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1.
Lymphocyte subset estimations by flow cytometry in population-based studies require transportation of samples from the field site to the laboratory. As samples arrive late in the day they have to wait overnight before being processed. The effect of two possible approaches, sample storage for 24 h before staining and immediate staining with analysis after 24 h and 48 h were evaluated. Two sets of experiments were performed with EDTA (ethylenediamine tetra-acetate) anticoagulated peripheral blood. In the first experiment, after collection, each sample was divided into two portions. One portion was stained at the time of blood collection and the other 24 h later after keeping it at room temperature (38–45°C). In the second experiment, blood samples were stained within 1–2 h. Each sample was analyzed immediately upon completion of staining process and subsequently after 24 h and 48 h of storage at 4°C. Results suggest that blood collected in EDTA can be processed using whole blood lysis method, after storage at room temperature (38–45°C) for 24 h with some but not significant alteration in T-cell subsets. Storage at 4°C after staining for 24 h results in a lesser and insignificant loss of cells or alteration of T-cell subsets and may be the method of choice.  相似文献   

2.
利用抗HGH单克隆抗体建立了HGH的RIA和IRMA。方法学鉴定表明,这两种测定方法的灵敏度高(RIA为1.2ng/ml,IRMA为2ng/ml)、特异性强(与HPL及HPRL均无交叉反应)、重复性好。同时,还将这两种方法与HGH常规抗血清RIA进行了比较。  相似文献   

3.
Tumor necrosis factor-alpha (TNF-α) has been implicated in the pathogenesis of several non-infectious and infectious diseases including tuberculosis. In a prospective longitudinal study, TNF-α level in blood was estimated by sandwich ELISA using anti human TNF-α antibody, in 22 patients with active pleuro-pulmonary and lymphnode tuberculosis before and after chemotherapy and 8 healthy controls. Six patients and six controls had detectable levels (> 5 pg/ml) of TNF-α in blood. The mean TNF-α levels in controls and cases before and after treatment were 182.4pg/ml, 896.7 pg/ml and 678.7pg/ml pg/ml respectively. Though not statistically significant, there was a trend towards younger age, shorter duration of symptoms, presence of fever and anorexia, and high ESR, in patient with high serum TNF-α levels.  相似文献   

4.
A monoclonal antibody-based antigen detection system was used to detect the levels of circulating antigen in filarial patients before and after treatment with DEC and in normal individuals living in an area endemic forW. bancrofti infection in Chennai, India. The present study was to show the use of this assay as a means of efficient screening for filariásis in an endemic area where blood was absorbed onto the filter paper by finger prick during day time. The results of the antigen levels collected onto filter strips correlated with their corresponding plasma antigen levels (r=0.83). In microfilaraemics, DEC treatment did not alter the levels of circulating antigens upto a period of one month. We conclude that this monoclonal antibody based ELISA using filter strips may be used in daytime and can replace the existing routine night blood survey.  相似文献   

5.
In the present study, monoclonal gammapathy was identified in a total of 245 patients of plasma cell dyscrasias during period of 1987 to 2000. The monoclonal band was identified in serum by agar gel electrophoresis in all the cases and in urine in a few cases. Characterization of paraprotein (monoclonal immunoglobulin class and light chain type) was carried out by employing immunoelectrophoresis and/or immunofixation electrophoresis using heavy chain specific gamma, alpha, mu, delta and epsilon and light chain specific kappa (K), lambda (λ) antisera. Serum immunoglobulins Ig G, Ig A, and Ig M were estimated by immunoturbidometry. Serum urea, creatinine, uric acid, alkaline phosphatase, total proteins, albumin, calcium and phosphorus were estimated by using routine biochemical methods. Among the 245 cases, 73.1% monoclonal gammapathies were of secretory type and 7.3% were non-secretory. Monoclonal gammapathies were associated with 80.4% of multiple myeloma, 8.9% of solitary plasmacytoma, 4.1% of extra-medullary plasmacytoma, 3.3% of lymphoma and 2.9% of plasma cell leukemia. Classification of secretory monoclonal immunoglobulin revealed monoclonal immunoglobulin Ig G in 74%, Ig A 15% and Ig M in 2.9% cases.  相似文献   

