B细胞表位作图研究进展

抗原-抗体的特异性结合是由抗体表面的抗原决定簇与抗原表面的表位基序间的特异性互补识别决定的。B细胞表位作图既包括B细胞抗原表位基序的鉴定(即确定抗原分子上被B细胞表面受体或抗体特异性识别并结合的氨基酸基序),也包括绘制抗原蛋白的全部或接近全部的B细胞表位基序在其一级或高级结构上的分布图谱的过程。B细胞表位作图是研发表位疫苗、治疗性表位抗体药物和建立疾病免疫诊断方法的重要前提。目前,已经建立了多种B细胞表位鉴定或绘制抗原蛋白B细胞表位图谱的实验方法。基于抗原-单抗复合物晶体结构的X-射线晶体学分析的B细胞表位作图和基于抗原蛋白或抗原片段的突变体库筛选技术的B细胞表位作图可以在氨基酸水平,甚至原子水平上揭示抗原分子上与单抗特异性结合的关键基序;其它B细胞表位作图方法(如基于ELISA的肽库筛选技术)常常只能获得包含B细胞表位的抗原性肽段,因而,很少用于最小表位基序的鉴定;而改良的生物合成肽法多用于B细胞表位的最小基序鉴定和精细作图。鉴于每种B细胞作图方法都存在各自的优势与不足,B细胞表位作图往往需要多种作图方法的有机结合。本文对目前常用的B细胞表位作图的实验方法及其在动物疫病防控中的应用进行综述,以期为研究者设计最佳的表位作图方案提供参考。     

Structure and function of beta-blucosidases in Dictyostelium discoideum.

There are two isozymes of beta-glucosidase in developing cells of Dictyostelium discoideum. A procedure for screening large numbers of clones for beta-glucosidase activity was utilized to obtain mutations which directly affect the activity. We recovered seven strains which lack both isozymes and four strains with residual activity in which enzymatic and physical properties of both isozymes are altered. Beta-Glucosidase appears to act as a block to selfing in macrocyst formation as shown by the fact that ssite mating type to form macrocyst-like structures. Immunological evidence utilizing antisera prepared against purified beta-glucosidase-1 demonstrates that most of the glycosidases in Dictyostelium discoideum share a common antigenic determinant which appears to be added post-translationally. The two isozymes of beta-glucosidase share common protein subunits but the antigenic determinant is either lacking or masked in beta-glucosidase-2. This may account for some of the enzymatic and physical differences between the two isozymes.     

Quantification of the presence and activity of specific microorganisms in nature

Traditional techniques for assessment of microbial numbers and activity generally lack the specificity required for risk assessment following environmental release of genetically engineered microbial inocula. Immunological and molecular-based techniques, such as DNA probing and genetic tagging, were initially used to determine the presence or absence of microorganisms in environmental samples. Increasingly they are being developed for quantification of populations of specific organisms, either indigenous or introduced, in the environment. In addition, they are being used to quantify the activity of particular organisms or groups of organisms, greatly extending the range of techniques available to the microbial ecologist. This article reviews the use of traditional techniques for the quantification of microbial population size and activity and the application of molecular techniques, including DNA probing, genetic marking, use of fluorescent probes, and quantitative PCR, in combination with advanced cell detection techniques such as confocal laser scanning microscopy and flow cytometry.     

Conventional culture for water quality assessment: is there a future?

Conventional culture for the detection, enumeration and identification of micro-organisms has been the traditional tool of the microbiologist. It is, however, time-consuming and labour-intensive and confirmed results often require several days of analysis. Culture may not grow the organisms being sought and for enumeration may only detect a small proportion of the total population. However, it does have the advantage of being simple to use and relatively inexpensive. It is also a direct means of assessing cell viability. Novel fluorogenic dyes and fluorgenic and chromogenic substrates have overcome some of these problems by providing a means of rapid and specific detection and enumeration whilst removing the need for subculture and confirmation tests. Immunological tests such as ELISA have significantly reduced analysis time by providing specific target organism detection. Molecular techniques have removed the need for culture. Improvements in sensitivity, and removal of the inhibitory nature of sample matrices, have allowed analysts to detect low levels of micro-organisms but the questions of viability and comparability with cultural techniques still remain. Are we about to see a change of culture in water quality assessment, or can cultural techniques be developed that reduce analysis time to a few hours and can rapid methods be used for detecting the presence and viability of organisms?     

