The use of immunodeficient male (CBA/N x BALB/c) F1 mice to produce monoclonal antibodies directed to proteins of Leptospira interrogans rather than to immunodominant lipopolysaccharides.

A method, using an immunodeficient mouse strain, for the production of monoclonal antibodies directed exclusively against the proteins in an antigen mixture also containing immunodominant LPS, is described. Male (CBA/N x BALB/c) F1 mice were immunized with an outer envelope antigen mixture from Leptospira interrogans strain Wijnberg containing both lipopolysaccharides and proteins. The immune response in these mice was shown to be predominantly directed against protein antigens. Hybridoma cell lines were generated by fusing spleen cells from a (CBA/N x BALB/c) F1 mouse with BALB/c Sp2/0 plasmacytoma cells. Hybridoma cell lines producing monoclonal antibodies reacting with the outer envelope preparation were identified by ELISA. All epitopes recognized by the monoclonal antibodies are sensitive to proteinase K degradation and resistant to oxidation by periodate indicating that they are located on proteins. All epitopes are located on a 35 kDa protein and specific for the pathogenic L. interrogans species.     

The use of maleimidocaproyloxysuccinimide to prepare malarial peptide carrier immunogens. Immunogenicity of the linking region

Malarial peptides synthesized with an added N terminal cysteine were conjugated to purified diphtheria toxoid (DT) protein using the bifunctional reagent maleimidocaproyloxysuccinimide (MCS). The molar ratio of peptide to carrier was determined by subtractive sulphydryl titration and confirmed by sodium dodecyl sulphate (SDS) electrophoresis. For enzyme-linked immunoabsorbent (ELISA) analysis of sera from animals immunized with the DT conjugates, peptides were conjugated to bovine serum albumin (BSA) using MCS as well as a glutaraldehyde based coupling procedure. Western blotting analysis shows that both DT and BSA adducts were recognized by monoclonal antibodies (Mabs) directed against the peptide epitopes in the native sequences. Animals immunized with the DT-peptide conjugates produced antibodies to the coupling reagent (MCS) as well as diphtheria toxoid and peptide specific antibodies. This MCS specificity could be largely abolished by pre-incubation of sera with a soluble MCS homologue, a thiosuccinimidocaproylamide (TSC).     

Production of monoclonal antibodies for the determination of s‐triazines with enzyme immunoassays

Two triazine derivatives, ametryn sulphoxide (2‐ethylamino‐4‐isopropylamino‐6‐methyl‐sulphoxide‐1,3,5‐triazine) and dichloroatrazine (2,6‐dichloro‐4‐isopropylamino‐1,3,5‐triazine) were conjugated to bovine serum albumin (BSA) and used for the immunization of BALB/c mice. Hybridomas were produced by cell fusion of immune spleen and mouse myeloma cells (PAI‐B₃AG8.I). After screening with a competitive enzyme‐linked immunosorbent assay (ELISA), four anti‐triazine monoclonal antibodies from permanent hybridoma cell lines were selected for further characterization. Cross‐reaction studies with the antibodies developed against the ametryn sulphoxide conjugate showed strong affinities to terbutryn and prometryn. ELISAs with antibodies from clone AS‐K1F4 reached a detection limit of approximately 0.1 μg/l for terbutryn, and with AS‐K1A11 antibodies a limit of 0.3 μg/l for prometryn was attained. Cell lines from mice immunized with the dichloroatrazine‐conjugate produced antibodies with the highest affinities to aziprotryn. The detection limit of the corresponding ELISA was 1 μg/l (clone DC‐C3K5). The scope of the monoclonal antibodies for use in pesticide residue analysis is discussed with respect to polyclonal antibodies.     