6.
彭开丽  彭可茂  席利卿 《资源科学》2012,34(12):2224-2233
农地除了能够依靠其生产功能而具有经济产出价值外,在空气和水的净化、生物多样性的维持、景观的提供及社会保障与安全稳定等方面也发挥着重要的作用。但由于市场机制的自身缺陷,人们对农地价值缺乏充分认识,导致农地资源得不到应有的保护。本文在将农地功能划分为经济生产、社会保障和生态服务的基础上,以2009年为评估期,尝试构建柯布-道格拉斯生产函数(C-D函数)和用近似市场法对我国1994年-2009年30个省份农地资源的经济产出价值、社会保障价值和生态服务价值进行了动态估算。结果显示:无法通过市场价格表现的社会保障价值与生态服务价值均远远大于农地经济产出价值,农地的社会保障价值在农地总价值中占有相当大的比重,其比例呈现出逐年增加的趋势。因此,农地的社会保障价值和生态服务价值无疑是农地价值中不容忽视的重要组成部分,应当引起土地管理者和农地非农转用决策者的充分重视。  相似文献   

7.
Building on recent breakthroughs in the field of microfluidic-based capture of rare cancer cells circulating in the blood, the present article reports on the use of Herceptin functionalized PDMS devices designed to efficiently capture from blood cancer cells, overexpressing the tyrosine kinase human epidermal growth factor receptor (HER2). The identification of patients overexpressing HER2 is critical as it typically associates with an aggressive disease course in breast cancer and poor prognosis. Importantly, HER2 positive patients have been found to significantly benefit from Herceptin (Trastuzumab), a humanized monoclonal antibody (MAb) against HER2. Disposable PDMS devices prepared using standard soft lithography were functionalized by the plasma polymerization of an epoxy-containing monomer. The epoxy-rich thin film (AGEpp) thus created could be conjugated with Herceptin either directly or through a polyethylene glycol interlayer. The properties and reactivity toward the monoclonal antibody conjugation of these coatings were determined using x-ray photoelectron spectroscopy; direct conjugation provided a good compromise in reactivity and resistance to biologically nonspecific fouling and was selected. Using the breast cancer cell line SK-BR-3 as a model for cells overexpressing HER2, the immunocapture efficacy of the Herceptin functionalized PDMS was demonstrated in model studies. Validation studies confirmed the ability of the device to efficiently capture (～80% capture yield) HER2 positive cells from full blood.  相似文献   

8.
The introduction of ultra-sensitive labels for immunoassays has exposed some of the limitations inherent in them. Thus, for instance a major problem with acridinium esters used, as chemiluminescence label is the formation of the so called “pseudobases” at an alkaline pH, which if not suppressed can affect the rate of the chemiluminescence reaction. Chemiluminescence labels such as luminol can also be problematic when attached to antibodies and small molecules to the extent that the sensitivity of the assay can be reduced by the decrease in the intensity of chemiluminescence. The increased use of molecular methods such as polymerase chain reaction (PCR) and post PCR methods to study mutations have various pittalls, which if unrecognized and uncontrolled can lead to incorrect results and misinterpretation. Patients who are exposed to mouse immunoglobulins through imaging or therapeutic techniques can develop antibodies to mouse immunoglobulins (human anti-mouse antibodies or HAMA) which can be a major problem for non optimized immunoassays using murine monoclonal antibodies. The types of therapy such as anticoagulant used in anticoagulant therapy, blood substitute and drug therapy can impact on the measurements of some of the biochemical and other analytes. One must separate the transient, physiological effects introduced by therapy from long-term biochemical alterations due to disease.  相似文献   

9.
The attempt is made to produce recombinant factor VIII but the first step in producing such product is production and purification of rabbit’s polyclonal antibody against factor VIII. The second and third steps involve monoclonal antibody and recombinant factor VIII production. Factor VIII is one of the most important coagulating factor where its deficiency leads to diseases like hemophilia type A or classic. It is an inherited disease. Previously, it was obtained through fractionation of blood plasma of blood donors. After processing, factor VIII could be used to manage such patients. Due to transfer of viral disease like hepatitis and HIV through factor VIII obtained by fractionation, high cost of production, insufficiency of the donors and the process of virus removal, thus production of factor VIII through recombinant technology can be useful and helpful. The reaction between antibody and antigen is one the most specific reaction; therefore, such reaction can be employed to identify factor VIII. Thereby, rabbits were injected several times with adjuvant-linked antigen to produce antibody. The antibody was separated from the blood sample, purified and used to identify factor VIII in the research.  相似文献   

