抗大肠杆菌curli菌毛CsgA蛋白特异性抗体的制备及鉴定

目的 为进一步研究curli菌毛主要蛋白csgA的功能,利用MBP-CsgA融合蛋白免疫兔子制备抗体.方法 用PCR方法扩增大肠杆菌MHCA4100株的csgA基因,在大肠杆菌中表达MBP-CsgA融合蛋白,免疫家兔以制备抗血清,用Western blotting和免疫电镜鉴定抗MBP-CsgA融合蛋白抗体的特异性.结果 PCR扩增出约476 bp的esgA基因,在大肠杆菌XL1-Blue 中成功地表达了Mr约为58 000的MBP-CsgAm融合蛋白,Western blotting和免疫电镜检测证明,针对MBP-CsgA融合蛋白的抗血清,不仅可特异性地识别MBP-CsgA,也可识别源于大肠杆菌的esgA蛋白,抗血清的最高滴度达1:50 000.结论 亚克隆csgA 基因,成功表达MBP-CsgA融合蛋白,制备并获得其特异性抗curli菌毛抗体.     

Preparation of specific antisera to bradykinin and their investigation by ELISA

Research Institute of Medical Enzymology, Academy of Medical Sciences of the USSR, Moscow. Crimean Medical Institute, Simferopol'. (Presented by Academician of the Academy of Medical Sciences of the USSR, I. P. Ashmarin.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 107, No. 6, pp. 727–729, June, 1989.     

Preparation of antisera against the S antigen of influenza A virus by immunization of guinea pigs with internal S antigen

Summary Internal S-antigens obtained by ether destruction of different influenza A viruses were administered intraperitoneally to guinea pigs at three day intervals. Three to five doses evoked S-antibodies in the course of the second and third week after the beginning of immunization; at that time the sera were free of V-antibodies. In a minor proportion of animals (19 out of 94) V-antibodies were detected later, when S-antibodies were starting to decrease. The preliminary evidence obtained indicated that the development of V-antibodies in these sera was independant of the level of S-antibodies as well as of the number of S-antigen doses administered. The S-antisera prepared in the way described were found to be potent enough to be used in the fluorescent antibody technique.     

Specific antisera produced by immunization with precipitin lines

Individual plasma proteins were precipitated, identified and isolated on the basis of line immunoelectrophoresis. A monospecific antibody response was induced by immunization of rabbits with less than 50 ng of precipitated antigen. Preservation of monospecificity was obtained by reimmunization with precipitates developed against the specific antisera.     

Characterization of monoclonal antibodies against prostate specific antigen produced by genetic immunization

Prostate specific antigen (PSA) is the most important marker for prostate cancer. Antibodies against minor variants of PSA may be useful in the development of novel diagnostic tests for prostate cancer, but it has been difficult to produce such antibodies by protein immunization. In this study, we have compared the characteristics of monoclonal antibodies (MAbs) obtained by genetic immunization with those obtained by protein immunization. The whole coding region of PSA-cDNA was cloned in a mammalian expression vector pCDNA-3. Six mice were immunized four times by intra-muscular (i.m.) injection of the PSA-pCDNA3 plasmid. The MAbs produced were characterized with respect to subclass, epitope specificity, binding to various molecular forms of PSA and affinity. After intra-muscular injection of DNA, anti-PSA antibodies were detected in the serum of all mice, but the antibody titers were markedly lower than after protein immunization. After fusion of the spleen cells from the mice, five hybridomas producing MAbs to PSA were obtained. The MAbs were of IgG1 and IgG2a isotype and they all recognized equally different forms of free PSA, namely enzymatically active, nicked and proPSA. Epitope mapping showed that these MAbs reacted with the same antigenic regions as those obtained by protein immunization. Thus, genetic immunization leads to production of anti PSA MAbs with similar characteristics to those obtained by immunizing with PSA protein. As applied in the present study, it is less efficient than protein immunization, but it is a useful technique when the antigen is not available in the quantities needed for immunization.     

