The use of monoclonal antibodies for studying the biological properties of Staphylococcus aureus endo-β-N-acetylglucosaminidase

Abstract Staphylococcus aureus endo- β - N -acetylglucosaminidase (SaG) has been suggested to function as a virulence determinant which interferes with the host cellular immune response. To further characterize the biological properties of SaG, monoclonal antibodies (mAbs) were raised against purified SaG. Four IgG₁ subclass mAbs were obtained, none of which reacted with the reduced, sodium dodecyl sulphate pretreated or boiled enzyme. The ability of the mAbs to react with the enzymes present in supernatants obtained from 197 S. aureus strains indicated that they recognized epitopes which are highly conserved; bacteriolytic enzymes produced by staphylococci other than S. aureus did not show any cross-reactivity. After pretreatment of SaG with mAbs (mAb-SaG molar ratios varying from 1 to 20), it was shown that all selected mAbs caused, at a mAb: SaG molar ratio of 10, a 90% inhibition of SaG bacteriolytic activity and a statistically significant reduction of its ability to interfere with phagocytosis to human polymorphonuclear leukocytes. All selected mAbs reacted with several commercially available exo- β - N -acetylglucosaminidases; mAb C1/10–11 also reacted with chicken and turkey egg muramidases and, at a mAb:SaG molar ratio of 10, inhibited their bacteriolytic activity by 97%. This suggests that one or more epitopes present in the above exo-glucosaminidases and muramidases share some degree of homology with others present in SaG.     

Genetic control of immune response to staphylococcal nuclease. XII: Analysis of nuclease antigenic determinants using anti-nuclease monoclonal antibodies

Summary SJL mice, which are high responders to Staphylococcal nuclease (nuclease), were immunized and used to produce hybridoma cell lines secreting anti-nuclease monoclonal antibodies (mAb). Ten stable clones were derived from a single fusion. Seven of these produced antibodies of the IgG_1, isotype and were more precisely characterized for antigenic specificity. Only one hybridoma cell line (54-10-4) produced anti-nuclease antibodies capable of inhibiting enzymatic activity of nuclease. Binding inhibition analyses strongly suggest that the other monoclonal antibodies, which failed to inhibit nuclease activity detect two different antigenic regions, or epitopes, of the molecule: epitope cluster 1 domain is defined by hybridomas 54-2-7, 54-5-2, 54-9-8, and 54-10-8; epitope cluster 2 by 54-5-1 and 54-1-9. Because of its capacity to inhibit nuclease enzymatic activity mAb 54-10-4 was considered specific for a third epitope of the nuclease molecule called epitope 3. Binding studies of these monoclonal antibodies were extended to peptide fragments of the nuclease molecule in order to examine possible cross-reactions with such fragments, as has previously been reported for antibodies purified from polyclonal antisera. Monoclonal antibodies specific for epitope cluster 1 on the native molecule also bound to the fragments 1–126 and 49–149 but failed to bind to fragment 99–149, suggesting that the corresponding epitope(s) is determined by amino acids localized between residues 49 and 99. The epitope clusters 2 and 3 appeared to be expressed only on the native molecule. Monoclonal antibodies of different clusters exhibited very different migration patterns on isoelectric focusing while monoclonal antibodies of the same cluster were indistinguishable, which suggests that they may have originated from the same B cell precursor. Taken together these data suggest that this panel of monoclonal antibodies detects at least three distinct epitopes of the nuclease molecule, one of which could be involved in the determination of the enzymatic site.     