Enhancing effect of human thyroid stimulating hormone (hTSH) monoclonal antibody in the hTSH immunoradiometric assay (IRMA) system

Different monoclonal antibodies, both commercial and indigenously produced, were evaluated in various combinations to optimize an immunoradiometric assay (IRMA) system for human thyroid stimulating hormone (hTSH). During these studies, it was observed that mixing one of the indigenously produced hTSH monoclonal antibody (2B11) in the hTSH IRMA system using Immunotech (Beckman Coulter, Czech Republic) kit reagents, led to an overall increase in the assay binding and sensitivity (from 0.025 mIU/L to 0.015mIU/L) of this IRMA system. This is not a general property of all monoclonal antibodies against hTSH. The mechanism for this enhancement can be attributed to the formation of a multicomponent complex.     

Use of monoclonal antibodies in an ELISA for the diagnosis of bovine leukaemia virus infection.

An ELISA diagnostic test for detection of bovine leukaemia virus (BLV) infected animals was developed. The test is based on the use of a mixture of monoclonal antibodies (MAbs) against envelope glycoprotein and against viral structural protein p24. The sensitivity and specificity of the test were found to be dependent on the relative proportions of MAbs of the appropriate epitope specificity. Polystyrene microtitre plates, wells or sticks were firstly coated with a mixture of purified MAbs and then non-purified viral antigens were adsorbed from tissue culture fluid obtained from BLV-producing cells. The optimal conditions for adsorption of MAbs and viral antigens as well as for the ELISA procedure were established. The test is more sensitive and cheaper (no need for virus antigen purification) than the routinely used ELISA using purified virus antigens. The assay is highly specific, rapid, practical and could be easily automated. It is suitable for the detection of BLV-antibodies in blood serum or milk in the large-scale screening programs for BLV-infected animals.     

Evidence for polyreactivity seen with monoclonal antibodies produced against type II collagen

Specific type II collagen monoclonal antibodies are needed for the quantification of articular cartilage collagen. In this study we produced and characterized 29 type II collagen monoclonal antibodies. Hybridomas were generated from mice immunized with rat type II collagen, selected for high antibody production against type II collagen using ELISA. Antibodies from selected and cloned hybridoma cells were purified by affinity chromatography and their reactivity tested by ELISA against a panel of antigens including actin, thyroglobulin, and single stranded DNA, all of which have been used to characterize the ‘naturally occurring antibodies’. It was found that many of the anti-type II collagen monoclonal antibodies reacted to more than one antigen. The monospecific antibodies had higher affinity to type II collagen than the antibodies which demonstrated multireactivity. Because of the prevalence of polyreactive anti-type II collagen antibodies, it is advisable to employ highly selective methodologies to isolate high affinity monospecific antibodies to type II collagen.     

Enzyme immunoassay analysis of antibody specificities present in the circulating immune complexes of selected pathological sera

Using immobilized anti-C3 antibody and an enzyme immunoassay, sera from 26 patients (eight with systemic lupus erythematosus (SLE), four with Hashimoto's thyroiditis, eight haemophiliacs and six with post-hepatitis cirrhosis) containing high levels of circulating immune complexes (IC) were selected. The IC were precipitated with 2.5% polyethylene glycol, washed, treated with acid buffer, neutralized and tested using an enzyme immunoassay in parallel with the original sera for antibody activity against a panel of antigens: human myosin and thyroglobulin, mouse actin and tubulin, calf thymus DNA and trinitrophenyl coupled to bovine serum albumin (TNP/BSA). It was found that all the isolated IC may contain IgG, IgA and IgM antibodies reacting with actin tubulin and TNP/BSA and also, depending upon the disease, antibodies reacting with some of the other antigens of the panel. By comparison to the antibodies present in the original sera, higher titers of antibodies were found in the isolated IC while some antibody specificities not detected in a given serum were occasionally noted in the isolated IC. The antibodies present in the IC seem to possess characteristics similar to those of polyreactive human natural autoantibodies. It is concluded that natural autoantibodies participate actively in the formation of IC found in pathological sera.     

