Isolation and characterization of the immunoglobulin-binding protein from Yersinia pseudotuberculosis

A high molecular weight immunoglobulin-binding protein localized on the surface of bacterial cells has been isolated from the protein fraction of the outer membrane of Yersinia pseudotuberculosis, and its properties are described. The immunoglobulin-binding protein is a trypsin-resistant and temperature-sensitive -structured protein. As shown by MALDI-TOF mass spectrometry, after heating at 100°C the molecular weight of the protein constituted 37.5 kD. The native protein is capable of interacting with human and rabbit IgG but looses the ability to bind the immunoglobulins after the temperature denaturation. The immunoglobulin-binding protein binds to the Fc-fragments of the immunoglobulins and binding depends on the presence of calcium ions.     

Does a monospecific hybridoma always secrete homogeneous immunoglobulins?

The fusion of splenocytes (from mice immunized with the beta 2 subunit of E. coli tryptophan synthase) with myeloma cells which do not produce immunoglobulins gave rise to a clone secreting immunoglobulins with two distinct isotypes : gamma 1 and gamma 2b (Djavadi-Ohaniance et al. (1984) Biochemistry, 23, 97-104). Analysis of the immunoglobulins secreted by this clone indicates that these two isotypes are carried by two distinct heavy chains which are able to randomly associate to form hybrid molecules. In addition, two classes of light chains are able to randomly and to form heterologous associations with both the gamma 1 and gamma 2b heavy chains. Only the association between the gamma 2b heavy chains with one of the two classes of light chains leads to a combining site specific for the binding of the antigen beta 2.     

激活补体试验检测静注人免疫球蛋白Fc段生物学活性的优化

优化激活补体试验的条件,完善静注人免疫球蛋白Fc段生物学活性的检测方法。方法采用补体活化经典途径的原理,分别探讨致敏红细胞密度和贮存时间对人免疫球蛋白Fc段生物学活性检测结果的影响;比较手工法和微孔板分光光度法检测Fc段生物学活性的检测结果。结果在致敏红细胞A541 nm=1.3和致敏红细胞贮存5 d时,检测结果较稳定。微孔板分光光度法检测优于手工法。结论完善了人免疫球蛋白Fc段生物学活性的检测方法,检测结果准确、重复性好。     

Leporid immunoglobulin G shows evidence of strong selective pressure on the hinge and CH3 domains

Immunoglobulin G (IgG) is the predominant serum immunoglobulin and has the longest serum half-life of all the antibody classes. The European rabbit IgG has been of significant importance in immunological research, and is therefore well characterized. However, the IgG of other leporids has been disregarded. To evaluate the evolution of this gene in leporids, we sequenced the complete IGHG for six other genera: Bunolagus, Brachylagus, Lepus, Pentalagus, Romerolagus and Sylvilagus. The newly sequenced leporid IGHG gene has an organization and structure similar to that of the European rabbit IgG. A gradient in leporid IgG constant domain diversity was observed, with the CH1 being the most conserved and the CH3 the most variable domain. Positive selection was found to be acting on all constant domains, but with a greater incidence in the CH3 domain, where a cluster of three positively selected sites was identified. In the hinge region, only three polymorphic positions were observed. The same hinge length was observed for all leporids. Unlike the variation observed for the European rabbit, all 11 Lepus species studied share exactly the same hinge motif, suggesting its maintenance as a result of an advantageous structure or conformation.     

Trypanosoma cruzi: immunoglobulin isotype profiles during the acute phase of canine experimental infection with metacyclic or blood trypomastigotes

A detailed investigation has been carried out about the serological profiles of groups of dogs experimentally infected with metacyclic (MT) or blood (BT) trypomastigotes of Berenice-78 Trypanosoma cruzi strain. Peripheral blood was collected from infected dogs and uninfected controls, weekly during 35 days following the acute phase of infection, and immunoglobulin profiles were determined by ELISA. Dogs infected with BT exhibited unaltered levels of IgG2, increases in IgM, IgE, IgA, IgG and IgG1. In contrast, dogs infected with MT presented unaltered levels of IgE and IgG1 and an increase in IgM, IgA, IgG and IgG2 levels. Compared with the MT group, animals infected with BT showed significant increases in IgM on days 7, 14 and 28, in IgA on days 7, 14 and 21, in IgE on days 7 and 14, in IgG on days 14 and 28, and in IgG1 on days 7, 14 and 21. Parasitemia levels of the infected animals were measured over the same time period. No correlations were found between the immunoglobulin profiles and the parasitemia levels. The results demonstrated that the inoculum source (BT or MT) influence the immunoglobulin isotype profile that may drive distinct outcome of acute canine Chagas disease.     

Determination of protease activity in blood and microorganisms

A protease activity may be determined by means of immunoglobulins. Since proteolytic products apparently do not retain antigenic determinants of the initial substrate, the monitoring of enzymatic process may employ ELISA methods. The ELISA determination of functional activity of specific IgA1 protease has been used not only for detection of this enzyme, but also for measurement of its inhibition constants. IgG adsorbed onto a microplate was used for evaluation of total proteolytic activity. Varying pH values of the reaction medium it is possible to measure activity of neutral, alkaline and acid proteases. This approach was used for estimation total proteolytic activity of neutral proteases in blood serum. Due to high sensitivity of this method it was possible to dilute serum up to the level when serum inhibitors had not blocked enzyme activity. Assay of serum enzyme activity at acidic pH results in activation of pepsinogens and determination of pepsin activity. Measurement of a total level of serum pepsinogen activity may have diagnostic importance in gastroenterology, due to decisive contribution of pepsinogen I to the detectable activity.     

Removal and inactivation of human immunodeficiency virus (HIV-1) by cold ethanol fractionation and pasteurization during the manufacturing of albumin and immunoglobulins from human plasma

Viral safety is a prerequisite for manufacturing clinical albumin and immunoglobulins from human plasma pools. This study was designed to evaluate the efficacy of cold ethanol fractionation and pasteurization (60°C heat treatment for 10 h) for the removal inactivation of human immunodeficiency virus type 1 (HIV-1) during the manufacturing of albumin and immunoglobulins. Samples from the relevant stages of the production process were spiked with HIV-1, and the amount of virus in each fraction was quantified by the 50% tissue culture infectious dose (TCID₅₀). Both fraction IV fractionation and pasteurization steps during albumin processing were robust and effective in inactivating HIV-1, titers of which were reduced from an initial 8.5 log₁₀ TCID₅₀ to undetectable levels. The log reduction factors achieved were ≥4.5 and ≥6.5, respectively. In addition, fraction III fractionation and pasteurization during immunoglobulins processing were robust and effective in eliminating HIV-1. HIV-1 titers were reduced from an initial 7.3 log₁₀ TCID₅₀ to undetectable levels. The log reduction factors achieved in this case were ≥4.9 and ≥5.3, respectively. These results indicate that the process investigated for the production of albumin and immunoglobulins have sufficient HIV-1 reducing capacity to achieve a high margin of safety.     

免疫球蛋白糖链结构异常和自身免疫性疾病

免疫球蛋白在人体体液免疫中发挥巨大作用,而其均为糖蛋白.免疫球蛋白中与蛋白质相连的寡糖链结构及组成对其功能有很大影响,当寡糖链糖基化异常时,可导致一些自身免疫性疾病.从IgA肾病和类风湿性关节炎的结构基础、分子机制、酶学基础、临床意义等方面对这两类自身免疫性疾病的发病机制与糖基化异常之间的密切相关性予以详细论述,为这两类疾病在临床上建立一种特异、灵敏的血清学检测方法提供了理论基础,开辟了一条新途径.