Successful selection of an infection‐protective anti‐Staphylococcus aureus monoclonal antibody and its protective activity in murine infection models

Recent clinical trials to develop anti‐methicillin‐resistant Staphylococcus aureus (MRSA) therapeutic antibodies have met unsuccessful sequels. To develop more effective antibodies against MRSA infection, a panel of mAbs against S. aureus cell wall was generated and then screened for the most protective mAb in mouse infection models. Twenty‐two anti‐S. aureus IgG mAbs were obtained from mice that had been immunized with alkali‐processed, deacetylated cell walls of S. aureus. One of these mAbs, ZBIA5H, exhibited life‐saving effects in mouse models of sepsis caused by community‐acquired MRSA strain MW2 and vancomycin‐resistant S. aureus strain VRS1. It also had a curative effect in a MW2‐caused pneumonia model. Curiously, the target of ZBIA5H was considered to be a conformational epitope of either the 1,4‐β‐linkage between N‐acetylmuramic acid and N‐acetyl‐D‐glucosamine or the peptidoglycan per se. Reactivity of ZBIA5H to S. aureus whole cells or purified peptidoglycan was weaker than that of most of the other mAbs generated in this study. However, the latter mAbs did not have the protective activities against S. aureus that ZBIA5H did. These data indicate that the epitopes that trigger production of high‐yield and/or high‐affinity antibodies may not be the most suitable epitopes for developing anti‐infective antibodies. ZBIA5H or its humanized form may find a future clinical application, and its target epitope may be used for the production of vaccines against S. aureus infection.     

黄芩素抑制金黄色葡萄球菌生物被膜的形成

细菌生物被膜的形成是导致细菌耐药和引起持续性感染的主要原因之一。本文通过检测黄芩素对金黄色葡萄球菌26112菌株（Staphylococcus aureus 26112,SA26112）多糖细胞间黏附素（polysaccharide intercellular adhesion, PIA）的合成和胞外DNA（extracellular DNA,eDNA）释放量的影响,及其对icaA和cidA基因表达量的影响,探讨黄芩素对金黄色葡萄菌生物被膜形成的抑制作用及其机制。结果显示,黄芩素能抑制SA26112生物被膜的形成,其抑杀SA26112的最低抑菌浓度和最低杀菌浓度均为0.04 mg/mL。0.16 mg/mL黄芩素和256 μg/mL环丙沙星单独作用时,均不能杀死其成熟生物被膜内的SA26112细菌,而当二者联用时则可杀死成熟生物被膜内的细菌。黄芩素能显著抑制SA26112菌株PIA的合成、eDNA的释放量及icaA和cidA基因的相对表达量。其中,0.04 mg/mL黄芩素作用SA26112菌株24 h,与对照组相比,eDNA的释放量减少97%,icaA和cidA基因的相对表达量分别减少62%和41%。上述结果表明,黄芩素能抑制SA26112菌株生物被膜的形成,其作用机制可通过降低icaA和cidA的基因表达量,进而影响PIA的合成和eDNA的释放,来抑制金黄色葡萄球菌生物被膜的形成。     

Identification of a heat-labile cellular nuclease in Staphylococcus aureus with properties similar to the extracellular nuclease (EC 3.1.4.7)

Besides the well-known heat-stable extracellular staphylococcal nuclease (EC 3.1.4.7) and cell surface bound nuclease, one more nuclease, which is heat-labile, has been identified and purified on phosphorylated cellulose column and characterized. Analyses by Sephadex G-75 gel chromatography indicates that the heat-labile cellular nuclease has molecular weight of about 16,000 similar to those of extracellular and cell-surface bound nucleases. Like the heat-stable nucleases, the heat-labile enzyme acts on both DNA and RNA, is more active on heat-denatured DNA, requires Ca²⁺ ions for activity and maximum catalytic activity is observed at pH 9.8–10 and at 45°C. The results suggest that the three enzymes have properties strikingly similar to one another and therefore may be related structurally.     

