Development of an immunochromatographic strip for detection of antibodies against porcine reproductive and respiratory syndrome virus

A simple and rapid immunochromatographic test strip incorporating a colloidal gold-labeled recombinant Nsp7 antigen probe was successfully developed for the detection of anti-porcine reproductive and respiratory syndrome virus (PRRSV) antibodies in swine. Recombinant Nsp7 protein of PRRSV labeled with colloidal gold was dispensed on a conjugate pad for use as the detector. Staphylococcal protein A and purified porcine anti-Nsp7 antibodies were blotted on a nitrocellulose membrane to form test and control lines, respectively. A comparison of the strip with standard diagnostic tests, enzyme-linked immunosorbent assays and immunoperoxidase monolayer assay, was also performed. The immunochromatographic test strip was shown to be of high specificity and sensitivity. Furthermore, the strip assay is rapid and easy to perform with no requirement for professional-level skills or equipment. It is suggested that the immunochromatographic test strip can be used to quickly and accurately detect PRRSV antibody and to be suitable for diagnostic purposes in the field.

Morphological and Immunohistochemical Identification of Villous M Cells in the Small Intestine of Newborn Piglets / Identificación Morfológica e Inmunohistoquímica de los Epiteliocitos Microplegados de las Vellosidades en el Intestino Delgado de Lechones Recién Nacidos

Microfold (M) cells act as antigen-sampling sites for initiating antigen specific mucosal immune responses, but they may also provide a gateway for enteropathogen entry. In this study we demonstrated villous M cells by morphological and immunohistochemical methods to be present in the three regions of the small intestine from newborn piglets. Immunohistochemical analysis, using anti- cytokeratin 18 (CK18) primary antibodies, showed a gradually decreased density of M cells from the lower crypt epithelium to the upper villous epithelium. The proportion of villous M cells was greater in the ileum than in the duodenum or the mid-jejunum. Ultrastructural observation revealed that villous M cells were mainly columnar in shape in the duodenum and the mid-jejunum, and appeared as more pocket-like structure in the ileum. These villous M cells exhibited short and irregular microvilli, rich vesicles and reduced glycocalyx, but lacked the lymphocyte-containing basolateral invagination. Our results support evidence that M cells can develop in the small intestinal villous epithelium of newborn piglets, implying that villous M cells may begin developing in the pig's small intestine during fetal stages, which depends neither on the influence of the mucosal lymphoid tissue nor the antigen from the intestinal lumen stimulation. In addition, the variable morphology and heterogeneity distribution of villous M cells in the three regions of the small intestine may be indicative of its different functional properties. This information extent our understanding of the diversity of M cells and provides important basic knowledge for further research on the actual role of villous M cells in neonate.

Los epiteliocitos microplegados (células M) actúan como receptores de antígeno para iniciar la respuesta inmune específica de las mucosas, pero también pueden proporcionar una puerta de entrada para enteropatógenos. En este estudio, se demostró por métodos morfológicos e inmunohistoquímicos que los epiteliocitos microplegados de las vellosidades están presentes en las tres regiones del intestino delgado de lechones recién nacidos. Se utilizaron anticuerpos primarios de citoqueratina 18 (CK18) para el análisis inmunohistoquímico, el cual mostró una disminución gradual de la densidad de los epiteliocitos microplegados desde el epitelio de las criptas inferiores hasta el epitelio de las vellosidades superiores. La proporción de los epiteliocitos microplegados, fue mayor en el íleon que el duodeno o yeyuno medio. La observación ultraestructural reveló que los epiteliocitos microplegados fueron principalmente de forma columnar en el duodeno y el yeyuno medio. Además, mostraron microvellosidades cortas e irregulares, muchas vesículas y glucocáliz reducidos, pero carecían de invaginaciones basolaterales contenedoras de linfocitos. Nuestros resultados apoyan la evidencia de que los epiteliocitos microplegados pueden desarrollarse en el epitelio de las vellosidades intestinales de los lechones recién nacidos, lo que implica que estas células pueden comenzar a desarrollarse en el intestino delgado del cerdo durante las etapas fetales, y no dependen ni de la influencia del tejido linfoide de las mucosas ni del antígeno para la estimulación del lumen intestinal. Además, la morfología y heterogeneidad de distribución de los epiteliocitos microplegados en las tres regiones del intestino delgado pueden ser indicativas de sus diferentes propiedades funcionales. Esta información mejora nuestra comprensión de la diversidad de los epiteliocitos microplegados y proporciona conocimientos básicos importantes para la investigación sobre el papel de los epiteliocitos microplegados en las vellosidades del neonato.

Recovery of IgG binding capability of human FcγRⅡa refolded by rapid dilution expressed in E. coli / 中华微生物学和免疫学杂志

Objective To study the effect of soluble, refolded, recombinant extracellular domain of the human Fc gamma receptor Ⅱ a (huFcγRⅡa) on the binding of human IgG to cells. Methods Extra-cellular domain of the huFcγRⅡ a gene was amplified from recombinant plasmid pe3huR Ⅱ by PCR and then cloned into pET-28a vector. The recombinant plasmid pETshuR Ⅱ was transformed into E. coli BL21 (DE3) after identified by PCR and doubly digested. The inclusion bodies of fusion protein were extracted and purified by washing, dissolved in 6 mol/L guanidine buffer, and refolded by rapid dilution technique. The refolding protein activity was tested by ELISA and flow cytometry. Results Extraceilular domain of the huFcγRⅡa gene was successfully cloned into pET-28a. The results of SDS-PAGE showed that the molecular mass (Mr) of the expressed protein was 24.8 × 103, and the expression rate was 30%. The purity of recom-binant shuR Ⅱ was up to 90% after washing. ELISA showed that the recombinant shuR Ⅱ was able to bind human IgG in a dose dependent manner, shuRⅡ could competitively inhibit the binding of human IgG to huFcγRⅡa expressed on the surface of COS-7 cells by flow cytometry. Conclusion The results demon-strate that it is possible to obtain large quantities of recombinant shuR Ⅱ which has comparable binding prep-erties to those of the whole membrane bound huFcγR Ⅱ a.