Enzyme-linked immunosorbent assay for secalonic acid D

A competitive enzyme-linked immunosorbent assay was developed for the xanthone dimer secalonic acid D. The immunogen and enzyme marker were prepared by direct reaction of secalonic acid D with bovine serum albumin and horseradish peroxidase, respectively. The resultant conjugates were characterized by UV/VIS spectra and thin layer chromatography. The hapten:protein ratios in the conjugates were estimated by difference UV/VIS spectra and by fluorescent techniques. Immunization procedures were conducted utilizing New Zealand rabbits over a period of 12 weeks. The competitive enzyme-linked immunosorbent assay on microtiter plates showed that secalonic acid D was detectable within a range of 250–25 000 ng/assay.     

苄非他明人工抗原合成

目的: 通过化学方法对苄非他明进行结构修饰并保留抗原决定簇,将结构改造后的产物与载体偶联合成苄非他明抗原。方法: 苄非他明经化学修饰后,增加活性基团连接上一类可用的经化学修饰的连接臂,使用碳二亚胺法与载体蛋白偶联成苄非他明人工合成抗原。该抗原通过紫外吸收光光谱扫描技术、SDS-PAGE电泳法及胶体金免疫层析法进行偶联效果和抗原活性的鉴定。结果: 苄非他明半抗原结构与载体偶联成功,该抗原具有较高的纯度和活性,与苄非他明抗体反应表现出较高的特异性。结论: 该方法合成的苄非他明抗原可用于免疫检测方法,也可作免疫原制备相关抗体。     

金霉素单克隆抗体的制备及检测方法的建立

采用羰基二咪唑法,将半抗原金霉素(AM)分别与牛血清白蛋白(BSA)和卵清蛋白(OVA)偶联制备金霉素免疫抗原AM-BSA和检测抗原AM-OVA,通过紫外光谱扫描检测偶联产物。采用细胞杂交瘤技术,制备抗金霉素单克隆抗体杂交瘤细胞株,建立了金霉素竞争ELISA检测方法,其灵敏度达到50ng/ml,且呈现良好的线性关系(r=0.9812),并且与其他抗生素无交叉反应。     

Antigen recognition in delayed-type hypersensitivity.

It is still uncertain if cell-mediated immune reactions are more or less specific than antibody-mediated reactions. Accordingly, hapten and carrier specificity were examined in delayed hypersensitivity in guinea pigs. Hapten specificity was demonstrated with 2,4-dinitrophenyl (DNP)-guinea pig albumin (GPA), 2,6-DNP-GPA, 2,4,6-trinitrophenyl (TNP)-GPA, and dansyl (DNS)-GPA. Guinea pigs immunized with each of these conjugates were tested 7 days later with the immunogen and the other conjugates. Strong delayed skin responses were highly specific for the immunogen; there were some weak cross-reactions among the nitrophenyl conjugates, no crossre-actions between the DNS and nitrophenyl conjugates, and no responses to unconjugated GPA. Conjugates carrying different numbers (1–45) of 2,4-DNP groups per molecule were all able to elicit specific responses to 2,4-DNP.Carrier specificity in delayed hypersensitivity was confirmed by immunizing with 2,4-DNP-GPA, and challenging with the immunogen, with 2,4-DNP coupled to bovine albumin (BSA), rabbit IgG, ovalbumin, and hemocyanin. Strong responses were seen to the immunogen, a weak response to 2,4-DNP-BSA, and no response to the other conjugates. Specific immune recognition of both hapten and carrier determinants is therefore required for expression of delayed hypersensitivity. These cell-mediated reactions thus appear to be more specific than those of antibody-mediated reactions in solution.     

