein

The purpose of this study was to develop a fluorescence chromatography method for the detection of cartilage oligomeric matrix protein (COMP) in the auxiliary diagnosis of rheumatoid arthritis (RA). The principle of double antibody sandwich method was used to prepare immunochromatographic test strips, and the performance evaluation and methodological comparison were carried out. Through the detection of clinical samples, a receiver operating characteristic (ROC) curve was obtained, and the sensitivity, specificity, positive and negative predictive values of the test strip were calculated. The linear range was 0.39-50.00 ng/mL. The coefficients of variation inter and intra batches were less than 15%. The test strip was stable at 37 ℃ for 20 days, and the variation range of fluorescence signal intensity was within 15%. There was no cross reaction with rheumatoid factor (RF) and anti-cyclic citrulline peptide (anti-CCP) antibody. Forty-eight clinical serum samples were detected in parallel with ELISA kit, and the correlation was good. The test strip prepared in this study was used to detect the sample, the cut-off value of COMP between RA patients and healthy people was 22.55 ng/mL (sensitivity 0.821, specificity 0.842, positive predictive value 0.741, negative predictive value 0.895). At the same time, the same sample was tested with ELISA kit, the sensitivity and specificity of the two methods reached more than 80%. A quantitative COMP fluorescence chromatography test strip was developed, which has the advantages of celerity, simplicity and sensitivity, and may provide rapid auxiliary diagnosis for RA patients.

Sandwich fluorometric method for dual-role recognition of Listeria monocytogenes based on antibiotic-affinity strategy and fluorescence quenching effect

In this work, a sandwich fluorometric method for dual-role recognition of L. monocytogenes was developed based on antibiotic-affinity strategy and fluorescence quenching effect for sensitive and rapid detection of L. monocytogenes in ham samples. Vancomycin (Van) was conjugated with magnetic nanoparticles (MNPs) to recognize and capture target bacteria. Biotinylated aptamers were used to bind specifically to L. monocytogenes through the cell wall. The two agents recognized target bacteria at different binding sites showing satisfied specificity. The upconversion fluorescence response signal could be enlarged by using the inner filter effect (IFE) between the colored products produced by enzyme-catalyzing substrate and upconversion nanoparticles (UCNPs). The change in fluorescence intensity could represent the concentration of target bacteria over 102–2 × 108 CFU mL−1. The developed sandwich fluorimetric method achieved a low detection limit (LOD) of 2.8 × 102 CFU mL−1. Overall, the constructed fluorometric sensor could provide a simple and reliable method for the detection of L. monocytogenes.

A Raman immunosensor based on SERS and microfluidic chip for all-fiber detection of brain natriuretic peptide

Early diagnosis of chronic heart failure (CHF) relies on the ultra-sensitive detection assay of its main biomarker, Brain natriuretic peptide (BNP). To meet such demand, we designed a near-infrared immunoassay biosensor, utilizing a fiber-embedded microfluidic chip for surface-enhanced Raman scattering (SERS) detection. A SERS probe, consisting of metal–organic framework and Au-HEPES coupling nanoparticles (AuHPs), was used to enhance the Raman signal of the toluidine blue (TB) dye. The probe anchored with TB is used as a SERS tag, which is linked to BNP through sandwich method. To enhance the Raman signal further, we embedded a SERS substrate at the bottom of the microfluidic channel, which serves an enhancer when the SERS tagged BNP passes through the microfluidic chip. The amplified Raman signal was collected by an optical fiber probe of a simple and portable Raman detection system with an all-fiber connected configuration. Such design resulted in the lowest detection limit of pg/mL, indicating that the proposed platform can be used for rapid and highly sensitive detection of BNP. In addition, our platform also has the advantages of low sample consumption and high throughput rate, providing an effective scheme for the early diagnosis of CHF.