10.
The incidence of autoimmune disorders that includes the connective tissue diseases has seen a rise in India in recent times. Antinuclear antibodies, the telltale sign of systemic autoimmune response, thus can be used as a screening tool and also to support the diagnosis of systemic autoimmune disease. The present retrospective cross- sectional analysis aimed to study the antinuclear antibodies profile (patterns and specific antibody reactivity) amongst suspected cases of auto-immune disorders at a tertiary care teaching hospital. The study retrieved and reviewed reports of 644 patients sent for ANA testing by indirect immunofluorescence assay over a period of 1 year by different specialty departments. Positive samples were further processed for anti-ds-DNA antibody and antibodies to extractable nuclear antigen. Data collected was statistically analysed. ANA pattern positivity was observed in 31% of cases and a positive antibody reactivity was seen in 66% of them. Female predominance (82%) was noted in both pattern positivity and antibody reactivity. High levels of pattern positivity and antibody reactivity was found in the young adults (45.9%). Amongst the ANA patterns, the nuclear homogenous pattern was found the commonest. The common antibodies associated with this pattern were anti-dsDNA and U1 Sm/RNP antibodies. A stronger fluorescence intensity on initial screening showed a higher confirmation rate for specific antibodies on immunoassay. High occurrence of positive ANA patterns in autoimmune disorders suggests its utilization as a screening tool for them and would also play an adjuvant to the diagnosis. Early knowledge about future autoimmunity will earn better prognostic achievements through better treatment interventions.  相似文献   

11.
Inflammatory bowel disease (IBD) is a heterogeneous group of chronic inflammatory disorders of the gastrointestinal tract with two main distinguishable entities, Crohn’s disease (CD) and ulcerative colitis (UC). IBD-unclassified (IBD-U) is a diagnosis that covers the “grey” zone of diagnostic uncertainty between UC and CD. Current diagnosis of IBD relies on the clinical, endoscopic, radiological, histological and biochemical features, but this approach has shortcomings especially in cases of overlapping symptoms of CD and UC. The need for a diagnostic tool that would improve the conventional methods in IBD diagnosis directed the search towards potential immunological markers, since an aberrant immune response against microbial or endogenous antigens in a genetically susceptible host seems to be implicated in IBD pathogenesis. The spectrum of antibodies to different microbial antigens and autoantibodies associated with IBD is rapidly expanding. Most of these antibodies are associated with CD like anti-glycan antibodies: anti-Saccharomices cerevisiae (ASCA) and the recently described anti-laminaribioside (ALCA), anti-chitobioside (ACCA), anti-mannobioside (AMCA), anti-laminarin (anti-L) and anti-chitin (anti-C) antibodies; in addition to other antibodies that target microbial antigens: anti-outer membrane porin C (anti-OmpC), anti-Cbir1 flagellin and anti-I2 antibody. Also, autoantibodies targeting the exocrine pancreas (PAB) were shown to be highly specific for CD. In contrast, UC has been associated with anti-neutrophil cytoplasmic autoantibodies (pANCA) and antibodies against goblet cells (GAB). Current evidence suggests that serologic panels of multiple antibodies are useful in differential diagnosis of CD versus UC and can be a valuable aid in stratifying patients according to disease phenotype and risk of complications.  相似文献   

12.
Nebulizers have considerable advantages over conventional inhalers for pulmonary drug administration, particularly because they do not require coordinated breath actuation to generate and deliver the aerosols. Nevertheless, besides being less amenable to miniaturization and hence portability, some nebulizers are prone to denature macromolecular drugs due to the large forces generated during aerosolization. Here, we demonstrate a novel portable acoustomicrofluidic device capable of nebulizing epidermal growth factor receptor (EGFR) monoclonal antibodies into a fine aerosol mist with a mass median aerodynamic diameter of approximately 1.1 μm, optimal for deep lung deposition via inhalation. The nebulized monoclonal antibodies were tested for their stability, immunoactivity, and pharmacological properties, which confirmed that nebulization did not cause significant degradation of the antibody. In particular, flow cytometry demonstrated that the antigen binding capability of the antibody is retained and able to reduce phosphorylation in cells overexpressing the EGFR, indicating that the aerosols generated by the device were loaded with stable and active monoclonal antibodies. The delivery of antibodies via inhalation, particularly for the treatment of lung cancer, is thus expected to enhance the efficacy of this protein therapeutic by increasing the local concentration where they are needed.  相似文献   

13.
Determination of blood glucose levels is very important to know the physiological condition of the human beings as the hormonal imbalance may cause abnormalities in glucose metabolism. The traditional methods of glucose estimation by colorimetric and titrimetric methods were involved with huge expenditure and time. The modified colorimetric microwell reader method proposed in the present study was performed with small quantities of sample and reagents with the same linearity that was observed in the normal colorimetric analysis. The modified method not only reduces the cost of the test to almost one third of the normal colorimetric method but also provide an opportunity to screen the large number of samples in a short duration of time.  相似文献   