筛选出一株可识别多种变异HBsAg的单克隆抗体及其应用

目的 制备筛选可识别变异表面抗原(hepatitis B surface antigen,HBsAg)的单克隆抗体(monoclonal antibody, mAb).用筛选出的单克隆抗体建立检测变异HBsAg的ELISA实验方法.方法 用血源HBsAg免疫Balb/c小鼠,通过杂交瘤细胞融合技术制备抗-HBs单克隆抗体.不同单克隆抗体包被酶标反应孔,检测真核细胞表达的野生及变异HBsAg,了解各种单克隆抗体的反应模式 .筛选出可以较好识别变异HBsAg的单克隆抗体Hb1,优化该抗体ELISA检测HBsAg的方法,与 8种HBs Ag检测试剂比较检测变异HBsAg的能力.结果 经过筛选,得到一种可以较好识别包括G145R在内大多数变异HBsAg的单克隆抗体.检测变异HBsAg的能力优于市售HBsAg 诊断试剂.结论 用本实验制备的单克隆抗体可以用于ELISA检测变异HBsAg,减少HBsAg变异株的漏检率.     

Monoclonal autoantibodies from insulin-dependent diabetic patients: autoantibodies against beta-cell surface or cytoplasmic antigens.

To investigate the target antigen(s) recognized during the autoimmune process in insulin-dependent diabetes mellitus (IDDM), we produced human monoclonal antibodies by Epstein-Barr virus transformation of peripheral blood lymphocytes from a large number (n = 50) of newly diagnosed IDDM patients. Screening by indirect immunofluorescence assay, using the RINm5F rat insulinoma cell line and eight other human or rat tumour cell lines, was performed to identify monoclonal antibodies that reacted with either membrane or cytoplasmic antigens. Eighteen IgM monoclonal antibodies reacting with cytoplasmic antigens of RIN cells were obtained; 14 of them also showed a staining of the cytoplasm of various non-beta-cell lines, while four displayed a binding restricted to beta-cells among the panel tested. However, among three monoclonal antibodies reacting with the membrane of RIN cells, one (HMD-1) produced an IgG antibody with a binding restricted to the membrane of beta-cells (RIN, HIT, and normal rat islet cells). The membrane antigens of HMD-1 were identified in Western blotting as proteins with molecular weights of 64 and 70 kD. This antibody had no apparent cytotoxic effect on RIN cells. These data suggest that, apart from 'natural autoantibodies,' it is feasible to obtain human monoclonal antibodies from IDDM patients that bind specifically to the beta-cell cytoplasm or to the beta-cell membrane.     

Production of mouse monoclonal antibodies to Pasteurella multocida type A and the immunological properties of a protective anti-lipopolysaccharide antibody

Eight monoclonal antibodies (MAbs) were produced from mice immunised with whole cells of heat-killed Pasteurella multocida type A which had been cultured under iron-restricted conditions. The MAbs were selected by an enzyme-linked immunosorbent assay (ELISA) in which the antigen consisted of whole bacteria of the immunising strain. Their reactivity was investigated further by immunoblotting, indirect haemagglutination, a complement-mediated bactericidal assay and passive protection of mice. One of the eight MAbs was shown by immunoblotting to react with lipopolysaccharide (LPS), was bactericidal, and completely protected mice against homologous challenge with 10 LD50 of live bacteria. This MAb was selected for further study. Its reaction with LPS of 17 type-A strains and of single strains of types B, D and E was investigated by immunoblotting. Strains that reacted with the anti-LPS MAb in immunoblots were susceptible to its bactericidal activity and gave high ELISA absorbances. Those that did not react were not susceptible to its bactericidal activity and gave low ELISA readings. The relation between bactericidal activity and ELISA absorbance was highly significant (p less than 0.001). Five of the strongly reacting heterologous strains and one non-reacting strain were selected as challenge organisms in a passive protection experiment: only the mice receiving the reacting strains were protected.     

