A Monoclonal Antibody Specific to Surface Antigen on Candida krusei

A monoclonal antibody (MAb; MAb 6B3) which reacts specifically with a cell wall antigen found in all strains or isolates of Candida krusei was developed. MAb 6B3 was extensively tested by immunofluorescence assay for cross-reaction with many Candida, Cryptococcus, Saccharomyces, Trichosporon, and Rhodotorula species and was found to react only with the species C. krusei. The specific epitope is expressed on the surface of fungal cells and appears to reside on a protein moiety. Taking into account the increasing importance of fluconazole-resistant strains in nosocomial fungal infections, the very high degree of specificity of this MAb for C. krusei could be useful for the routine detection of C. krusei in culture or in tissue samples.     

抗日本血吸虫SEA特异性IgY应用于循环抗原检测的研究

本研究应用抗日本血吸虫可溶性虫卵抗原(SEA)的鸡卵黄免疫球蛋白(IgY)建立敏感、特异的检测循环抗原的双抗体夹心酶联免疫吸附试验( ELISA).用SEA皮下多点注射法免疫海蓝鸡,水稀释法制备IgY抗体,以辣根过氧化物酶标记纯化的IgY抗体(IgY-E)和兔抗IgG抗体(IgG-E)分别作为检测抗体,IgY抗体和兔抗...     

双特异抗体体外模拟特异B细胞抗原递呈作用

用化学偶联的双特异性抗体复合物－αＴＴ－α－ＩｇＭ通过体外实验，观察其抗原递呈能力。结果显示：αＴＴ－αＩｇＭ的对照组低１０００倍左右，与抗原特异性Ｂ细胞的抗原递呈效应相似。抑制试验显示：羊抗鼠ＩｇＭ，鼠ＩｇＭ能抑制αＴＴ－αＩｇＭ对非特异性Ｂ细胞抗原递呈作用的增强效应，表明Ｂ细胞膜ＩｇＭ在抗原递呈中起关键性作用。本研究结果表明：αＴＴ－αＩｇＭ的存在确能模拟抗原特异性Ｂ细胞的高效抗原递呈功能。     

Preparation of a Relatively Acid-Stable Antigen (CEA) by Disruption of its Specific Precipitate with Antibody

The carcinoembryonic antigen (CEA) of intestinal tract cancers is presently purified by extraction from a homogen-ate of such tissues with 1.0M perchloric acid (PCA), followed by two steps of chromatography and one of electrophoresis. In this study, rabbit antisera prepared against such purified CEA, and absorbed with normal human plasma, liver, kidney, spleen and brain, gave a conventional precipitin curve with a water homogenate or with a dialyzed PCA extract of intestinal tract cancers. The immune precipitate was almost completely soluble in PCA. Following dialysis of this solution against cold water, a new precipitate composed mainly of denatured antibody appeared. The remaining solution contained purified antigen. Recovery of this antigen (CEA-R), as judged by radioimmunoassay, appeared optimal when 3.0M PCA was used for disruption of the immune precipitate, and presence of 1.8M urea was helpful. When injected into rabbits, CEA-R was not as strongly antigenic as CEA. Comparison by immunodiffusion and immunoelectrophoresis against their specific rabbit anti-sera indicated that CEA and CEA-R share some specificities but are not identical.     

Preparation of a Relatively Acid-Stable Antigen (CEA) by Disruption of its Specific Precipitate with Antibody

The carcinoembryonic antigen (CEA) of intestinal tract cancers is presently purified by extraction from a homogen-ate of such tissues with 1.0M perchloric acid (PCA), followed by two steps of chromatography and one of electrophoresis. In this study, rabbit antisera prepared against such purified CEA, and absorbed with normal human plasma, liver, kidney, spleen and brain, gave a conventional precipitin curve with a water homogenate or with a dialyzed PCA extract of intestinal tract cancers. The immune precipitate was almost completely soluble in PCA. Following dialysis of this solution against cold water, a new precipitate composed mainly of denatured antibody appeared. The remaining solution contained purified antigen. Recovery of this antigen (CEA-R), as judged by radioimmunoassay, appeared optimal when 3.0M PCA was used for disruption of the immune precipitate, and presence of 1.8M urea was helpful. When injected into rabbits, CEA-R was not as strongly antigenic as CEA. Comparison by immunodiffusion and immunoelectrophoresis against their specific rabbit anti-sera indicated that CEA and CEA-R share some specificities but are not identical.     