沙丁胺醇人工抗原的合成及抗体制备

沙丁胺醇是一种β-兴奋剂,常被很多畜禽水产养殖户非法用于动物养殖。为建立沙丁胺醇在食品中残留的快速检测方法,研究了沙丁胺醇免疫原的合成和抗体的制备方法。采用对氨基苯甲酸法合成了沙丁胺醇（SAL）免疫原SAL-cBSA,采用重氮化法合成的克伦特罗（CL）偶合物CL-cOVA作为包被抗原,用紫外光谱法分析了所合成免疫原和包被抗原。用免疫原SAL-cBSA免疫新西兰大白兔获得多克隆抗体,抗体效价达到32000。采用间接ELISA法检测抗体IC50值为8.79ng/ml,SAL的浓度在1ng/ml~100ng/ml区间时,SAL与对抗体的竞争结合力呈直线关系。表明所制备的沙丁胺醇免疫原具有良好的免疫原性,所制备的抗体拥有很高的灵敏度。     

Isotype-specific resistance against tolerance induction in SJL mice

Age-related changes in antibody response of SJL mice were examined in terms of isotype expression after treatment with immunogen or with immunogen, preceded by the molecule in normally tolerogenic form. We report here that tolerance induction and resistance to down regulation are isotype specific. Tolerance can be induced in terms of all detectable isotypes at the age of 5 weeks. In older SJL mice, tolerance to the carrier is found in IgM antibody, whereas there is resistance against down regulation in terms of IgG2a and IgG2b isotypes, and sensitization in terms of IgG3, IgG1, and IgA antibody. Furthermore, the degree of down regulation is determinant dependent. This was observed when older SJL mice, pretreated with the carrier in a normally tolerogenic form, were immunized with haptenated carrier and tested for their response to hapten and carrier determinants. In this case, IgA antibody shows tolerance to the hapten and sensitization by carrier determinants.     

Role of antigen and,antibody in the regulation of the immune response

The enzyme dextranase could degrade antigenic dextran in vivo even when given 6-15 d after the antigen. Dextranase injected after the antigen suppressed the immune response when given 24 but not 48 h after the antigen, indicating that the antigen must interact with the immune system for 48 h to initiate a response. Thereafter, the B cells are independent of further antigen stimulation. To show whether antibody-mediated suppression of the immune response was determinant specific FITC-conjugated SRC were applied as immunogen and antibodies were raised both against the carrier (SRC) and the FITC hapten. When these antibodies were injected 1-3 h after the immunogen they only suppressed the immune response to the corresponding determinant. Anti-carrier antibodies usually enhanced the response to the hapten. Therefore, antibody-mediated suppression of the immune response is determinant-specific and cannot be mediated in vivo to a detectable extent by the Fc part of the antibodies.     

紫杉醇免疫检测方法的研究进展

紫杉醇是一种有效的抗肿瘤药物,广泛应用于治疗卵巢癌、乳腺癌和肺癌等癌症。紫杉醇在紫杉树皮中的含量极低（仅为0．01％）,而且紫杉醇是一种对蛋白质有着高亲和力的小分子,在体液中约有98％的分子与蛋白质结合,因此需要一种高灵敏度、高通量的检测方法对紫杉醇进行鉴定。在分析紫杉醇检测方法的基础之上,综述了紫杉醇免疫学检测方法的研究进展,包括紫杉醇半抗原的分子修饰、蛋白偶联物的构建和鉴定以及免疫学检测方法在植物组织和病人血浆中紫杉醇定性和定量中的应用。     

Homogeneous enzyme immunoassay of diosgenin and its glycosides

Homogeneous enzyme immunoassay has been used as a tool for the determination of diosgenin and its glycosides in plants. Diosgenin antisera was found to inhibit the activity of diosgenin hemisuccinate-horseradish peroxidase conjugate which was reversed by the addition of free diosgenin or its glycosides. The increase of enzyme activity was proportional to the quantity of the hapten over a certain range of hapten concentration. Thus, a minimum of 2.5 micrograms/ml of diosgenin and 11.5 micrograms/ml of diosgenin glycosides could be determined by this method. The results were comparable with those obtained by high-performance liquid chromatography and gravimetric methods.     

Photoreversible antigen-antibody reactions

A monoclonal antibody (Z1H01) for an oligopeptide carrying an azobenzene group, was prepared under conditions where the azobenzene group is in the trans form. The antibody bound the hapten peptide effectively when the hapten peptide is in the trans form (K = 5 x 10(7) M-1), but the antibody released the hapten under irradiation with UV light where the hapten is in the cis form. The antibody bound the hapten again, when the hapten reverted to the trans form after irradiation with visible light.     