Characterization of monoclonal antibodies against human thyrotropin and use in an immunoradiometric assay and immunohistochemistry

Monoclonal antibodies were prepared against human thyrotropin. 13 different antibodies were characterized. Ten antibodies were of the IgG1 subclass. The affinities of the antibodies were in the range 10(9)-10(11) mol-1 X l. Four of them were specific for hTSH and did not react with hLH, hFSH, hCG or alpha hCG. Four reacted with these hormones and recognized the alpha subunit of hCG. One cross-reacted only with hFSH. The remaining four antibodies recognized the holo-hTSH only, and thus were designated as anti-conformational determinants. Monoclonal antibodies reacting with different antigenic determinants on the hTSH molecule defined seven clusters. Two of them were used to develop a simplified two-site sandwich radioimmunoassay in which one monoclonal antibody was immobilized on tubes (anti-beta TSH) and another (anti-alpha) labelled with 125I. This assay was highly specific and demonstrated a sensitivity level of 0.1 microIU/ml. Two monoclonal antibodies were used in immunohistochemistry and their quality and specificity was assessed in the detection of hTSH immunoreactivity in human pituitary biological sections.     

ELISA for detection of Mycoplasma pneumoniae antigens using monoclonal antibodies

Seven monoclonal antibodies directed against major antigens of Mycoplasma pneumoniae were selected for the development of an antigen detection assay. Three of these were directed to the 170,000-dalton adhesin of M. pneumoniae. The test was an antigen-capture enzyme immunoassay using the different monoclonal antibodies for capture of antigen and a polyclonal rabbit antiserum as detection reagent. With three of the monoclonal antibodies a detection limit of approximately 2 ng M. pneumoniae protein was obtained, as determined by titration of M. pneumoniae organisms in buffer. The detection limit of the assays was only slightly less when the other four monoclonal antibodies were used. In artificially infected nasopharyngeal aspirates the detection limit was approximately 10 times lower. The fact that no significant differences in the detection limit of the assays were recorded using monoclonal antibodies directed against different antigens indicates that these antigens were available for reaction with antibodies irrespective of their location in intact M. pneumoniae cells. In the assay there were no significant cross-reactions with a number of bacterial species potentially colonizing the respiratory tract, except for a protein A-positive strain of Staphylococcus aureus. Our test is equally sensitive to another recently described ELISA using polyclonal antibodies. In comparison with other recommended methods such as immunoblot and culture-amplified antigen detection assays, the ELISA is more rapid and less laborious.     

Interaction of monoclonal antibodies with the IgE-receptor on rat mast cells and rat basophilic leukemia (RBL) cells

The close relation between rat mast cells and rat basophilic leukemia (RBL) cells with regard to the presence of receptors for IgE and Fc led us to generate monoclonal antobodies directed against cell surface antigens. Hybridomas were obtained by the fusion of NS1 mouse myeloma cells with murine spleen and lymph node cells. The culture supernatants were assayed by two ELISA techniques: a) for the production of mouse immunoglobulin in general and b) for antibodies directed against surface antigens of RBL cells. For this purpose RBL cells were attached to polyvinyl chloride microtitre plates. Eight hybrids produced antibodies directed against surface antigens on RBL cells. Hybrids were cloned and characterized with regard to their isotype and light chains. All eight clones secreted IgM with K light chains. Immunofluorescence studies performed with RBL cells revealed that all eight antibodies were able to show a specific fluorescence. Furthermore, four of these eight antibodies also showed a specific fluorescence with purified rat mast cells. These four antibodies were analyzed as to their ability of interacting with the IgE-receptor on RBL cells and purified rat mast cells. They reduced the binding rate of radiolabelled rat IgE to RBL and rat mast cells. A mutual inhibition of the passive cutaneous anaphylaxis (PCA) reaction in the rat by either mixing mouse reaginic serum directed against 2,4-dinitrophenol bovine serum albumin (DNP-BSA) or by mixing monoclonal mouse anti-DNP IgE with the monoclonal mouse anti-cell surface (rat basophilic leukemia, rat mast cell) IgM was determined. The histamine release of rat mast cells obtained from Nippo-strongylus brasiliensis infected rats was markedly reduced in the presence of two monoclonal anti cell surface antibodies. Our data suggest that monoclonal antibodies with specificity for the IgE receptor (inhibition of IgE-binding) can be distinguished from those which bind closely to the IgE-receptor but also modulate the histamine release from presensitized mast cells.in partial fulfilment of G.M. M.D.-thesis supported by DFG Kö 427-7/2     