Detection ofAspergillus andPenicillium extracellular polysaccharides (EPS) by ELISA: using antibodies raised against acid hydrolysed EPS

Species of the fungal generaAspergillus andPenicillium produce immunologically active extracellular polysaccharides (EPS) in which galactofuranose residues are immunodominant. The antigenic determinant of the EPSA. fumigatus, A. niger andP. digitatum could be removed by acid hydrolysis. Due to the hydrolysis of the EPS the immunological reaction between IgG anti-native EPS and hydrolysed EPS disappeared. Antibodies raised in rabbits against the acid hydrolysed EPS revealed new antigenic determinants that were exposed as a result of the acid hydrolysis. Immunological inhibitory experiments showed that the antibodies were no longer directed to galactofuranose residues.Enzyme Linked Immunosorbent Assay, carried out with antibodies raised against the acid hydrolysed EPS showed that the antibodies against the acid hydrolysed EPS were more species specific in comparison with the antibodies against the native EPS.     

Dendritic cells: immunological sentinels with a central role in health and disease

Immunological effector cells must be sensitive to the antigens or environmental signals that indicate that a pathogen is present. To this end, a group of cells known as the professional antigen-presenting cells have the ability to educate T, B and NK cells as to the fingerprints of specific infections. The most adept of these cells are a closely related family termed dendritic cells (DC). A subset of these act as peripheral sentinels, specializing in the uptake, processing and presentation of antigenic material combined with an ability to detect a wide variety of 'danger' signals. These 'danger' or activation signals induce profound changes in dendritic cell physiology, facilitating the efficient stimulation of both adaptive and innate immunity. In the present review, a number of recent advances in the understanding of DC biology are discussed. These advances offer insights into the pathogenesis of a wide variety of diseases and point towards future strategies for immunotherapy.     

Molecular relationships of the human B cell alloantigens, MT2, MB3, MT4, and DR5

The human class II, HLA-linked, B cell alloantigens include the HLA-DR, MB, MT, and Te determinants. Interest in the molecular relationships of these antigens has recently intensified because of their homology to the murine Ia antigens and their possible importance in disease predisposition and transplantation. We have used alloantisera with carefully defined immunochemical as well as serologic specificity, and two immunochemical techniques, sequential immunoprecipitation with analysis by SDS-PAGE and two-dimensional gel electrophoresis, to explore the molecular relationships of the MT2, MB3, MT4, and HLA-DR5 antigenic determinants. The data presented here indicate that 1) all class II molecules that bear the DR5 antigenic determinant also bear the MT2 antigenic determinant; (2) the homozygous DR5 cell line, Swei, expresses at least two structurally distinct class II molecules, both of which bear MT2: one bears the MT2, MB3, and MT4 antigenic determinants, and the second bears the MT2, but not the MB3 or MT4 antigenic determinant; and (3) the DR5 determinant is located on at least one and possibly both of these distinct class II molecules.     

Characterization of Colicin Ia and Colicin Ib: Antigenic Homology

Immunological techniques have been employed to determine antigenic homology between colicin Ia and colicin Ib. Results of these experiments demonstrate that the two colicins are antigenically similar.     

Separation of two Salmonella O-antigen polysaccharides by means of a glutaraldehyde-concanavalin-A polymer. Primary studies on the separated fractions.

The Salmonella zuerich (1,9,12,(46),27) cell wall O-polysaccharides have been characterized as a mixture of two immunologically distinct fractions differing in the presence or absence of the antigenic determinant 1 usually carried by side chains of an alpha-D-glucosyl residue. In this paper, we describe the use of concanavalin A in separating the two O-polysaccharides. The procedure involves polymerization of concanavalin A with glutaraldehyde, followed by selective adsorption of the polysaccharide containing O factor 1 and elution with D-glucose. Immunological and chemical analyses showed that the separation was successful and demonstrated chemical differences between these two fractions in relation to their immunological behaviour.     