Early Expansion of Secondary B Cells after Primary Immunization with Antigen Complexed with IgE

IgE antibodies have potent immunoregulatory effects in vivo , and mice immunized with IgE–antigen (IgE/Ag) complexes exhibit a several hundred-fold higher humoral Ag-specific response than mice immunized with non-complexed Ag. In vitro studies indicate that this is a result of efficient endocytosis of the IgE/Ag complexes via the low-affinity receptor for IgE (CD23) on B cells, leading to efficient antigen presentation to T cells. Previous studies of IgE-induced Ab responses in vivo have only measured serum responses. The authors have now studied the up-regulated response as the number of IgG-, IgA-, IgE- and IgM-secreting single B cells in spleen, lymph nodes and bone marrow of mice immunized with IgE-anti-TNP + BSA-TNP (2,4,6-trinitrophenylated bovine serum albumin). IgE and Ag induced a greater than 500-fold increase of specific IgG-secreting spleen cells with the peak of the response 6 days after primary immunization. The response of other Ab isotypes and the response in other lymphoid organs was marginal. The rapid increase in the number of IgG-secreting cells in the spleen suggests that IgE/Ag complexes induce a secondary type of antibody response without requirement for conventional priming.     

Differences in Specific Antibody Responses of Human Tonsillar Cells to an Oral and a Parenteral Antigen

Tonsillar lymphocytes stimulated in vitro with either β-lactoglobulin or with tetanus toxoid were shown to produce specific antibodies by a direct plaque assay and by radioimmuno-precipitation of the culture supernatants. There was a sixfold increase in the number of IgA-secreting cells in response to β-lactoglobulin; no such effect was seen in response to tetanus toxoid, where a fivefold rise in IgG-secreting cells occurred. These differences in antibody response are probably due to the route of initial antigen presentation. Those antigens priming the mucosa-associated lymphoid system stimulate mainly IgA-producing cells, in contrast to parenteral antigens, which elicit a predominantly IgM and IgG response.     

An Echinococcus multilocularis Antigen B3 Proteoform That Shows Specific Antibody Responses to Active-Stage Alveolar Echinococcosis

Alveolar echinococcosis (AE), caused by the Echinococcus multilocularis metacestode, represents one of the most frequently fatal zoonoses. Early diagnosis significantly reduces morbidity and mortality associated with AE. Diagnosis of AE largely depends on a combination of imaging and serological tests due to its minimal clinical manifestations. Several antigens derived from the whole worm and protoscolex have been targeted for AE serodiagnosis, while the antigenic properties of E. multilocularis hydatid fluid (EmHF) are unclear. We observed two AE-specific 6- and 8-kDa antigen proteoforms through an immunoproteome array of the EmHF. We identified these proteins as representing an E. multilocularis antigen B3 (EmAgB3) isoform, and the proteins were shown to be encoded by the same gene. We cloned the gene and expressed the recombinant EmAgB3 protein (rEmAgB3) in Escherichia coli. rEmAgB3 exhibited sensitivity of 90.9% (80/88 cases) and specificity of 98.5% (597/606 samples) by immunoblotting. The positive and negative predictive values were 89.9% and 98.6%, respectively. The protein did not show antibody responses to 33 AE sera collected during posttreatment follow-up monitoring. Mouse sera experimentally infected with AE protoscoleces began to demonstrate specific antibody responses to native and recombinant EmAgB3 6 months after infection. At that stage, fully mature metacestode vesicles that harbored the brood capsule, primary cell, and protoscolex were observed within an AE mass(es). The response declined along with worm degeneration. Our results demonstrate that the immune responses to this EmAgB3 isoform were highly correlated with worm viability accompanied with AE progression. rEmAgB3 is a promising biomarker for serological assessment of AE patients.     

Peroxidase Arthritis: I. An Immunologically Mediated Inflammatory Response with Ultrastructural Cytochemical Localization of Antigen and Specific Antibody.

Synovitis was produced in rabbits by daily intra-articular injections of the heterologous antigen horseradish peroxidase. The resulting “peroxidase arthritis” resembled rheumatoid arthritis histologically. Many of the subsynovial plasma cells, plasmablasts and immunoblasts contained specific antibody to horseradish peroxidase; the remainder appeared to contain immunoglobulins of other specificities. Peroxidase arthritis has unique advantages for the study of the cellular and subcellular events in the pathogenesis of the local immune inflammatory response to heterologous antigen. Antigen and specific antibody can be localized precisely by ultrastructural cytochemical technics. The reaction can be terminated at any stage, permitting observation of the early events in its pathogenesis.     