Immunization of breast cancer patients using a synthetic sialyl-Tn glycoconjugate plus Detox adjuvant

We have synthesized various formulations that have potential for active specific immunotherapy (ASI) of human cancers. Sialyl-Tn (STn) is a potentially important target structure for ASI because its expression on mucins is a strong, independent predictor of poor prognosis, suggesting that it may have functional significance in the metastatic process. In this first pilot study of synthetic sialyl-Tn hapten conjugated to keyhole limpet hemocyanin (STn-KLH), with Detox. adjuvant, toxicity and humoral immunogenicity were assessed in 12 patients with metastatic breast cancer. Toxicity was minimal, restricted to local cutaneous reactions (apart from transient nausea and vomiting following single low-dose cyclophosphamide treatment). Using STn-conjugated human serum albumin in a solid-phase enzyme-linked immunosorbent assay, it was shown that all patients developed IgM and IgG specific for the synthetic STn hapten. Following immunization, most patients were shown to develop increased titres of complement-mediated cytotoxic antibodies, partially inhibited by synthetic STn hapten, but not by the related TF hapten. We also detected IgM and IgG antibodies reactive with natural STn determinants expressed on ovine submaxillary mucin, the STn specificity of this reactivity being confirmed by hapten inhibition. Evaluation of clinical efficacy in a small pilot study is difficult. Five patients are alive 12 or more months after entry, and another 4 patients are alive 6 or more months after entry into the study. All 3 patients with known widespread bulky disease progressed despite ASI, 2 having died from widespread cancer. Two patients had partial responses, each lasting 6 months. While several patients had disease stability for 3–10 months, 1 patient with pulmonary metastases remains stable 15 months after entry into the program.     

Reaginic antibody formation in the mouse. X. Possible role of suppressor T cells in transient IgE antibody responses.

The kinetics of IgE antibody response to alum-absorbed dinitrophenyl derivatives of ovalbumin (DNP-OA) was dependent on the dose of immunogen. A persistent IgE antibody response was obtained when high responder BDF1 mice were immunized with a minimum (0.05 microgram) dose. An increase of the immunogen to 10 microgram depressed IgE antibody responses but enhanced IgG antibody responses of both hapten and carrier specificities. Determination of T helper cell activity and B memory cells after immunization with different doses of antigen indicated that minimum immunogen was favorable for developing helper activity, whereas 1 to 10 microgram immunogen were more favorable than a 0.05-microgram dose for developing both IgE and IgG B memory cells. Nevertheless, neither helper T cells nor B memory cells in the spleen explains a transient IgE antibody response to a high (10 microgram) dose of DNP-OA. Evidence was obtained that immunization with 10 microgram OA induced generation of antigen-specific suppressor T cells, which were not detectable after immunization with 0.05 microgram OA. Transfer of suppressor T cells to DNP-OA-primed mice depressed both anti-hapten and anti-carrier IgE antibody responses. The results suggested strongly that suppressor T cells are involved in a transient IgE antibody response to a high-dose immunogen.     

Synthesis and application of organomercury haptens for enzyme-linked immunoassay of inorganic and organic mercury

Two organomercury haptens were synthesized via the classical oxymercuration reaction. An intramolecular oxymercuration reaction was the strategy employed to prepare a structurally simple, but chemically robust, organomercury hapten that was conjugated to chicken immunoglobulin G (IgG). The resulting immunogen afforded mouse anti-mercury antibodies that were evaluated in an enzyme-linked immunosorbent assay (ELISA). Antibodies demonstrating high titers were obtained, and various immunoassay parameters were investigated. The sensitivity and selectivity of the resulting antibodies were evaluated by exploring different cross-coupling chemistries and solid-phase synthetic variations. A second hapten was prepared with the intermolecular oxymercuration reaction, and the resulting compound, once coupled to carrier protein, afforded a solid-phase conjugate that revealed the versatility of the mouse anti-mercury antibody. The anti-mercury antibody developed in this study was capable of detecting both mercury(II) salts and organomercury compounds.     