Absolute values of ras p21 defined by direct binding liquid competition radioimmunoassays

Several distinct and high-conserved genes comprise the ras gene family of rodents and humans, i.e., rodent Harvey and Kirsten, and human Harvey, Kirsten and neuroblastoma. Transformation, either by a point-mutation resulting in a change in one amino acid of the 21 kDa ras gene product (p21), or by increased expression of ras p21, has been demonstrated to be mediated by members of this gene family. We report here the development of direct binding liquid competition radioimmunoassays for the detection and quantitation of the ras oncogene and proto-oncogene products. Using these radioimmunoassays and ras p21 purified from Escherichia coli containing the full-length T24 mutant human Harvey ras gene protein product as a standard, we have defined the actual amount of ras p21 per micrograms of total cellular protein, or per cell, in various ras transformed and 'normal' mammalian cell lines. One of the radioimmunoassays developed is group-specific, since the antigenic determinant recognized is shared by both the point-mutated and proto-forms of Harvey, Kirsten and neuroblastoma members of the ras gene family, while the second may be termed type-selective, since it recognizes an antigenic determinant localized primarily on the Harvey ras p21. Both radioimmunoassays are interspecies, since they detect a ras p21 antigenic determinant common to cells of human and rodent origin. These studies thus describe the first means for defining absolute values of any oncogene or proto-oncogene protein product. The assays described, when used in combination with specific c-DNA probes to define specific ras proto-oncogenes or point-mutated oncogenes being expressed, will now permit truly quantitative analyses of ras p21 expression in experimental cell culture systems, animal models and human biopsy material.     

A new antiserum with conformational specificity for LHRH: usefulness for radioimmunoassay and immunocytochemistry

We have produced and characterized a new high titer, highly specific antiserum for luteinizing hormone-releasing hormone (LHRH), and demonstrated its usefulness for radioimmunoassay (RIA) and immunocytochemistry. The antiserum can be used at a final dilution of 1:500,000 to 1:600,000 for RIAs with a sensitivity of 0.2 pg/tube. Both the amino and carboxy terminal ends of the LHRH molecule are required for antibody recognition, and the antigenic determinant appears to be part of a three-dimensional structure of LHRH. Fragments of LHRH and other brain peptides are not recognized by the antiserum. Using immunocytochemical techniques, we have localized LHRH-containing neurons in the medial basal hypothalamus of the rhesus monkey, guinea pig, and rat. Staining of LHRH fibers and cell bodies was eliminated by preabsorbtion of the immune serum with synthetic LHRH. This antiserum should be useful in studies that require quantification of very low amounts of LHRH and in studies that require correlation between immunocytochemical localization and tissue content or secretion of LHRH.     

A simplified high speed multicolor immunoblotting method

We describe a simplified technique for the rapid and specific detection of antigenic determinants within a mixed population of antigens. Each determinant develops its own characteristic color in a single Western blot or dot blot. This multicolor immunostaining technique can be achieved with as little as a one-step incubation, involving a mixture of different primary and developing antibodies, and a one-step substrate reaction, involving a mixture of different substrates. The time required can be reduced to short periods of time, ranging from minutes to about 1 h. This can represent an increase in the speed of detection by one to two orders of magnitude when compared with conventional methods. The simplified protocols may facilitate the automation of routine analyses.     

Immunological relation between 14 S dynein and 30 S dynein from the cilia of Tetrahymena pyriformis.