An Engineered Human Antibody Fab Fragment Specific for Pseudomonas aeruginosa PcrV Antigen Has Potent Antibacterial Activity

Pseudomonas aeruginosa is an opportunistic pathogen that can cause acute lung injury and mortality through the delivery of exotoxins by the type III secretion system (TTSS). PcrV is an important structural protein of the TTSS. An engineered human antibody Fab fragment that binds to the P. aeruginosa PcrV protein with high affinity has been identified and has potent in vitro neutralization activity against the TTSS. The instillation of a single dose of Fab into the lungs of mice provided protection against lethal pulmonary challenge of P. aeruginosa and led to a substantial reduction of viable bacterial counts in the lungs. These results demonstrate that blocking of the TTSS by a Fab lacking antibody Fc-mediated effector functions can be sufficient for the effective clearance of pulmonary P. aeruginosa infection.Pseudomonas aeruginosa is an opportunistic pathogen that causes both acute and chronic infections in compromised individuals. It is a frequent causative agent of bacteremia in burn victims (32) and immunocompromised patients (18). It is also the most common cause of nosocomial gram-negative pneumonia (7, 25), especially in mechanically ventilated patients (25), and is the most prevalent pathogen in the lungs of individuals with cystic fibrosis (CF) (10, 17, 20). In CF, P. aeruginosa infection follows a well-established pattern of recurrent pulmonary infection in early childhood leading to the establishment of chronic infection in older CF patients, where it is a major contributing factor in the progressive decline in lung function and disease exacerbations leading to respiratory failure (10, 17).Morbidity and mortality associated with P. aeruginosa infections remain high despite the availability of antibiotics to which the bacterium is sensitive, and antibiotic resistance is an increasingly common problem in nosocomial infections (6).The type III secretion system (TTSS) is an important virulence determinant of P. aeruginosa in animal models of infection (15) and is required for the systemic spread of P. aeruginosa in a mouse pulmonary challenge model (31). The expression of a functional TTSS also correlates with poor prognosis in clinical infections (26, 27). This needle-like structure comprises a complex secretion and translocation machinery to inject a set of up to four different exotoxins (ExoS, ExoT, ExoU, and ExoY) directly into the cytoplasm of eukaryotic cells (9, 33, 34). Various strains of P. aeruginosa secrete different exotoxins. In addition, the TTSS can mediate direct cytotoxicity toward macrophages and neutrophils in the absence of exotoxins, a process called “oncosis,” requiring bacterial swarming in response to macrophage factors and leading to a direct perforation of the cell membranes (3, 4). In all of these functions of the TTSS, the needle tip protein, PcrV, is an essential component of the translocation apparatus.Antisera raised against PcrV in rabbits have been shown to block the translocation of Pseudomonas exotoxins into mammalian cells (12, 28) and to protect against lethality in a mouse model of acute pulmonary Pseudomonas infection (28, 29). Polyclonal anti-PcrV antibodies have also been shown to reduce lung damage and protect against bacteremia and septic shock in rat and rabbit pulmonary infection models (29) and to protect burned mice from infection (22). A mouse monoclonal anti-PcrV antibody, monoclonal antibody (MAb) 166, with potent neutralizing activity in mouse and rat models of Pseudomonas infection has also been described (11). This antibody inhibits the function of the TTSS in cell-based assays (11, 12). The MAb acts to prevent sepsis and mortality in an acute pulmonary infection model in mice when delivered either systemically or by intratracheal administration (11) and reduces lung damage due to Pseudomonas in a rat model (8). The antibody has activity when dosed either prophylactically or therapeutically in these models both as whole immunoglobulin G (IgG) and as a Fab fragment, indicating that the inhibition of TTSS function is sufficient to inhibit lung damage in pulmonary Pseudomonas infections.Here, we identify an engineered human antibody Fab fragment specific for the P. aeruginosa PcrV protein which competes with MAb 166 for binding to the same epitope on PcrV. The human Fab shows potent TTSS-neutralizing activity, equivalent to the activity of MAb 166, in cellular cytotoxicity assays. This Fab shows potent in vivo activity in protecting mice from potentially lethal doses of P. aeruginosa. In addition, the Fab mediates a substantial clearance of bacteria from the lungs of infected mice, suggesting that antagonism of the TTSS is sufficient not only to prevent damage to the pulmonary epithelium but also to restore normal immunological clearance mechanisms for the effective resolution of Pseudomonas infection in the complete absence of antibody effector functions.     