Characterization of human-human hybridoma monoclonal anti-Ro(SS-A) autoantibodies derived from normal tonsil lymphoid cells

Human-human hybridomas obtained from the separate fusion of tonsillar lymphoid cells from three different normal individuals to the lymphoblastoid cell line GM 4672 were screened by ELISA for the presence of autoantibody to Ro(SS-A). Those anti-Ro(SS-A) reactive hybridomas were then cloned by limiting dilution. Nineteen monoclonal IgM anti-Ro(SS-A) antibodies were obtained, which showed specificity to Ro(SS-A) by ELISA and Western blotting (60 kDa). Some of these monoclonal anti-Ro(SS-A) antibodies showed reactivity to DNA (2/19), cardiolipin (9/19), Sm/RNP (15/19) by ELISA, and to IgG (12/19) and La(SS-B) (19/19) by ELISA and Western blotting. None showed reactivity to the unrelated proteins casein and BSA, nor to RNA. Inhibition studies revealed that the binding to Ro(SS-A) of both IgM hybridoma monoclonal and SLE serum polyclonal IgM anti-Ro(SS-A) antibodies was inhibited with Ro(SS-A), La(SS-B) and Sm/RNP but not with IgG, DNA, RNA and BSA. These data indicate that (1) normal humans have the genetic potential to express antibodies to Ro(SS-A) and (2) the normally derived monoclonal and SLE serum IgM anti-Ro(SS-A) antibodies share similar antigen binding properties and therefore may possibly originate from a common pool of precursor B cells.     

Antithyroglobulin monoclonal and autoantibodies cross-react with an orbital connective tissue membrane antigen: a possible mechanism for the association of ophthalmopathy with autoimmune thyroid disorders.

The possibility that Graves' ophthalmopathy and autoimmune thyroid disorders may be associated because of autoimmune reactions against antigens shared between human orbital and thyroid tissues was investigated using anti-thyroglobulin (Tg) monoclonal and autoantibodies. Eleven of 16 mouse monoclonal antibodies (MCAB) tested reacted, in an enzyme-linked immunosorbent assay (ELISA), with an antigen in human orbital connective tissue membranes (OCTmem), but not with the OCT soluble fraction, or with membrane or soluble fractions of human eye muscle, lacrimal gland or skin connective tissue. The anti-OCTmem activity was absorbed by OCTmem and Tg, but not by liver membranes or bovine serum albumin (BSA). In preliminary studies four out of 113 human MCAB against thyroid or orbital tissue antigens showed reactivity restricted to Tg and OCTmem. Sera from approximately 50% of patients with autoimmune thyroid disorders, with or without ophthalmopathy, also reacted with OCTmem. The autoantibody activity correlated closely with serum titres of antithyroglobulin but not with the presence, duration, or severity of the eye disease. The OCTmem reactivity was absorbed by Tg, thyroid membranes, and OCTmem but not liver membranes, membranes prepared from other orbital tissues, or BSA. The OCTmem-Tg shared antigen site appeared not to be native thyroglobulin since, (i) MCAB and serum autoantibodies did not react with the cytosol fraction of OCT, and (ii) because the membrane antigen was not solubilizable. Because not all patients with ophthalmopathy have detectable anti-Tg antibodies and, conversely, because not all patients with detectable anti-Tg antibodies develop ophthalmopathy it is unlikely that autoimmunity against a OCTmem-Tg shared antigen is the primary mechanism of Graves' ophthalmopathy, although this possibility has not been excluded. On the other hand the reaction of anti-Tg autoantibodies with OCT membranes may be a model for other autoimmune reactions against other thyroid-orbital tissue-shared antigens. While the pathogenesis of Graves' ophthalmopathy is likely to be multifactorial, humoral and cellular reactions against primary orbital antigens, thyroid-orbitol tissue shared antigens, or both, are likely to play important roles.     