The immunological relation between 14 S dynein and 30 S dynein obtained from Tetrahymena cilia was investigated by using antisera specific for each dynein subunit or some dynein subunits separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Although 14 and 30 S dynein main subunits have different electrophoretic mobilities, our immunodiffusion tests showed that there exists a close immunological relation between them. At least three immunologically different polypeptides designated polypeptides A, B and C are included in the 30 S dynein main band which has been recognized as a single component by electrophoresis, and that the polypeptides designated A',B' and C' are included in the 14 S dynein main bands. Polypeptides A and A',B and B', or C and C' appeared to have a certain common antigenic determinant(s). Polypeptide C of 30 S dynein was shown to possess a certain antigenic determinant(s) specific for 30 S dynein, besides the determinant common with that of polypeptide C' of 14S dynein. The second main component of 30 S dynein proved to be a specific polypeptide of 30 S dynein but not to be a degraded product of the main polypedtide. All antisera reacted with native dynein molecules to some extent, but did not inhibit dynein ATPase (ATP phosphohydrase, EC 3.6.1.3) activity significantly.     

Three-dimensional structure and antigen binding specificity of antibodies.

A number of specific Fab and Fv fragments and their complexes with antigens (avian lysozymes), haptens, and anti-idiotopic Fabs have been studied by immunochemical and crystallographic techniques. Antigen and antibody interact through closely complementary contacting surfaces, without major conformational changes. An idiotopic determinant of a monoclonal antibody is shown to include parts of most of its complementarity determining regions. The specificity of antigen recognition resides in the close complementarity of the antigenic determinant with the antibody combining site.     

Serological Characterization of Actinobacillus pleuropneumoniae Biotype 1 Strains Antigenically Related to both Serotypes 2 and 7

Nine Danish Actinobacillus pleuropneumoniae biotype 1 isolates were shown by latex agglutination and indirect haemagglutination to possess capsular polysaccharide epitopes identical to those of serotype 2 strain 1536 (reference strain of serotype 2) and strain 4226 (Danish serotype 2 strain). Immunodiffusion confirmed the antigenic relationship with serotype 2 and further demonstrated an antigenic relationship with strain WF83 (reference strain of serotype 7). SDS-PAGE with LPS from strains 1536, 4226, WF83 and strain 7317 (representative of the 9 isolates examined) showed that strains WF83 and 7317 had an identical smooth ladder pattern whereas LPS from strains 1536 and 4226 showed a distinctly different pattern. The antigenic similarities of the LPS of strains WF83 and 7317 were confirmed by immunoblots using rabbit or pig antisera prepared against the 3 strains. No antigenic similarities in the LPS of strains 1536 and 7317 were revealed. Since an antigenic determinant specific for the 9 isolates could not be demonstrated with the methods used, the strains are proposed to be designated K2:O7.     

The cellular location of a foreign B cell epitope expressed by recombinant bacteria determines its T cell-independent or T cell-dependent characteristics.

We have targeted two foreign B cell antigenic determinants to different locations in the Escherichia coli cell to examine what effect this had on antibody responses elicited by the recombinant bacteria. The two epitopes were the 132-145 peptide from the PreS2 region of hepatitis B virus and the C3 neutralization epitope of poliovirus type 1. They were each expressed in two forms either on the surface, as part of the outer-membrane protein LamB, or soluble in the periplasm, as part of the periplasmic protein MalE. When live bacteria expressing the foreign epitope at the cell surface were used for immunization of mice, they induced T cell-independent antibody responses characterized by a rapid induction of IgM and IgG antibodies. In contrast, when the same foreign epitope was inserted into the MalE protein, the antibody response was only detectable after 3 wk, belonged only to the IgG class and was strictly T cell dependent. This study has therefore identified two major pathways by which epitopes expressed by bacterial cells can stimulate specific antibody responses. The first pathway is mediated by direct activation of B cells by bacterial cell-surface Ag and does not require T cell help. The second pathway is T cell dependent and concerns Ag that can be released from the bacteria in a soluble form. We have also studied the effect of the exact position of the B cell antigenic determinant within the LamB protein and with respect to the outer membrane by comparing the immunogenicity of the PreS epitope inserted at three different permissive sites of LamB. The data indicated that to obtain an antibody response with intact bacteria, the epitope must be protruding sufficiently from the outside of the outer membrane. In contrast, when semipurified hybrid proteins were used as immunogen, the exact position of the B cell antigenic determinant within solubilized LamB protein does not influence its immunogenicity.     