双烯雌酚人工抗原的合成及特异性抗体的制备

目的制备抗双烯雌酚(DIEN)特异性抗体,为进一步研制DIEN免疫检测试剂盒打基础。方法采用4-溴丁酸乙酯对DIEN进行活化,以活泼酯法与BSA偶联制备免疫原(DIEN-CP-BSA);经紫外扫描和飞行时间质谱扫描鉴定偶联情况;以DIEN-CP-BSA免疫Balb/c小鼠制备特异性抗体,间接(竞争)ELISA评价抗血清效价及特异性。结果本试验获得较高效价的DIEN抗血清,其效价达1∶64 000,双烯雌酚半抑制浓度(IC50)为62 ng/ml;抗血清与结构类似物己烷雌酚的交叉反应率仅为0.34%,与己烯雌酚和17-β雌二醇无交叉反应;利用该抗体建立的间接竞争ELISA检测法,双烯雌酚在10～300 ng/ml呈线性关系。结论本研究制备了抗双烯雌酚(DIEN)特异性抗体,为研究畜产品中DIEN残留及开发DIEN免疫检测试剂盒奠定了基础。     

Preparation of specific antisera to the opacity factors of group-A streptococci

Type-specific antibody to the opacity factor (OF) of group-A streptococci can be demonstrated in human sera but the multiplicity of antibodies to different serotypes limits their usefulness in anti-OF typing. The antibody response in rabbits is inconsistent; only 61 of 138 (44%) of rabbit anti-M sera tested contained OF antibody. Of these only about half had titres of greater than 16, and usable sera to only 11 of 23 OF-positive serotypes were obtained. On the other hand good anti-OF sera (titres greater than 16) to 27 of 28 serotypes resulted from the interperitoneal and subcutaneous inoculation of heat-killed whole-cell vaccines in guinea-pigs and the frequency of response in groups of animals given injection of the same vaccine was 100% for all but three serotypes. Antibody response was not obtained with M-type 13. A comparison of routes of inoculation for M-type 25 showed that the subcutaneous route alone could probably be used in the routine production of anti-OF typing sera. Use of the set of 27 sera in OF-inhibition tests confirmed the remarkable specificity of OF antigens and their parallelism with M-antigen specificity, with the exception fo a reciprocal cross reaction between M-type 61 and provisional type PT3875.     

Generation of high titer antisera in rabbits by DNA immunization

Mesothelin is a GPI-linked, membrane-associated differentiation antigen that is over-expressed in several forms of human cancers. Intradermal injection into rabbits of plasmid DNA encoding full length mesothelin resulted in antisera with titers as high as 1:100,000. Each immunization consisted of 320 microg of DNA delivered into 4 sites. After the initial three injections antisera titers were moderate (between 10 to 30,000) and fell over the course of about 7 weeks. When the titers had fallen, an injection of a booster dose of DNA resulted in very high titers of antisera. These antisera contained IgGs that could bind to both recombinant mesothelin made in Eschericha coli and to mesothelin present on human cells in Western blots and in immunofluorescence assays. These observations indicate that simple intradermal DNA immunization of rabbits can result in high titers of antibodies that can be used for a variety of purposes.     

Production of effective luteinizing hormone antisera by two immunization methods

This paper addresses the production of effective luteinizing hormone antisera by two different immunization methods; the traditional and modified methods. The main difference between these two methods is in the immunization procedure. In the modified method, an additional injection of emulsion with complete Freund's adjuvant and only one booster are applied at the third and 28th day from the first injection, respectively. The results of the study indicated the possibility of producing the antiseria using the modified method in short time and better quality than those produced by the traditional methods. The details of both methods are presented together with the results obtained from the antibodies detection. Comparison between these two methods is also presented along with discussions.     

Improvement of antisera raised against complex antigen mixtures by the use of heterologous sources of antigen for immunization

Polyspecific antisera against antigen mixtures are important tools for many experimental purposes. However, following immunization with extracts containing a great number of different proteins, many antigens remain non-immunogenic. Therefore, the improvement of the antibody spectrum of antisera is an essential goal in maximising the power and applicability of many serological analyses. In the present report we demonstrate that the additional use of heterologous (i.e., antigenically related, but not identical) antigen mixtures for immunization increases the variety of antibodies in polyspecific antisera as well as the antibody titer against weak immunogens.     