冬凌草甲素对金黄色葡萄球菌生物膜的抑制机制

【背景】金黄色葡萄球菌是一种常见的食源性致病菌,易在食品及加工器具表面形成生物膜,引起食品腐败和疾病的传播,威胁食品安全。【目的】研究冬凌草甲素抑制金黄色葡萄球菌生物膜形成的作用机制。【方法】使用结晶紫染色法和扫描电镜观察冬凌草甲素对金黄色葡萄球菌生物膜形成的抑制作用,刚果红平板法定性检测冬凌草甲素对细胞间多糖黏附素(polysaccharideintercellular adhesion,PIA)合成的影响,分光光度法测定冬凌草甲素对供试菌株胞外DNA (eDNA)释放量的影响,RT-PCR技术检测冬凌草甲素对供试菌株ica A、cid A、agr A和sar A基因表达量的影响。【结果】冬凌草甲素对金黄色葡萄球菌生物膜形成有较强的抑制作用;冬凌草甲素能显著抑制PIA的合成,且呈浓度剂量依赖;冬凌草甲素能抑制供试菌株e DNA的释放量,其中1/4最小抑菌浓度(minimum inhibitory concentration,MIC)的冬凌草甲素作用金黄色葡萄球菌16 h后,与对照组相比,e DNA的释放量降低了48.62%;冬凌草甲素可显著抑制金黄色葡萄球菌生物膜形成相关基因的表达,其中1/2MIC的冬凌草甲素作用金黄色葡萄球菌16 h后,ica A、cid A、agr A和sar A基因的表达量分别比对照降低了91.6%、94.7%、77.6%和70.4%。【结论】冬凌草甲素通过抑制ica A和cid A基因的表达,影响PIA的合成和eDNA的释放,进而干预生物膜的形成。     

Defining the immune mechanism with monoclonal antibodies

Summary It has long been realized that only the study of homogeneous antibodies or cell populations could enable a definitive understanding of much of the immune mechanism. Hybridoma technology has greatly facilitated such approaches. Hybridoma antibodies have been used to delineate both B cell and T cell subpopulations. T cell studies per se have been accomplished by the use of T cell hybridoma cell lines producing a variety of factors. Anti-idiotypes against B cell hybridoma antibodies have been used to characterize T cell receptors and factors. B cell studies have been facilitated by hybridomas that have made available the immunoglobulin of pre-B cells or defective B cell lines. Hybridoma antibodies have also been used to dissect closely related antibody families and the potential for responsiveness against a variety of antigenic determinants. Finally, hybridomas have provided a primary source of material for protein and DNA sequence analysis. In our laboratories hybridoma antibodies derived against the murine H-2 locus have demonstrated the ability of B cell antibodies to discriminate amongst H2 mutants—a capacity previously attributed only to T cell specificities. Hybridoma antibodies have also been generated by fusions with antigen stimulated neonatal B cells to provide homogeneous antibodies reflective of the earliest developmental immunoglobulin readout. Such probes should increase our understanding of the processes involved in the generation of both the T and B cell repertoires. Presented in the symposium on the Biology of Hybridomas at the 32nd Annual Meeting of the Tissue Culture Association. Washington, D.C., June 7–11, 1981. This work was supported by United States Public Health Service Grants AI 15797 and AI 15710. This symposium was supported in part by the following organizations: Bethesda Research Laboratories, Cetus Corporation, Hybritech Incorporated, MAB-Monoclonal Antibodies, Inc., National Capital Area Branch of the Tissue Culture Association, New England Nuclear Corporation, and Ortho Pharmaceutical Corporation.     