Potentiation of antibody responses by specific IgM: specificity and thymus dependency

Modulation of antibody responses induced by IgM directed against the immunogen was investigated. When IgM directed against ox erythrocytes (ORBC) was given together with trinitrophenyl (TNP)-ORBC, the subsequent antibody response to the carrier, ORBC, as well as the response to the hapten, TNP, was potentiated. In contrast, IgG with carrier specificity inhibited both responses. The hapten-specific potentiation was found in both direct and indirect plaques, and was antigen-dose dependent, i.e., no potentiation was found with the lowest antigen doses. The response to 2,4-dinitrophenyl (DNP)-labeled proteins was potentiated by a monoclonal IgM with specificity for the hapten. The effects were observed both in primary and secondary responses. One strict requirement for IgM potentiation to occur was observed. The determinant against which potentiation was achieved had to be physically linked to the determinant against which the IgM was directed, be it hapten or carrier determinants. Thus, irrelevant IgM-antigen complexes were incapable of potentiating the responses. Similar specificity requirements were found for IgG induced suppression of antibody responses. Experiments with nude mice and their euthymic littermates showed that IgM potentiation of antibody production is T-cell dependent. Furthermore, passive transfer of carrier-primed spleen cells together with antigen challenge suggests that IgM potentiation of secondary antibody responses is dependent on specific carrier-primed immune T cells.     

Preparation and characterization of antibodies against 6-O-alpha-D-xylopyranosyl-beta-D-glucopyranose (beta-isoprimeverose), the disaccharide unit of xyloglucan in plant cell-walls

The p-aminophenyl beta-glycoside of 6-O-alpha-D-xylopyranosyl-D-glucopyranose (isoprimeverose), the disaccharide unit of plant xyloglucan, was coupled to bovine serum albumin, and the resulting glycoconjugate was used as an immunogen for the immunization of a rabbit. The immunochemical specificities of the rabbit antiserum raised against the glycoconjugate were characterized by immunodiffusion, quantitative precipitation, and hapten inhibition. After removal of anti-bovine serum albumin antibodies, the antiserum exhibited a specificity for the introduced disaccharide unit of the artificial antigen. The antibody-combining site was also shown to recognize the aglycon portion of the introduced hapten. The antiserum interacted with some xyloglucans, such as those from tamarind seed and the cell wall of pea stem. beta-Isoprimeverose and alpha-D-xylopyranosides were good inhibitors of the xyloglucan-antibody precipitation system, indicating that the antibodies recognize the beta-isoprimeverose unit of the xyloglucan.     

Use of polystyrene-supported 2-isobutoxy-1-isobutoxycarbonyl-1,2-dihydroquinoline for the preparation of a hapten–protein conjugate for antibody development

Polystyrene-supported 2-isobutoxy-1-isobutoxycarbonyl-1,2-dihydroquinoline (PS-IIDQ), a polymer-supported covalent coupling reagent, was successfully employed for the first time in the bioconjugation of an example hapten (phytanic acid derivative) to a carrier protein (bovine serum albumin (BSA)) within the context of immunogen preparation for antibody development. The ability of the prepared example phytanic acid derivative–BSA conjugate to bind an anti-phytanic acid antibody was confirmed using an enzyme-linked immunosorbent assay (ELISA).     

铅螯合物人工抗原的制备与鉴定

以双功能螯合剂异硫氰酸苄基乙二胺四乙酸(ITCBE)螯合铅离子,制备得半抗原Pb-ITCBE,然后再分别与载体蛋白KLH或BSA偶联制备得免疫原Pb-ITCBE-KLH与包被抗原Pb-ITCBE-BSA,ITCBE-BSA.用二喹啉甲酸法测3种抗原的浓度,分析半抗原、抗原与载体蛋白的紫外吸收光谱,利用SDS-PAGE对3种抗原的分子量进行鉴定,用三硝基苯磺酸法检测3种抗原中的赖氨酸残基的ε-NH2被半抗原替换的程度,用石墨炉原子分光吸收法检测抗原中铅的含量.研究结果表明,免疫原与包被抗原制备成功,Pb-ITCBE-KLH、Pb-ITCBE-BSA、ITCBE-BSA的浓度依次为6.47± 0.08 mg/ml,6.68± 0.06 mg/ml,5.57± 0.05 mg/ml;抗原与载体蛋白的紫外吸收光谱的特征各不相同;SDS-PAGE的结果显示3种抗原的分子量均不同于各自的载体蛋白;抗原中载体蛋白ε-氨基的替换程度依次为1.86± 0.74 %、55.53± 1.13%、54.19± 1.34%;铅的含量依次为15.64± 0.11 μg/ml,17.33± 0.15 μg/ml,0 μg/ml.     