Expression of NDV HN protein in rice and development of a semi-quantitative rapid method for detection of antibodies

The aim of this study was to develop a semi-quantitative immunochromatographic method for rapid detection of Newcastle disease virus (NDV) antibodies by expressing HN protein in rice endosperm bioreactor. The recombinant plasmid pUC57-HN was digested by MlyⅠ and XhoⅠ to retrieve the HN gene, while the intermediate vector pMP3 containing promoter, signal peptide and terminator was digested by NaeⅠ and XhoⅠ. The HN gene and the linearized pMP3 were purified and ligated to form a recombinant plasmid pMP3-HN1. Subsequently, pMP3-HN1 and plant vector pCAMBIA1300 were digested by EcoRⅠ and Hind Ⅲ, and the HN1 gene was cloned into pCAMBIA1300. The recombinant plasmid pCAMBIA1300-HN1 was introduced into Agrobacterium tumefaciens EHA105 by electrotransformation, and the pCAMBIA1300-HN1 was transferred into rice callus by agrobacterium-mediated method. After dark culture, callus screening, differentiation, rooting and transplanting, transgenic rice seeds were obtained 4 months later. PCR identified that the HN gene has been inserted into the rice genome. SDS-PAGE and Western blotting indicated that the HN protein was successfully expressed in the positive rice endosperm. The purity of the HN protein was more than 90% by SP cation exchange chromatography and gel filtration chromatography. According to the national standards for the diagnostic techniques of Newcastle disease HI test (HI≥4log2, positive antibody reaction), a colloidal gold labeled purified HN protein was used to prepare a semi-quantitative test strip by double-antibody sandwich method for rapid detection of NDV antibody. The results showed that the test strip did not cross-react with positive sera against other viruses, and the sensitivity of the test strip reached 1:102 400 for standard positive sera of Newcastle disease. Testing of a total of 308 clinical sera showed that the compliance rate of the test strip with HI test was 97.08%, and the Kappa value was 0.942. In conclusion, high purity recombinant HN protein was obtained from rice endosperm, and a simple, rapid, highly sensitive and highly specific semi-quantitative immunochromatographic strip was developed. The test strip could be used for immune evaluation of the Newcastle disease vaccine.

Establishment and Clinical Application of a Highly Sensitive Time-Resolved Fluorescence Immunoassay for Tumor-Associated Trypsinogen-2.

To establish a rapid and highly sensitive assay for tumor-associated trypsinogen-2 (TAT-2) based on the time-resolved fluorescence immunoassay (TRFIA) and evaluate its potential clinical value in patients with lung cancer. The double-antibody sandwich method was used in detecting TAT-2 antigen concentrations, and two types of TAT-2 antibodies (coating antibodies and Eu3+ labeled antibodies) were used. A TAT-2-TRFIA method was then established, evaluated, and used in detecting the serum TAT-2 levels of healthy subjects and patients with lung cancer. The linear range of the TAT-2-TRFIA method was 1.53-300ng/mL, the intra-assay coefficient of variation (CV) were between 1.67% and 8.42%, and the inter-assay CV were between 4.29% and 11.44%. The recovery rates of TAT-2-TRFIA were between 99.17% and 107.06%. The cross-reactivities of trypsin and T-cell immunoglobulin mucin 3 were 0.02% and 0.82%, respectively. The serum TAT-2 levels of patients with lung cancer were higher than those of healthy subjects (P < 0.001). Combined with TAT-2, the sensitivity and specificity of CEA and CA-125 for lung cancer improved significantly. Conclusion: We successfully established a highly sensitive TAT-2-TRFIA method, which was able to facilitate the timely diagnosis of lung cancer.

The roles of Single Nucleotide Polymorphism (SNP) Endoplasmic Reticulum Aminopeptidase 1 (ERAP 1) gene in axial spondyloarthritis Indonesian adults