香芹酚抑制金黄色葡萄球菌生物被膜的形成

【背景】生物被膜是细菌的一种自我保护形式,可以增强细菌对药物及宿主免疫应答的抵抗力,引起细菌耐药性和持续性感染。【目的】探究香芹酚对金黄色葡萄球菌生物被膜的作用机制,为开发新型抗生物被膜药物提供可靠的理论依据。【方法】通过结晶紫染色法检测香芹酚对供试菌株生物被膜形成的抑制和对成熟生物被膜的清除作用;使用刚果红平板法探究香芹酚对供试菌株生物被膜形成过程中细胞间多糖黏附素(polysaccharide intercellular adhesion,PIA)合成的作用;通过分光光度法检测香芹酚对胞外DNA (extracellular DNA,eDNA)分泌的抑制作用;利用RT-PCR技术检测香芹酚对供试菌株的生物被膜相关基因icaA、cidA和sarA转录水平的影响。【结果】香芹酚对生物被膜形成的抑制和生物被膜的清除均有较强作用效果。256μg/mL香芹酚抑制PIA合成和e DNA释放的效果显著。香芹酚可通过抑制相关基因转录从而抑制生物被膜的形成,当64μg/mL的香芹酚作用后,sarA的转录水平降低了60.44%±2.91%,cidA的转录水平降低了76.48%±1.67%,icaA的转...     

白背飞虱单克隆抗体的制备及其特性的研究

应用杂交瘤技术,制备出4株高度特异性的白背飞虱单克隆抗体,分别命名为WPH-1H9、WPH-2B6、WPH-2E12和WPH-3F12。这些抗体与其它8种昆虫未发生交叉反应,其中WPH-2B6可与白背飞虱所有虫态发生反应,其余3株只与卵和雌成虫发生反应。应用免疫双扩散法鉴定抗体类型及亚类,结果表明：WPH-2B6为IgG_2b亚类,其余均为IgG₁亚类。SDS-聚丙烯酰胺凝胶电泳和Western blot印迹分析表明,白背飞虱抗原主要由分子量分别为182、116、66.2及40 kD的4个多肽组成,其中WPH-2B6与182、116 kD的多肽结合,其余3株的单抗只与116 kD的多肽具有亲和性。最后对这些单克隆抗体在捕食作用研究中的应用潜力进行了讨论。     

治疗耐甲氧西林金黄色葡萄球菌感染的抗体药物

耐甲氧西林金黄色葡萄球菌(methicillin-resistant staphylococcus aureus, MRSA) 作为超级细菌的典型代表,严重威胁人民群众的健康。目前经典的抗生素治疗和疫苗主动预防都无法有效控制MRSA的感染。近年来,随着抗体药物产业的兴起,诸多药物研发公司和研究人员致力于研究治疗性抗体,以期控制MRSA的感染并已经取得了很多突破。就治疗MRSA感染的抗体药物研究进展作一简要综述。     

An immobilized hybridoma culture perfusion system for production of monoclonal antibodies

A fixed bed perfusion system for hybridoma cell immobilization is presented. The system consists of a culturing vessel (300 ml total volume) in which polyurethane (PU) sponges in the form of small cubes of about 5 mm sides are packed. Cells are immobilized by physical entrapment in the foam matrix. By entrapment of the cells in the pores of the matrix high cell concentration can be maintained in a mechanically protected environment. Medium is continuously circulated by an airlift pump mounted in the cell-free chamber (700 ml total volume).Medium flow rate, feeding rate, dissolved oxygen, pH, nutrient uptake and waste product formation can be easily monitored and controlled. Steady state conditions are established with medium dilution rates of 1.0–1.5 reactor volume per day. The steady state is characterized by a constant cell density, constant culture volume and constant glucose and lactate levels. Cell-free supernatant is collected continuously in a cold room adjacent to the 37°C culture room. Monoclonal antibodies (MAb) are produced at a concentration of 150–200 g/ml for several weeks. An important feature of the system is the capacity to maintain a population of cells after the growth phase in a non-proliferating state for extended time periods expressing high titers of MAb.Abbreviations DO Dissolved Oxygen - FBS Fetal Bovine Serum - FBR Fixed Bed Reactor - MAb Monoclonal Antibody - PU polyurethane     