Cascade immunization: a method of obtaining polyspecific antisera against crude fractions of antigens

Between 20 and 30 precipitation lines are usually obtained by crossed immune electrophoresis of an Escherichia coli cytoplasmic extract against antisera produced against that extract in individual rabbits. With a combination of several such antisera, the number of precipitation lines increases to 30-40. Nevertheless, extracts as used in this work contain many antigens in addition to those thus detected. Intermolecular immune competition may be avoided by removing strong immunogens from the extracts. The remaining antigens which give no immune response in the primary immunization are used for further immunization. New antibody production against 8-14 additional antigens occurs after one such 'cascade immunization' step. Separation of strong from weak immunogens is performed by preparative CIE and the use of immuno-affinity columns. The procedure is called cascade immunization because it involves repeated removal of antigens and production of further antisera directed against antigens in the remainder.     

Detection of porcine endogenous retrovirus (PERV) using highly specific antisera against Gag and Env

Porcine endogenous retroviruses (PERV) are considered an obstacle to the safe use of cells, tissues, and organs from pigs in the course of xenotransplantation. Thus, the detection of viral proteins and of a potential PERV infection is of major interest. Recently, we have published the generation of a highly specific antiserum directed against the nucleocapsid (p10) of PERV (Xenotransplantation 7 (2000), 221). Here we present new peptide-antisera specific to the capsid protein (p30) and the surface molecule of PERV class B (SU, gp70(B)) as well as the transmembrane moiety of the envelope protein (TM, p15E) of PERV which showed functionality in several immunological assays, such as immunoblots, immunofluorescence, and immunogold staining. Thus, these antisera can be used as tools for the identification of viral proteins in basic research as well as clinical trials.     

Preparation of immune sera against carcinoembryonic antigen (CEA).

CEA antigen obtained by the Wroc?aw team and CEA standard antigen from Chester Beatty Cancer Research Institute, London were used for immunization of goats. Two immunization methods were adopted: one with decreasing antigen doses beginning from 500 microgram and the other with equal, small doses of 10 microgram. Anti-goat IgG1 + IgG2 immune serum was obtained by immunizing a horse with 10 mg of the antigen with complete Freund's adjuvant. Anti-CEA immune sera obtained after immunization with small doses were highly active; in the RIA test they showed half maximum CEA binding at 1:40,000 and 1:80,000 dilutions. Likewise, the anti IgG1 and IgG2 serum from the horse proved to be highly active.     

Preparation of typing antisera specific for O antigens of Pseudomonas aeruginosa.

Results of serotyping 966 clinical isolates of Pseudomonas aeruginosa showed that 72% agglutinated specifically in one or another of the 16 typing antisera, but 28% agglutinated in two or more and often in as many as 10 antisera; this polyagglutinability correlated with a high incidence of cross-reactivity among the antisera. Absorption of each typing antiserum with either cell suspensions of five O-type strains or with a suspension of a particular polyagglutinable strain (SMC 247) abolished cross-reactivity in the typing antisera without significantly reducing titers against the homologous strains. All but four of the polyagglutinable strains agglutinated specifically in one or another absorbed antisera. The cross-reactions of unabsorbed antisera were interpreted to have been caused by antibodies directed not against specific O antigens but against thermostable specificities that remain undefined.     

Histamine release from human lung by specific antisera

Several specific antisera have been tested for ability to release histamine from samples of washed, normal human lung in vitro. Goat anti-rat serum, sheep anti-rabbit serum, goat anti-human IgA and goat anti-human IgM were inactive at the dilutions tested. Goat anti-human IgG and Coomb's reagent appeared to have some histamine releasing activity. Sheep anti-human IgE was the most active of the antisera tested. The significance of these results is discussed.     

A new specificity, SH7 (Bw75)*, defined by antisera produced by planned immunization

SH7 specificity was defined by a group of antisera developed in Shanghai, China by planned immunization. SH7 antibody is cross-reactive with B5 and B15, as demonstrated by the serological reaction patterns obtained during planned immunization. Using 9th and 10th International Histocompatibility Workshop sera, we were able to confirm SH7, TS-1 and BISKemp as identical specificities. They are equivalent to BW62S and B15Short-Thai. In view of the cross-reaction patterns obtained during planned immunization, SH7 appears to belong to the B5, B15, B35 and Bw70 cross-reactive groups.