BackgroundAxial Spondyloarthritis (AxSpA) is chronic inflammatory arthritis involving the axial joint whose pathogenesis is related to the SNP ERAP1 gene, HLA B27, and cytokine proinflammatory (IL-17A and IL-23). ObjectiveAnalyzed the role of SNP gene ERAP1 on disease activity and proinflammatory cytokines. MethodsThis study comprised of two phases including a cross-sectional study and an in-vitro experiment in post-test with a control-group design. Participants underwent a PCR investigation searching for HLA-B27. Disease activities were measured by Ankylosing Spondylitis Disease Activity Score-Erythrocyte Sedimentation Rate (ASDAS-ESR) and modified Stokes Ankylosing Spondylitis Spinal Score (mSASSS). Subjects with HLA-B27 positive underwent PCR ERAP1 gene rs27434, genome-sequencing, and analysis. ELISA sandwich method was used to measure ERAP-1, IL-17, and IL-23 levels with lipopolysaccharide and IFN-γ induction. Analysis using independent t-test, Mann Whitney, and Pearson correlation test with p < 0.05. ResultsThe average ASDAS-ESR was 3.33 ± 0.89 and the average mSASSS was 26.53 ± 9.90. In HLA B27 positive group, SNP ERAP1 gene rs 27434 in which alleles A changed to G and A/G with genotypes AA to AG/GG was observed. SNPs of the ERAP1 gene had a correlation on mSASSS (r = 0.553; p < 0.05) and no correlation on ASDAS-ESR (r = 0.232; p = 0.235). There were significant differences observed in the SNP ERAP1 gene on ERAP1 and IL-17A levels in subjects with lipopolysaccharide and IFN-γ induction (p = 0.05) but no significant difference in IL-23 levels (p > 0.05). ConclusionThe SNP ERAP1 gene affects mSASSS value, ERAP1 levels, and IL-17A levels whereas ASDAS-ESR value and IL-23 level were not associated.

Somethings About Biological Prostheses

Finite element models of the female biofidel were developed using a specific combination of segmentation with computed tomography and solid modeling tools capable of representing bone physiology and structural behavior. This biofidel finite element (FEM) model is used to evaluate the change in the physiological distribution of stress in the femoral prosthesis and to evaluate the new design criteria for biopsy. Biomimetics, biomechanics, and tissue engineering are three multidisciplinary fields that have been considered in this research to achieve the goal of improving the reliability of prosthetic implants. The authors took these studies to gather the untapped potential of such advanced materials and design technologies by developing finite models of Biofidel elements capable of correctly mimicking the biomechanical behavior of the femur. The new remodeling of the tetrahedral elements was performed in 3Matic looking for the congruence of the node at the bone-implant interfaces, where the material was defined for the new configuration of the finite elements. The evaluation of the mechanical properties was made taking into account the mechanical characteristics of the cortical and trabecular bone. For biomechanical integration of the implant, a custom material with an improved combination of strength and rigidity that matches the bone should be used. This greater biomechanical compatibility will avoid weakening the implant and increase lifespan, avoiding additional surgery for revision and allowing good biological integration (bone growth). Innovative biomimetic materials for tissue engineering based on hydrophilic polymers were developed by our research group and presented attractive physical, biological, and mechanical properties for biomedical applications. For use with metal prostheses, the authors have developed a hybrid biocompatible material, extremely biocompatible, based on hydrophilic chemicals and hydroxy-ethyl-methacrylate type. The structural metal composition of the new prostheses will be made of titanium alloys using additive technology based on melting thin layers of titanium powder (about 50 microns) on each other until the desired component is obtained (sandwich method). Then, the biomaterial and osteoconductive nanostructured material developed in our previous studies can cover the titanium structural prosthetic skeleton. These hybrid biological prostheses, which are made using synthetic materials capable of inducing the growth of biological networks and structural steel scaffolding, may favor the emergence of new classes of orthopedic hybrids in the medical field. The new hybrid bio-prosthesis could drastically reduce protection against stress while providing an advantageous improvement in the life of the prosthesis compared to traditional solutions. Recovering optimal joint functionality will improve the patient's quality of life, which perceives a significant reduction in the risk of the new surgery. The requirement to predict potential structural changes that could be induced by improper use of biologically compatible prostheses in bone structure and morphology has forced our studies to evaluate fictitious models that could be considered for efficient bone distribution and orthotropic behavior.