Radiolabeling of Anti-human Prostatic Specific Membrane Antigen Antibody with 99Tcm and its Biodistribution in Nude Mice Bearing Human Prostate Cancer

目的 研究99Tcm-抗前列腺特异性膜抗原(PSMA)抗体(J591)对前列腺癌细胞体外结合性能、在荷人前列腺癌裸鼠体内生物分布.方法 用改进的Schwarz方法进行99Tcm标记,经Sephadex G-50柱分离纯化;用纸层析法和三氯醋酸法测定标记率与放化纯;用流式细胞术测定抗体与肿瘤细胞在体外的结合性能;PSMA阳性的C4-2前列腺癌裸鼠为实验组,PSMA阴性的PC3前列腺癌裸鼠为对照组,裸鼠静脉注射7.5 MBq(25μg/只)99Tcm-J591,分别于2、6、12及24h测定体内放射性分布,计算每克组织百分注射剂量率(％ ID/g)及T/NT比值.结果 表明99Tcm - J591标记率为(78.9±6.2)％,放化纯＞90％.J591与99Tcm - J591在体外能结合PSMA阳性的C4-2细胞,与PSMA阴性的PC3细胞不结合,显示出J591具有良好的特异性.生物分布实验显示:实验组裸鼠肿瘤部位出现99Tcm浓聚,对照组则没有.肿瘤组织百分注射剂量率(％ ID/g)在12h达高峰,为(15.91±5.16)％ID/g,与对照组(3.22±1.33)％ID/g相比差异有统计学意义(P＜0.05),其余组织和器官的百分注射剂量率在两组间差异无统计学意义(P＞0.05).结论 J591具有良好的免疫活性和对前列腺癌的靶向定位性能,可望用于前列腺癌的导向诊断及导向治疗.     

Antigen Specific Immunotherapy of Multiple Sclerosis

The development of antigen specific therapy for multiple sclerosis (MS) involves specifically suppressing undesired immune responses targeting the myelin sheath and underlying axon. We have recently reported some success with altered peptide ligands for a major target of the autoimmune response in MS. Antigen specific therapy has the potential to suppress undesirable autoimmunity, while leaving the rest of the immune system intact. Induction of an antigen specific Th1-to-Th2 shift could achieve this aim, once side effects, such as allergic responses, are minimized with optimal dosing.     

Evaluation of Molecular Species of Prostate-Specific Antigen Complexed with Immunoglobulin M in Prostate Cancer and Benign Prostatic Hyperplasia

This study was aimed at defining molecular species of prostate-specific antigen (PSA) in immune complexes with immunoglobulin M (IgM). Having in mind the oligoreactivity of IgM and its preference for carbohydrate antigens, there is the possibility that it can selectively recognize known PSA glycoisoforms. PSA-IgM complexes and free PSA fractions were separated from the sera of subjects with prostate cancer (PCa) and benign prostatic hyperplasia (BPH) by gel filtration and subjected to on-chip immunoaffinity and ion-exchange chromatography. PSA-immunoreactive species were detected using surface-enhanced laser desorption/ionization time of flight mass spectrometry. The obtained spectra were analyzed for protein and glycan composition. The general pattern of the molecular species of PCa PSA and BPH PSA found in complexes with IgM was similar. It comprised major peaks at 17 kDa and minor peaks at 28 kDa, corresponding to the entire mature glycosylated PSA. The main difference was the presence of incompletely glycosylated 26.8 kDa species, having putative paucimannosidic structures, observed in PCa PSA-IgM, but not in BPH PSA-IgM. Characteristic PCa PSA-IgM glycoforms pose the question of the possible role of glycosylation as a framework for immune surveillance and may be of interest in light of recent data indicating mannose-containing glycans as cancer biomarker.     

Specific Inhibition of Antibody Production: II. Paralysis Induced in Adult Mice by Small Quantities of Protein Antigen

A state of immunological paralysis has been induced in adult CBA mice by intraperitoneal injections of small quantities of bovine γ globulin (BGG). The minimum paralysing dose of BGG has been found to be between 50 and 200 μg. A dose as small as 2 μg. has been found to have a slight paralysing effect. The time necessary for the induction of paralysis by 50 μg. to 2 mg. of BGG in CBA mice is 3–4 days.

Paralysis is induced by only one component of BGG; this component is incapable of inducing an antibody response unless an injection of adjuvant is made at the same time or slightly before the injection of the antigen. The BGG is centrifuged at an RCF of 20,000–30,000 g to remove particulate matter. Failure to remove the particulate matter leads to sporadic immune responses in groups of mice injected with the protein. Mice given a paralysing injection of BGG were subsequently challenged by an injection of BGG in Freund's adjuvant. The result of this challenge was tested by an injection of radioactively-labelled antigen and the elimination of this antigen from the circulation of the challenged mice was followed for several days. `Immune elimination' can easily be distinguished from `non-immune elimination'. The presence of antibody to the non-paralysing components of BGG in sera from paralysed mice was confirmed using the Ouchterlony geldiffusion technique.