Inhibition of Staphylococcus aureus by the commensal bacteria of human milk

AIMS: To study the bacterial diversity in expressed human milk with a focus on detecting bacteria with an antimicrobial activity against Staphylococcus aureus, known as a causative agent of maternal breast infections and neonatal infections. METHODS AND RESULTS: Random isolates (n = 509) were collected from breast milk samples (n = 40) of healthy lactating women, genotypically identified, and tested for antimicrobial activity against Staph. aureus. Commensal staphylococci (64%) and oral streptococci (30%), with Staph. epidermidis, Strep. salivarius, and Strep. mitis as the most frequent isolates, were the predominant bacterial species in breast milk. One-fifth of Staph. epidermidis and half of Strep. salivarius isolates suppressed growth of Staph. aureus. Enterococci (Ent. faecalis), isolated from 7.5% of samples, and lactic acid bacteria (LAB) (Lactobacillus rhamnosus, Lact. crispatus, Lactococcus lactis, Leuconoctoc mesenteroides), isolated from 12.5% of samples, were also effective against Staph. aureus. One L. lactis isolate was shown to produce nisin, a bacteriocin used in food industry to prevent bacterial pathogens and spoilage. CONCLUSIONS: Expressed breast milk contains commensal bacteria, which inhibit Staph. aureus. SIGNIFICANCE AND IMPACT OF THE STUDY: The strains inhibitory against the pathogen Staph. aureus have potential use as bacteriotherapeutic agents in preventing neonatal and maternal breast infections caused by this bacterium.     

Identification of the secreted macromolecular immunogens of Staphylococcus aureus by analysis of serum

The ability of sera to recognise secreted macromolecules of Staphylococcus aureus was examined by ELISA and Western immunoblotting. Individual secreted proteins were also studied using both human sera and sera from rabbits immunised with secreted macromolecules. Patients sera showed a wide range of IgG antibody titres to secreted macromolecules and whole bacteria. Controls showed a significantly lower IgG response. Western immunoblotting revealed that a significant number of secreted proteins were recognised by circulating IgG antibodies. Surprisingly, both the sera from controls and from patients recognised similar macromolecules including a number of potential virulence factors. The major difference was in the IgG binding to a 16-kDa component, which was recognised by the majority of the sera from infected individuals, but only by a small number of sera from healthy controls. The higher incidence of antibodies recognising the 16 kDa component may be related to our earlier finding that the major bone resorbing component of S. aureus is a heterodimeric protein containing a 16-kDa subunit, the activity of which could be blocked by sera.     

抗菌肽17BIPHE2对金黄色葡萄球菌生物被膜的抑制作用

【目的】研究抗菌肽17BIPHE2单独使用及联合抗生素对金黄色葡萄球菌(Staphylococcus aureus)生物被膜的抑制作用。【方法】采用刚果红平板测试法和结晶紫染色评估受试菌形成生物被膜的能力;微量肉汤稀释法和琼脂平板测试法测定金黄色葡萄球菌最小抑菌浓度(MIC)和最小杀菌浓度(MBC);利用抑制金黄色葡萄球菌黏附实验和生物被膜形成抑制实验观察17BIPHE2单独使用及联合抗生素对生物被膜黏附阶段和形成阶段的影响;通过扫描电子显微镜(SEM)观察17BIPHE2单独使用及联合抗生素对成熟生物被膜的清除作用。【结果】17BIPHE2的MIC为8μmol/L,1/2×MIC就可以有效抑制浮游菌的生长。单独使用17BIPHE2在细菌黏附阶段抑制率为40%,在生物被膜形成阶段抑制率达到35%。17BIPHE2联合抗生素使用较单独使用抗生素其抑制率均有所下降。生物被膜成熟阶段17BIPHE2于1/4×MIC浓度即可促进生物被膜崩解,1×MIC生物被膜崩解同时细菌黏附量有所下降,联合万古霉素促进生物被膜崩解同时细菌胞质大量外泄。【结论】抗菌肽17BIPHE2具有良好的抑制金黄色葡萄球菌生物被膜作用,联合抗生素其抗生物被膜作用进一步提高。这将为治疗由金黄色葡萄球菌生物被膜引起的相关感染提供了一个新思路。     

小鼠杂交瘤单克隆抗体快速制备技术研究进展

小鼠杂交瘤单克隆抗体来源稳定、后期易制备、产量高,是免疫学中使用最为普遍的抗体.传统的耗时费力的杂交瘤制备技术无法满足日益增长的市场需求.文中从抗原设计筛选、B细胞富集与筛选、骨髓瘤细胞的改造、融合技术的改进、阳性杂交瘤细胞筛选及单克隆抗体性能快速测定中所涉及的快速制备技术方面进行阐述,以期为系统化的小鼠杂交瘤单克隆抗...     