Presence of natural autoantibodies in hyperimmunized mice.

Mice were immunized with various antigens in complete Freund's adjuvant following various injection schedules. Hybridomas were produced from the spleens of these immunized mice and examined for production of antibodies directed against the antigen injected and against a panel of self (tubulin, actin, myosin, DNA) and non-self antigens (myoglobin, spectrin, peroxidase, trinitrobenzene). Two to five percent of the hybrids were found to secrete polyspecific antibodies able to react with two or more antigens of the panel. Several of these hybrids were subcloned and expanded into ascites. The monoclonal immunoglobulins they secreted were isolated and shown to be IgM (kappa) and to possess the polyspecific antibody function. Several hybrids were also found to secrete antibodies reacting with the immunizing antigen as well as one or more antigens of the panel. The antibody secreted by one subclone which reacts with both the immunizing antigen, prolactin and one of the panel antigens, TNP, has been isolated using a DNP-immunoadsorbent. The isolated antibody was found to be a monoclonal IgM (kappa) immunoglobulin and to react both with prolactin and TNP. The hypothesis is advanced that cells carrying polyspecific natural antibodies as receptors after a given antigenic stimulation proliferate into cells producing highly specific antibodies for epitopes of that given antigen; the cells with polyspecific receptors will be continuously replaced by new cells probably on bone-marrow origin.     

Towards an idiotype vaccine against mammary tumors. Induction of an immune response to breast cancer-associated antigens by anti-idiotypic antibodies

Previously we have reported on the production of two sets of human monoclonal antibodies reacting with mouse mammary tumor virus (MMTV) and human mammary tumor virus (HuMTV) cross-reacting antigens. B11 is a monoclonal IgG generated by the human-human hybridoma technique using axillary lymph node of breast cancer patients. 4.6/6 is a monoclonal IgG established by the mouse-human hybridoma procedure using peripheral blood lymphocytes of a healthy investigator working with the breast cancer cell line T47D which secretes HuMTV antigens. Two anti-idiotypic (Id) antibodies were produced by immunizing rabbits with B11 or 4.6/6. Following exhaustive adsorption on unrelated human-Ig, fetal calf serum and purification on B11 or 4.6/6 affinity columns, the anti-Id antibodies were shown to react specifically with their respective Id. The binding of these anti-Id antibodies to the respective Id was specifically inhibited by prior incubation of the Id with MMTV and/or HuMTV antigens. Rabbit anti-B11 and rabbit anti-4.6/6 anti-Id antibodies were employed to immunize female C3Heb mice. Following immunizations, humoral and cellular immune responses to MMTV-related antigens could be demonstrated. The sera of the mice contained anti-MMTV antibodies and delayed-type hypersensitivity was specifically expressed when irradiated cells of the Mm5MT line (which carry surface MMTV antigens) were injected. Our results support the notion that anti-Id antibodies harboring the internal image can immunize animals against tumor cells bearing on their surface viral-associated antigens.     

Enzyme-linked immunosorbent assay with a monoclonal antibody for detecting group A meningococcal antigens in cerebrospinal fluid.