Establishment and clinical application of time-resolved immunofluorescence assay of lipoprotein-associated phospholipase A2

AimThis study aimed to establish a highly sensitive time-resolved fluorescence immunoassay (TRFIA) for the detection of serum lipoprotein-associated phospholipase A2 (Lp-PLA2) and evaluate the clinical application value of Lp-PLA2 in patients with breast cancer. MethodsThe level of Lp-PLA2 was detected using the double-antibody sandwich method. First, the Lp-PLA2-TRFIA method was established, and the method was evaluated on the basis of linearity, sensitivity, precision, specificity, and recovery rate. Then, the fluorescence counts in serum of healthy subjects and patients with breast cancer were detected by Lp-PLA2-TRFIA, and the levels of Lp-PLA2 were calculated using a standard curve. ResultsLp-PLA2-TRFIA had a wide linear range (43.48–2000 ng/mL). The intra-assay precisions of Lp-PLA2-TRFIA ranged from 2.66% to 4.84% (<10%), and the inter-assay precisions were between 5.39% and 6.95% (<15%). No cross-reaction was observed among Lp-PLA2, Tumor-associated trypsinogen-2, and T-cell immunoglobulin mucin 3. In addition, the recovery rates were between 90% and 100%. The serum Lp-PLA2 levels of patients with breast cancer were significantly higher than those of healthy subjects. ConclusionsWe successfully established a highly sensitive Lp-PLA2-TRFIA method, and found serum Lp-PLA2 may be associated with dyslipidemia in breast cancer and could be used for auxiliary diagnose.

The differences in the levels of interleukin 1 beta (IL-1ß) and tumor necrosis factor alpha (TNF-Α) based on the storage of thrombocyte concentrates

Thrombocyte concentrate (TC) transfusion plays an important role in preventing bleeding in patients with severe thrombocytopenia. Febrile non hemolytic transfusion reaction (FNHTR) may occur after TC administration. IL-1ß and TNF-α cytokines released by thrombocytes and leucocytes during TC storage play important roles in the occurrence of FNHTR after TC transfusion. The purpose of this study was to analyze changes in levels of IL-1ß and TNF-α on the duration of TC storage. This was an observational analytical research with time series design carried out at the Clinical Pathology Laboratory and Blood Bank of the Dr. Soetomo Hospital Surabaya in September - October 2019. IL-1ß and TNF -α levels in 20 bags of Thrombocyte Concentrate blood components derived from Platelet Rich Plasma during storage for day 1, day 3 and day 5 were measured using ELISA Sandwich method. The statistical analysis was performed using the Subject Same Variant Test or Friedman Test. The results showed no significant differences in the levels of IL-1ß and TNF-α based on the storage duration of TCs on day 1, 3 and 5, p = 0.262 and p = 0.534 respectively. There was a significant difference of IL-1ß levels between day 1 and day 3 (p=0.032).

Re-Assessment of the Oral Salt Loading Test Using a New Chemiluminescent Enzyme Immunoassay Based on a Two-Step Sandwich Method to Measure 24-Hour Urine Aldosterone Excretion.

Since April 2021, the plasma aldosterone concentration has been measured by chemiluminescent enzyme immunoassay (CLEIA) in Japan. In the present study, we developed a new CLEIA using a two-step sandwich method to measure the 24-hour urine aldosterone level. We collected 115 urine samples and measured 24-hour urine aldosterone levels employing radioimmunoassay (RIA), CLEIA, and liquid chromatography–tandem mass spectrometry (LC-MS/MS). The results showed that the 24-hour urine aldosterone levels measured using CLEIA and LC-MS/MS were significantly correlated (ρ = 0.992, P < 0.0001). Based on the results of Passing–Bablok regression analysis, the slope was 0.992 and the intercept –19.3. The 24-hour urine aldosterone levels measured using CLEIA and RIA were also significantly correlated (ρ = 0.905, P < 0.0001). However, the aldosterone level measured by CLEIA was lower than that measured by RIA (slope, 0.729; intercept, 120.9). In Japan, a new guideline for primary aldosteronism has been announced, with changes in the aldosterone measurement method. The cutoff values for oral sodium loading test (OSLT) were changed, but clinical verification using real-world urine samples has not been performed. Therefore, we examined the cut-off value of the 24-hour urine aldosterone level after the OSLT. Receiver operating characteristic analysis revealed a cut-off value for primary aldosteronism of 3 μg/day.