     

Correlation of Pneumococcal Antibody Concentration and Avidity with Patient Clinical and Immunologic Characteristics

Purpose

The quality of an antibody response is determined by both the concentration and the strength of antigen-binding, or avidity, of the antibodies produced. Currently, only antibody concentration is routinely evaluated in the clinical assessment of humoral immunity. Here we studied correlations of avidities and concentrations of antibodies to pneumococcal polysaccharides with immunologic and clinical characteristics of patients with recurrent infections.

Methods

We measured concentration and avidity of antibodies to 12 pneumococcal serotypes in 78 children aged 0.6–18 years with recurrent bacterial respiratory infections, and in 80 individuals who were being tested for peanut allergy, ages 0.4–15 years, serving as a comparison group. Avidity was assessed by measuring antibody binding in the presence of thiocyanate.

Results

Antibody concentrations and avidities correlated positively for very few types contained in the conjugated pneumococcal vaccine (PCV7) in both patients and controls with some dependence on age; there were even fewer correlations for non-PCV7 types. Antibody concentrations and avidities negatively correlated with age for most of the PCV7 types. There was no consistent correlation of total IgG or IgG subclasses with either concentrations or avidities. Overall, antibody concentrations were higher and avidities were lower in patients compared to controls. Patients requiring chronic antibiotic use tended to have higher antibody concentrations and lower avidities for most serotypes than patients who did not. We identified several patients having many infections with apparent good antibody concentrations with low avidity for many types.

Conclusion

Antibody concentration and avidity correlate with patient clinical characteristics and distinguish patients from controls. Measurement of antibody avidity may provide another dimension for the clinical assessment of pneumococcal polysaccharide antibody response.     

Rapidity of Specific Antibody–Antigen Interactions

Analysis of humoral immune responses against viruses has concentrated on studies with serum dilutions, which reflect characteristics pertaining to the diluent buffer but not the serum environment. The majority of virus-specific antibody in serum from foot-and-mouth disease virus (FMDV)-vaccinated cattle bound to antigen within 10-60 s, whereas aspecific reactions evolved more slowly. Upon dilution of sera, the reaction characteristics no longer related to those obtained with the serum, particularly when individual animals were compared. Diagnostic enzyme-linked immunosorbent assay (ELISA) - employing diluted serum - identified little variation in the reactivity of serum samples from different vaccinated cattle. This related to previous analyses showing similar specific antibody titres. In contrast, analyses of serum reactivities over a 10- and 60-s incubation period demonstrated high variation between individual animals. Furthermore, when a challenge infection was performed on vaccinated animals, only those with the higher serum reactivities over a 10- and 60-s incubation were protected. These results demonstrate the importance of the specific serum antibody reactions, which will occur within seconds. Moreover, such qualitative characteristics would be overlooked when employing conventional assays with diluted sera and long incubation periods.     

Chemical Denaturation of Ovalbumin Abrogates the Induction of Oral Tolerance of Specific IgG Antibody and DTH Responses in Mice

We have examined the effects of ingestion of chemically denatured ovalbumin (OVA) in mice. Both 8 M urea-denatured OVA (UD-OVA) and carboxymethylated UD-OVA (CM-OVA) were purified by gel filtration. Specific IgG antibody and systemic delayed-type hypersensitivity (DTH) responses to OVA were not suppressed by CM-OVA fed prior to or after immunization with OVA in complete Freund's adjuvant (CFA). When CM-OVA was used instead of OVA for immunization, serum IgG and DTH responses to CM-OVA were orally tolerized by OVA, but not by UD-OVA or CM-OVA. Studies of antigen uptake in mice using sandwich ELISA tests showed that OVA, but not CM-OVA, was absorbed after antigen ingestion. In vitro studies further demonstrated that CM-OVA was digested much more rapidly than OVA. Moreover, studies using bovine serum albumin (BSA) demonstrated that both IgG and DTH responses to BSA were orally tolerant to BSA, but not to denatured BSA. Finally, studies using human gamma-globulin (HGG), a well-known tolerogen, also found that the IgG antibody response to HGG was not orally tolerized by denatured HGG. These results suggest that complete denaturation of globular proteins may affect their processing and absorption in the gut and thus abrogates oral tolerance induction.