14.
Monocytes represent a class of immune cells that play a key role in the innate and adaptive immune response against infections. One mechanism employed by monocytes for sensing foreign antigens is via toll-like receptors (TLRs)—transmembrane proteins that distinguish classes of foreign pathogens, for example, bacteria (TLR4, 5, and 9) vs. fungi (TLR2) vs. viruses (TLR3, 7, and 8). Binding of antigens activates a signaling cascade through TLR receptors that culminate in secretion of inflammatory cytokines. Detection of these cytokines can provide valuable clinical data for drug developers and disease investigations, but this usually requires a large sample volume and can be technically inefficient with traditional techniques such as flow cytometry, enzyme-linked immunosorbent assay, or luminex. This paper describes an approach whereby antibody arrays for capturing cells and secreted cytokines are encapsulated within a microfluidic device that can be reconfigured to operate in serial or parallel mode. In serial mode, the device represents one long channel that may be perfused with a small volume of minimally processed blood. Once monocytes are captured onto antibody spots imprinted into the floor of the device, the straight channel is reconfigured to form nine individually perfusable chambers. To prove this concept, the microfluidic platform was used to capture monocytes from minimally processed human blood in serial mode and then to stimulate monocytes with different TLR agonists in parallel mode. Three cytokines, tumor necrosis factor-α, interleukin (IL)-6, and IL-10, were detected using anti-cytokine antibody arrays integrated into each of the six chambers. We foresee further use of this device in applications such as pediatric immunology or drug/vaccine testing where it is important to balance small sample volume with the need for high information content.  相似文献   

15.
Immunoassay is one of the important applications of microfluidic chips and many methodologies were reported for decreasing sample∕reagent volume, shortening assay time, and so on. Micro-enzyme-linked immunosorbent assay (micro-ELISA) is our method that utilizes packed microbeads in the microfluidic channel and the immunoreactions are induced on the beads surface. Due to the large surface-to-volume ratio and small analytical volume, excellent performances have been verified in assay time and sample∕reagent volume. In order to realize the micro-ELISA, one of the important processes is the immobilization of antibody on the beads surface. Previously, the immobilization process was performed in a macroscale tube by physisorption of antibody, and long time (2 h) and large amount of antibody (or high concentration) were required for the immobilization. In addition, the processes including the reaction and washing were laborious, and changing the analyte was not easy. In this research, we integrated the immobilization process into a microfluidic chip by applying the avidin-biotin surface chemistry. The integration enabled very fast (1 min) immobilization with very small amount of precious antibody consumption (100 ng) for one assay. Because the laborious immobilization process can be automatically performed on the microfluidic chip, ELISA method became very easy. On-demand immunoassay was also possible just by changing the antibodies without using large amount of precious antibodies. Finally, the analytical performance was investigated by measuring C-reactive protein and good performance (limit of detection <20 ng∕ml) was verified.  相似文献   

16.
Serum thyroglobulin (Tg) and thyroid stimulating hormone (TSH) measurements have evolved as important analytes for monitoring the prognosis of patients with differentiated thyroid cancer, post-thyroidectomy. Individual analyte immunoassay is the current practice in clinical pathology, but the simultaneous assay for all relevant analytes for a given disease, can reduce assay costs, improve patient compliance and give the clinician more information for an unequivocal diagnosis. Microarray immunoassay (MI) can achieve this goal and, hence, we have developed and validated a immuno-radiometric MI for quantitation of serum TSH and Tg by using highly micro-porous polycarbonate (PC) track-etched membranes (TEM) to immobilize the monoclonal anti-TSH and polyclonal anti-Tg antibodies in ~1 mm diameter spots. Non-competitive immunoassays were performed using mixture of 125I labeled monoclonal anti-TSH and anti-Tg antibodies. Phosphorimager was used to quantify the bound radioactivity. TSH and Tg were detected with detection limit of 0.07 µIU/ml and 0.13 ng/ml respectively, which is lower than the clinically required cut-off level. The assay showed: acceptable intra-assay precision within 20 % and recovery in the range of 76–111.2 %. MI compared well with the established immunoradiometric assay (IRMA) with r = 0.98, p < 0.01 (n = 41). No cross-reactivity was seen between the immobilized antibodies. Although two hormones are addressed in this report, MI using PC TEM and isotopic/non-isotopic tracers has the potential for highly automated multiplexed analysis.  相似文献   