A Universal Bacterial Catcher Au-PMBA-Nanocrab-Based Lateral Flow Immunoassay for Rapid Pathogens Detection.

In traditional lateral flow immunoassays (LFIA) for pathogens detection, capture antibody (CA) is necessary and usually conjugated to Au nanoparticles (NPs) in order to label the target analyte. However, the acquisition process of the Au-CA nanoprobe is relatively complicated and costly, which will limit the application of LFIA. Herein, p-mercaptophenylboronic acid-modified Au NPs (namely Au-PMBA nanocrabs), were synthesized and applied for a new CA-independent LFIA method. The stable Au-PMBA nanocrabs showed outstanding capability to capture both Gram-negative bacteria and Gram-positive bacteria through covalent bonding. The acquired Au-PMBA-bacteria complexes were dropped onto the strip, and then captured by the detection antibody on the test line (T-line). Take Escherichia coli O157:H7 as an example, the gray value of T-line was proportional to the bacteria concentration and the linear range was 103-107 cfu·mL-1. This CA-independent strategy exhibited higher sensitivity than the traditional CA-dependent double antibody sandwich method, because detection limit of the former one was 103 cfu·mL-1 only by visual observation, which was reduced by 3 orders of magnitude. Besides, this platform successfully screened E. coli O157:H7 in four food samples with recoveries ranging from 90.25% to 107.25%. This CA-independent LFIA showed great advantages and satisfactory potential for rapid foodborne pathogens detection in real samples.

Efficacy and Safety of the Sandwich Method in Patients with Benign Prostate Hyperplasia: Bipolar Transurethral Resection with GreenLight Vaporization.

Benign prostate hyperplasia (BPH) refers to the nonmalignant enlargement of the transition zone of the prostate gland. While holmium laser enucleation of the prostate and open simple prostatectomy are effective in the management of patients with large prostates, they have some limitations. Thus, this study aimed to analyze the efficacy and safety of the sandwich method of bipolar transurethral resection of the prostate (B-TURP) and GreenLight photoselective vaporization of the prostate (GLPVP) in patients with large prostates. Patients diagnosed with BPH who underwent the sandwich method with B-TURP and GLPVP from 2015 to 2020 were included. Efficacy analyses included the change in the uroflowmetry results in both group A (prostate volume < 80 g) and group B (prostate volume ≥ 80 g), and complication analyses included perioperative complications, early postoperative complications at three months and late postoperative complications at 12 months. The cohort comprised 188 and 44 patients in groups A and B, respectively. The prostate volume of groups A and B were 50.83 ± 14.14 g and 102.03 ± 19.36 g (p < 0.001), respectively. The peak (Qmax) and average (Qavg) flow rates were comparable between the two groups. The only significant difference noted was in the postoperative post-void residual (PVR) urine. Improvement was seen in all the variables including the Qmax, Qavg and PVR urine in each group. No patient experienced perioperative complications. Analysis of the overall one-year complication rate showed no significant difference between the two groups. The sandwich method of B-TURP and GLPVP may be feasible for the management of patients with large prostate.

Selective Detection of Legionella pneumophila Serogroup 1 and 5 with a Digital Photocorrosion Biosensor Using Antimicrobial Peptide-Antibody Sandwich Strategy.

Rapid detection of Legionella pneumophila (L. pneumophila) is important for monitoring the presence of these bacteria in water sources and preventing the transmission of the Legionnaires’ disease. We report improved biosensing of L. pneumophila with a digital photocorrosion (DIP) biosensor functionalized with an innovative structure of cysteine-modified warnericin antimicrobial peptides for capturing bacteria that are subsequently decorated with anti-L. pneumophila polyclonal antibodies (pAbs). The application of peptides for the operation of a biosensing device was enabled by the higher bacterial-capture efficiency of peptides compared to other traditional ligands, such as those based on antibodies or aptamers. At the same time, the significantly stronger affinity of pAbs decorating the L. pneumophila serogroup-1 (SG-1) compared to serogroup-5 (SG-5) allowed for the selective detection of L. pneumophila SG-1 at 50 CFU/mL. The results suggest that the attractive sensitivity of the investigated sandwich method is related to the flow of an extra electric charge between the pAb and a charge-sensing DIP biosensor. The method has the potential to offer highly specific and sensitive detection of L. pneumophila as well as other pathogenic bacteria and viruses.