Characterization of an antiserum to guinea pig antithrombin III (AT III). II. Stimulation of T lymphocyte proliferation

A goat antibody specific for an antigenic determinant shared between guinea pig antithrombin III (AT III) and thymocytes was shown to be mitogenic for lymph node T lymphocytes in the presence of macrophages. Although the antiserum was not mitogenic for purified populations of B lymphocytes, B lymphocytes were as efficient as T lymphocytes in absorbing the mitogenic activity of the serum. The shared antigenic determinant appeared to be carbohydrate in nature in that native and guanidine-treated AT III, but not periodate oxidized AT III, were capable of inhibiting the mitogenic activity of the serum when added continuously to the cultures. The possibility that the plasma protease inhibitor AT III or an antigenically related membrane protein are involved in the regulation of T cell activation is discussed.     

Serological detection and immunogold localization of cross-reactive antigens shared by Camellia sinensis and Exobasidium vexans

AIMS: Pathogenicity of Exobasidium vexans, causal agent of blister blight of tea, was studied in 30 commercially cultivated tea varieties by analysing the antigenic patterns of host and pathogen using immunological techniques. METHODS AND RESULTS: Whole plant inoculation of tea varieties with E. vexans showed that T-78 and T-17/1/54 were most susceptible and most resistant respectively. Antigen preparations from tea varieties, pathogen, nonpathogen (Fusarium oxysporum) and of nonhosts (Glycine max, Leucaena leucocephala and Oryza sativa) were compared by indirect enzyme-linked immunosorbent assay and dot-immunobinding assay using polyclonal antibodies raised against the pathogen, nonpathogen, susceptible and resistant tea varieties. Cross-reactive antigens (CRA) were found among susceptible varieties and E. vexans isolates but not in resistant varieties, nonhosts or nonpathogen. Indirect staining of antibodies using fluorescein isothiocyanate indicated CRA were concentrated mainly around epidermal and mesophyll cells in compatible host (T-78). This was substantiated by ultrastructural studies using gold-labelled antibodies through transmission electron microscopy which showed specific localization in the chloroplasts and host cytoplasm. CONCLUSION: Pathogenicity of E. vexans to different tea varieties is therefore related to the level of antigenic similarity between host and pathogen. SIGNIFICANCE AND IMPACT OF THE STUDY: Immunological methods proved to be valuable in screening commercially cultivated tea varieties against E. vexans.     

Expression of an onco-developmental antegen among avian species.

Rabbit antisera directed against an onco-developmental antigen on chicken red blood cells have been serologically dissected through specific adsorptions. It is now possible to detect 13 antigenic determinants with the fractionated antisera. The onco-developmental antigen referred to as chicken fetal-leukemic antigen (CFA) is fetal-specific in the white Leghorn chicken, being present on the embryonic but not adult peripheral red blood cells of non-being present on the embryonic but not adult peripheral red blood cells of non-leukemic birds. However, one or more of the onco-developmental antigenic determinants have been detected on adult peripheral red blood cells of non-Gallus avian species, as well as on red blood cells from two adult chicken varieties. For phylogenetic purposes, red blood cells from avian species were characterized for their combinations of CFA determinants. Comparisons among species revealed specific patterns of antigenic expression within phylogenetic groups. Several CFA determinants were restricted in their occurrence to species within a single family, and one determinant was found in all cases where CFA was expressed. The distribution of CFA determinants was used to determine immunological distances among four Galliform species. These distances agreed with the immunological relationships established using different serological markers.     

Antigenic Components of Group A Arbovirus Virions

Three group A arboviruses, Sindbis (SIN), western (WEE) and eastern equine encephalitis (EEE), were selectively degraded with a nonionic detergent to yield a core particle and a soluble envelope component. Antigenic analysis by using radioimmune precipitation techniques revealed marked antigenic similarity among the core particles of the three viruses. The soluble envelope component exhibited antigenic specificity similar to that of intact virions. A close relationship between SIN and WEE envelopes was shown, whereas EEE envelope antigen appeared antigenically specific. These data indicate that nucleocapsids of group A arboviruses contain an antigenic determinant common to the group; the envelope contains virus-specific antigens as well as antigens which relate members of a subgroup.