17.
Detection of lgE and lgG antibodies in Aspergillosis is of diagnostic significance. The serological methods, such as agglutination, gel diffusion and counter immuno electrophoresis that are commonly used in the laboratories for diagnosis of Aspergillus induced infections, are less sensitive and high cross reactivity is often encountered. We carried out work on characterization and identification of diagnostically relevant antigens ofA. fumigatus. Well characterized antigens were used to develop an ELISA with 92% sensitivity and 89% specificity for detection of specific lgE and lgG in the sera of patients of allergic bronchopulmonary aspergillosis (ABPA), aspergilloma and invasive aspergillosis. Subsequently, a sample kit having “ready to use type” of dry reagents (powder/tableted buffers and lyophilized antigen, conjugate and reference sera) was formulated. The kit was validated with sera from patients of ABPA, related allergic disorders, tuberculosis, post-Kochs cases and thalassemic children receiving repeated blood transfusions. The performance of the kit was found to be satisfactory with coded sera.  相似文献   

18.
The seamless integration of reagents into microfluidic devices can serve to significantly reduce assay complexity and cost for disposable diagnostics. In this work, the integration of multiplexed reagents into thermoplastic 2D microwell arrays is demonstrated using a scalable pin spotting technique. Using a simple and low-cost narrow-bore capillary spotting pin, high resolution deposition of concentrated reagents within the arrays of enclosed nanoliter-scale wells is achieved. The pin spotting method is further employed to encapsulate the deposited reagents with a chemically modified wax layer that serves to prevent disruption of the dried assay components during sample introduction through a shared microchannel, while also enabling temperature-controlled release after sample filling is complete. This approach supports the arbitrary patterning and release of different reagents within individual wells without crosstalk for multiplexed analyses. The performance of the in-well spotting technique is characterized using on-chip rolling circle amplification to evaluate its potential for nucleic acid-based diagnostics.  相似文献   

19.
In this paper, we present a low cost and equipment-free blood filtration device capable of producing plasma from blood samples with mL-scale capacity and demonstrate its clinical application for hepatitis B diagnosis. We report the results of in-field testing of the device with 0.8–1 ml of undiluted, anticoagulated human whole blood samples from patients at the National Hospital for Tropical Diseases in Hanoi, Vietnam. Blood cell counts demonstrate that the device is capable of filtering out 99.9% of red and 96.9% of white blood cells, and the plasma collected from the device contains lower red blood cell counts than plasma obtained from a centrifuge. Biochemistry and immunology testing establish the suitability of the device as a sample preparation unit for testing alanine transaminase (ALT), aspartate transaminase (AST), urea, hepatitis B “e” antigen (HBeAg), hepatitis B “e” antibody (HBe Ab), and hepatitis B surface antibody (HBs Ab). The device provides a simple and practical front-end sample processing method for point-of-care microfluidic diagnostics, enabling sufficient volumes for multiplexed downstream tests.  相似文献   

20.
We developed an automated laser induced fluorescence system utilizing microfluidic chips for detection and quantification of immunoglobulins. Microchips were fabricated from polydimethysiloxane (PDMS) using the so-called "prepolymerization technique." The microchip structure helped minimize the effects of PDMS autofluorescence and light scattering. Furthermore, a thin and uniform PDMS layer forming the top of the microchip enabled proper focusing and collection of the excitation beam and the emitted fluorescence, respectively. The developed system was tested for the detection of mouse immunoglobulins. The capturing antibodies were immobilized on internal microchannel walls in the form of a polyelectrolyte. We clearly show that this immobilization technique, if correctly realized, gives results with high reproducibility. After sample incubation and washing, secondary antibodies labeled by fluorescein isothiocyanate were introduced into microchannels to build a detectable complex. We show that mouse antibodies can be quantified in a wide concentration range, 0.01-100 μg ml(-1). The lower detection limit was below 0.001 μg ml(-1) (6.7 pM). The developed laser induced fluorescence (LIF) apparatus is relatively cheap and easy to construct. The total cost of the developed LIF detector is lower than a typical price of plate readers. If compared to classical ELISA (enzyme linked immunosorbent assay) plate systems, the detection of immunoglobulins or other proteins in the developed PDMS microfluidic device brings other important benefits such as reduced time demands (10 min incubation) and low reagent consumption (less than 1 μl). The cost of the developed PDMS chips is comparable with the price of commercial ELISA plates. The main troubleshooting related to the apparatus development is also discussed in order to help potential constructors.  相似文献   

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