Sensitive and selective detection of carbamazepine in serum samples by bionic double-antibody sandwich method based on cucurbit[7]uril and molecular imprinted polymers

A novel bionic enzyme-linked immunosorbent assay (BELISA) based on double-antibody sandwich method is firstly designed for the detection of carbamazepine (CBZ) in human serum samples. In this BELISA system, cucurbit[7]uril (CB[7]) is employed as an artificial capture antibody (cAb), and molecularly imprinted polymers (MIPs) is used as an artificial detection antibody (dAb). Nanozymes (PdNPs) as signal generators are integrated with MIPs. This couple of bionic antibodies exhibits not only the excellent physical and chemical stability, but also the superior molecular recognition ability. Based on two bionic antibodies that can selectively recognize different sites of CBZ molecule, a new BELISA method has been constructed for the first time. The proposed BELISA method displays a good linear relationship ranging from 2 to 20 μg mL-1. The detection limit is 0.37 μg mL-1, which can well meet clinical testing demand. It provides a more stable and economical method for clinical therapeutic drug monitoring (TDM).

Establishment of Galectin-3 Time-resolved Fluoroimmunoassay and its Application in Idiopathic Membranous Nephropathy.

The aim of this study was to establish a time-resolved fluorescent immunoassay (TRFIA) for the detection of serum Galectin-3 (Gal-3) and apply this method to evaluate the clinical significance of serum Gal-3 in predicting Idiopathic Membranous Nephropathy (IMN) progression.The Gal-3-TRFIA was established using the double antibody sandwich method, with the capture antibodies coated on a 96-well microplate and the detection antibodies chelated with Europium (III) (Eu3+). Serum Gal-3 was detected in 81 patients with IMN and 123 healthy controls to further evaluate the value of the Gal-3 in staging of IMN.The sensitivity of the Gal-3-TRFIA assay was 0.85ng/mL, and the detection range was 0.85-1000ng/mL. The Gal-3 intra-batch and inter-batch coefficients of variation were 3.45% and 5.12%, respectively. The correlation coefficient (R) between the Gal-3-TRFIA assay and commercially available enzyme-linked immunosorbent assay kits was 0.83. The serum Gal-3 concentration was higher in patients with IMN (65.57 ± 55.90ng/mL) compared to healthy controls (16.29 ± 9.91ng/mL, P < 0.0001).In this study, a wide detection range Gal-3-TRFIA assay was developed using lanthanide (Eu3+) chelates for the detection of Gal-3 concentrations in serum. Gal-3 concentration is elevated in patients with IMN.

Isothermal and enzyme-free MicroRNA assay based on catalytic hairpin assembly and rare earth element labeled probes

MicroRNAs (miRNAs) are a category of short non-coding RNA molecules which play essential roles in regulating gene expression. Correspondingly, the aberrant expression of miRNAs is commonly related to the development of various diseases including cancers and cardiovascular diseases. Hence, miRNA is considered as an ideal biomarker for disease diagnosis and its quantification is highly demanded. Herein, an isothermal and enzyme-free miRNA assay based on catalytic hairpin assembly (CHA) amplification coupled with inductively coupled plasma mass spectrometry (ICP-MS) detection is proposed. Two hairpin probes are employed in CHA reaction with rare earth element tag on H1 for ICP-MS detection and biotin on H2 for immobilization on magnetic beads. In the presence of target miRNA, the two independent probes open up their stem-loop structure and hybridize. The target miRNA will be released after the hybridization to trigger another CHA reaction. Ultimately, one target miRNA molecule promotes a number of CHA cycles and produces several copies of hybridized complex leading to the enhanced ICP-MS signal. Based on this approach, miRNA-141 was successfully analyzed with high sensitivity and specificity. The limit of detection (LOD) was 88 fmol with a linear range 0.1–2 pmol. Compared with fluorescence mediated CHA method and ICP-MS mediated basic sandwich method, the current strategy displayed the lowest LOD. The developed method was also applied to analyze miRNA-141 and miRNA-21 simultaneously to demonstrate its multiple detecting capability and practical applicability. Furthermore, this miRNA assay has the potential to become a high-throughput method by employing more distinct rare earth elements.