Hybridomas were produced from spleen cells of BALB/c mice immunized with a membrane preparation from Neisseria meningitidis group A strain 4402 and S194/5.XXOBU.14 myeloma cells. The hybridomas were screened for secretion of antibodies suitable for an enzyme-linked immunosorbent assay (ELISA) diagnostic for group A meningococcal meningitis. One hybridoma antibody, 3G7, was directed against the pilus protein. This antibody bound to all six lipopolysaccharide and protein group A meningococcal serotyping strains, as well as to meningococcal strains from serogroups C, W135, and Y, but not to a strain of Escherichia coli, Haemophilus influenzae type b, or to two or more strains of Streptococcus pneumoniae, Neisseria gonorrhoeae, and Salmonella typhi. The ELISA used on antibody, antigen, antibody-conjugate sandwich. Rabbit anti-meningococcal serum was the coating antibody for the antibody sandwich, cerebrospinal fluids contained the bacterial antigens, and 3G7-alkaline phosphatase conjugate was the detecting antibody. The monoclonal antibody conjugate ELISA system was able to detect group A meningococcal antigens in 21 of 25 cerebrospinal fluid specimens that were positive in an immune rabbit serum conjugate ELISA; cerebrospinal fluid samples from patients with Haemophilus meningitis served as the controls. Counterimmunoelectrophoresis detected meningococcal antigens in 16 of the same 25 cerebrospinal fluid samples.     

抗弓形虫P30单克隆抗体的制备及其抗虫作用观察

制备抗弓形虫天然P30、重组P30的单克隆抗体(mAb),并研究它们对虫体的影响,为弓形虫病诊断及保护性抗原鉴定奠定基础。采用弓形虫天然P30、重组P30及全虫抗原(对照)分别免疫BALB/c小鼠,取免疫小鼠脾细胞与SP2/0骨髓瘤细胞融合,筛选稳定分泌高滴度mAb的杂交瘤细胞株。用ELISA法测定mAb亚类和效价;通过SDS-PAGE和West-ern blot进行特异性鉴定;Giemsa染色观察杂交瘤细胞的染色体数目;利用扫描电镜及间接荧光抗体试验(IFAT)观察mAb对弓形虫的杀伤作用及其靶抗原定位。结果表明,筛选出2株(2B3、1H6)特异性较好、能稳定分泌mAb的杂交瘤细胞株。Western blot结果显示,2B3、1H6产生的mAb与弓形虫全抗原的阳性反应带均在30 000 Mr处;mAb亚类均属IgM,其效价(ELISA)分别为:2B3 1∶102 400、1H6 1∶51 200;2株细胞的染色体数均在100条以上。电镜观察与mAb作用后的弓形虫虫体聚集、肿胀,膜变厚,表面出现缺口和空洞,甚至虫体变形、破碎。抗弓形虫天然P30的mAb能识别重组体pET-30a-P30的表达蛋白。成功制备了抗弓形虫天然P30、重组P30的mAb,可进一步用于弓形虫病诊断及保护性抗原鉴定研究。     

Monoclonal antibodies to outer membrane antigens of Vibrio cholerae.

Hybridoma-derived monoclonal antibodies were prepared against outer membrane antigens of four strains of Vibrio cholerae that were cultivated under iron-limited conditions, and these antibodies were partially characterized. We established a library of 66 hybridomas which produced monoclonal antibodies defining 16 different V. cholerae antigens. Two antigens (molecular weights, 18,000 and 112,000) were heat modifiable, whereas the reacting epitope of a third antigen (40,000-dalton-18,000-dalton doublet) was completely destroyed when it was heated at 100 degrees C. The 112,000-dalton heat-modifiable protein was an iron-regulated outer membrane protein. This protein bound 59Fe in vitro when it was combined with the V. cholerae siderophore-iron complex 59Fe-vibriobactin; it was also found in in vivo grown V. cholerae, as were three other antigens. A total of 26 hybridomas produced antibody to V. cholerae lipopolysaccharide. Of these, 12 were cross-reactive with lipopolysaccharides of other gram-negative bacteria, including 2 which recognized lipid A. Several of these anti-lipopolysaccharide monoclonal antibodies appeared to be lipopolysaccharide region specific. Some membrane antigens were strain specific, whereas others were common to both O group 1 and non-O group 1 vibrios.