Hybridoma monoclonal antibodies in the analysis fo human cell surface antigens

Summary In the present paper, we will summarize studies we have performed on two distinct human lymphocyte cell surface antigens defined by monoclonal antibodies: Leu-1 and HLA-DR. Presented in the symposium on The Biology of Hybridomas at the 32nd Annual Meeting of the Tissue Culture Association, Washington, D.C., June 7–11, 1981. This work was supported by USPHS-NIH Grants CA-21223, AI-11313, and CA-09302. This symposium was supported in part by the following organizations: Bethesda Research Laboratories, Cetus Corporation, Hybritech Incorporated, MAB-Monoclonal Antibodies, Inc., National Capital Area Branch of the Tissue Culture Association, New England Nuclear Corporation, and Ortho Pharmaceutical Corporation.     

Inhibition of methicillin-resistant Staphylococcus aureus by an in vitro continuous-flow culture containing human stool microflora

We used an in vitro continuous-flow culture model of human stool microflora to examine the ability of human stool microflora to inhibit growth of two methicillin-resistant S. aureus (MRSA) strains. Continuous-flow cultures consistently eliminated MRSA inocula of 10(6) cfu/mL within 4 days, and addition of continuous-flow culture resulted in elimination of a pre-established MRSA culture ( approximately 10(8) cfu/mL) within 6-8 days. Anaerobic or "aerobic" (i.e., continuous bubbling of room air to eliminate obligate anaerobes) cultures eliminated MRSA at similar rates. The MRSA strains were unable to replicate under anaerobic conditions in sterile filtrates produced from the continuous-flow culture, but rapid growth occurred when glucose was added. These data demonstrate that indigenous stool microflora efficiently eliminate MRSA colonization and obligate anaerobes are not essential for inhibition. Our findings also suggest that inhibition of MRSA in continuous-flow cultures is due to depletion of nutrients rather than production of inhibitory conditions.     

Plasmid curing in Staphylococcus aureus by antibiotics affecting the bacterial cell wall

Abstract Strains of Staphylococcus aureus were converted into L-forms with β-lactam antibiotics, vancomycin and lysostaphin. Reverted bacteria obtained after several transfers in protoplast forms (unstable L-forms) as well as stable L-forms lost their plasmids. Curing was obtained whatever the plasmid size (from 3.4 to 28.2 kb) but complete curing required cell division. Elimination of plasmids in wall-less organisms could be the result of an inhibition of new rounds of plasmid replication following the loss of DNA-envelope interactions.     

Immunological Recognition of Fibronectin-Binding Proteins of Staphylococcus aureus and Staphylococcus capitis,Strain LK499

Antibodies to fibronectin-binding proteins (FnBPs) of Staphylococcus aureus, including binding domain of FnBPA, the D region, or the A-C regions of FnBPB were produced in rabbits and mice. These antibodies were used to characterize cell-associated FnBPs of S. aureus strain Cowan I, S. aureus strain U320 and a coagulase-negative Staphylococcus capitis strain LK499 as well as extracellular FnBPs in culture supernatants of the strain U320. FnBPs of S. aureus were predominantly FnBPA, while FnBPB was hardly detected on the cells or in culture supernatant of these S. aureus strains. Moreover, S. capitis strain LK499 possessed different FnBP(s) compared to S. aureus because the antibodies to S. aureus FnBPs did not recognize FnBP(s) on S. capitis.