Phytotoxicity of plant extracts of Vismia japurensis cultivated in vivo and in vitro.

Plants that produce secondary metabolites with allelopathic activity or phytotoxicity can be biotechnologically important, serving as sources of allelochemicals, and thus contributing to the agroindustrial sector. Vismia japurensis (Hypericaceae) is an Amazonian species that grows in clumps called vismiais, from which most other plants are absent. Accordingly, the objective of this study was to identify possible phytotoxicity effects of hexane and methanol extracts of Vismia japurensis leaves and branches in vivo and from seedlings grown in vitro on Lactuca sativa. In addition, fresh and dry leaves were assayed by the sandwich method in order to determine their ability to release allelochemicals. The hexanic extract from in vitro seedlings reduced germination by 10%, while the methanol extract produced a 16% reduction in germination speed. Root growth of Lactuca sativa was inhibited by 64.7% when subjected to hexane leaf extract, by 39.3% under the influence of hexane branch extract, and by 96.09% for in vitro seedling hexanic extract. When analysed by thin layer chromatography and 1H nuclear magnetic resonance, extracts showed evidence of terpenes, anthraquinones and flavonoids, with greater intensity of signals in the aromatic region of in vitro seedling hexanic extract. Clearly, Vismia japurensis has a high biotechnological potential in terms of the production of substances of low polarity with capacity to interfere in plant development.

ELISpot Assay for the Detection of ASFV-Specific Interferon-Gamma (IFN-γ)-Producing Cells.

The enzyme-linked immunospot (ELISpot) assay is a technique of unparalleled sensitivity to determine the frequency of antigen-specific immune cells secreting an immunomodulatory mediator upon recall antigen stimulation, making it a valuable tool in vaccine research. Typically done in multi-well microplate format, it also allows a high-throughput analysis of numerous immune cell samples, e.g., from different donor subjects, especially with the help of automated plate readers and specialized software that currently exist in most laboratories. IFN-γ is a hallmark cytokine secreted especially by T-cell subsets in recall response to pathogens, and consequently the IFN-γ ELISpot assay is one of the most widely used. The cellular arm of the immune response is known to be fundamental in protection against virulent ASFV, and therefore this assay is frequently employed in ASFV vaccine research to evaluate the results from immunization experiments.The technique involves the use of plates with wells that have a membrane for base with a strong binding capacity for amino acids that thus can be densely coated with an antibody for IFN-γ. Upon adding cells and specific antigen or other control stimuli, responding cells will release IFN-γ that is captured by the antibody in close proximity and revealed using a second antibody (sandwich method) through either chromogenic or fluorescent methods, leading to the detection of a "spot" on the membrane for each positive cell. Here we detail our protocol to detect the frequency of ASFV antigen-specific IFN-γ-producing cells in immunized pig lymphocytes and give an example of a typical result using the technique.

Epidemiological Survey on Echinococcosis in Xinjiang Uygur Autonomous Region, People's Republic of China.