Comparative circular dichroism studies of an anti-fluorescein monoclonal antibody (Mab 4-4-20) and its derivatives.

This study presents circular dichroism (CD) spectra of a high-affinity monoclonal anti-fluorescein antibody (Mab 4-4-20), its Fab fragments, and corresponding single-chain antibody (SCA). In the region 200-250 nm, the differences in the CD spectra between these proteins reflect the uneven distribution of chromophores (tryptophan and tyrosine) rather than a major conformational change. On the basis of near-UV CD spectra, binding of the hapten fluorescein to these protein antibodies elicits an increased asymmetry in the microenvironment of the chromophoric residues in contact with the hapten and also perturbs the interface between VL and VH domains. The hapten-binding site provides a chiral microenvironment for fluorescein that elicits a pronounced induced fluorescein CD spectrum in both the visible and UV regions. In contrast to the parent molecules, SCA is thermolabile. Our results demonstrate that (1) UV CD spectra are useful for assessing the chromophoric microenvironment in the binding portion of antibodies and (2) the extrinsic fluorescein hapten CD spectra provide information about the interaction of hapten with the binding pocket.     

Tolerance susceptibility of newly generating memory B cells

Newly generating memory B cells rapidly accumulate somatic mutations that can alter their Ag-combining sites and potentially engender recognition of self determinants. To investigate the possibility that, during their emergence secondary B cells pass through a window of tolerance susceptibility, we have examined the in vitro generation of memory B cells in the presence or absence of tolerogen. The findings indicate that, before antigenic stimulation, precursors to memory B cells are resistant to tolerance induction. However, 2 to 7 days after T cell-dependent antigenic stimulation, newly emerging hapten-specific secondary B cells can be inactivated by the presence of hapten on a carrier not recognized by available Th cells. This inactivation can be blocked by the presence of free hapten and can be competed by the presence of immunogen. Inactivation of newly generating secondary B cells appears less specific than the tolerance induction of immature neonatal or bone marrow B cells because inactivation can be accomplished by cross-reactive determinants. Interestingly, the presence of tolerogen after primary stimulation did not preclude the generation of cells responsive to a third in vitro stimulation. Therefore, whereas newly emerging memory B cells are highly susceptible to inactivation, the progression of the clones of progenitors to memory B cells appears resistant to tolerance induction.     

Comparison of immune response potentiation and in vivo inflammatory effects of Freund's and RIBI adjuvants in mice.

Freund's adjuvant and the RIBI adjuvant system were compared for their immune potentiating and toxic effects. Each adjuvant was administered with benzo(a)pyrene (BaP), a nonimmunogenic hapten, conjugated to a bovine gamma globulin (BGG) carrier protein to 10 mice intraperitoneally. Complete Freund's adjuvant was used at initial immunization, while incomplete Freund's was used for booster immunizations. Five mice were given the immunogen conjugate (BaP-BGG) in saline as a control. Antibody titers were determined by ELISA to both hapten and carrier after each of the two booster immunizations. Titers to BaP were 2- and 27-fold higher for RIBI than for Freund's after each of two booster immunizations. Titers to bGG were 119 and 12-fold higher for RIBI compared with Freund's. Titers to both immunogens were markedly less when administered in saline. Body weights were monitored in all three groups for the duration of the study. No differences were observed among the three groups. Mice from each group were euthanized at regular intervals to assess pathology. Splenic weight:body weight ratios were determined at the time of necropsy, and no differences were noted among the three groups. Granulomatous inflammatory lesions were most severe in the Freund's immunized mice, less severe in those immunized with RIBI, and least with saline. Results indicate that the RIBI system was more effective in potentiating an immune response and elicited less tissue reaction than did Freund's adjuvant with this particular immunogen.