According to the latest data, the detection rate of echinococcus in Hoboksar Mongol Autonomous County was 3.7%. The objective of this study is to further investigate the epidemiology of echinococcosis in Ho-boksar Mongol Autonomous County of Xinjiang Uygur Autonomous Region, People's Republic of China and provide the scientific evidence for preventive and control measures. We performed ultrasound examination of 521 people in Hoboksar Mongol Autonomous County of Xinjiang Autonomous Region, and collected 508 serum samples, which were analyzed with enzyme-linked immunosorbent assay. Data were analyzed by t-test and multinomial logistic regression for risk factor analysis. We collected 126 pieces of herder's dog feces and used double antibody sandwich method to detect the positive rate of fecal antigen. The prevalence rate of human echinococcosis in this region was 4.4% (23/521), including 4.0% (21/521) for cystic echinococcosis (CE), 0.38% (2/521) for alveolar echinococcosis (AE). It was found that CE seropositivity was significantly different from gender, age, ethnic group, occupation, culture, area, income and awareness of this disease. The seroprevalence rate of people aged 41 - 65 (3.74%) was higher than of age 0 - 17 (0.197%) (p > 0.05); Female serological positive (4.921%) was higher than male (1.772%) (p > 0.005); Mongolian serological positive (5%) was higher than Han (0.197%) and Kazakhs (1.181%) (p > 0.05); The herdsmen serological positive (2.756% was higher than students (0.197%) (p > 0.05); The primary school students serological positive (2.559%) was higher than children before school 0% (p > 0.05); Chagankule serological positive (9.211%) was higher than Bayinaow (8.497%) (p > 0.05); The seroprevalence rate of people with income < 2,000 (3.74%) was higher than people with income over 5,000 (0.197%) (p > 0.05); The seroprevalence rate of people who had no disease awareness (4.724%) was higher than those who had awareness of Hydatid disease (1.969%) (p < 0.05). Multivariate logistic regression show age, ethic group and awareness of station are the influence factors of epidemiology of echinococcosis. Canine fecal antigen positive rate was 50% (p > 0.05). Narenhebuke (48.78%) was higher than chahet (20.00%), but there is no statistical difference (p > 0.05). The surveillance data and our study results tend to be consistent that echinococcosis has an increasing trend in Hoboksar Mongol Autonomous County. Efforts should be continued, in both animals and humans by increasing training campaigns and public awareness.

A study of Thyroid dysfunction in patients with chronic kidney disease undergoing maintenance haemodialysis among the patients of tertiary care hospital

Introduction: Thyroid hormones are necessary for the growth and development of the kidney and for the maintenance of water and electrolyte homeostasis. The kidney normally contributes to the clearance of iodine, primarily by glomerular filtration. Thus iodide excretion is diminished in advanced renal failure, leading sequentially to an elevated plasma inorganic iodide concentration and an initial increment in thyroidal iodide uptake. Materials and Methods: The present study was conducted in a tertiary care hospital in Belagavi, Karnataka State. 50 cases that were on regular maintenance hemodialysis treatment were selected and 50 controls were taken for study. Age & sex matched controls with normal renal function and no previous history of thyroid dysfunction was included in the study as controls. The quantitative determination of serum T3, T4 & TSH was done. The assay principle combines a one step enzyme immunoassay sandwich method with a final fluorescent detection (ELFA). Serum urea and creatinine was estimated by urease/glutamate dehydrogenase method and modified. Results: There was a significant difference between the control and study group with respect to serum TSH & T3 levels, serum T4 levels were found to be not statistically significant. The serum TSH level was increased in 8 patients (16%) among those with CKD; the mean serum TSH concentration was 5.49±11.03 in CKD patients which was significantly increased than in controls (3.27±2.06). Serum T3 concentration was less than the normal range in 10 of the 50 (20%) CKD patients, the mean serum total T3 concentration was 1.69±0.67 in CKD patients was significantly lower than that in the control subjects (2.03±1.2). Serum concentration of T4 was less in cases (81.34±23.87) than in controls (101.41±19.12) but the results were statistically not significant (P=0.083). Conclusion: The present study finds thyroid dysfunction to be very common in CKD patients and reveals the significant association between CKD progression and thyroid dysfunction and mean of T3, T4 decreases and TSH increases significantly in cases as compared to controls. The diagnosis of hypothyroidism can be easily missed in Haemodialysis patients. Timely diagnosis and treatment of hypothyroidism may prevent deterioration of the patient’s condition and prolong survival.