Monoclonal Antibodies Raised against Native Major Capsid Proteins of Lactococcal c2-Like Bacteriophages

Phage Q38, a representative member of the c2 species, was purified by CsCl gradient and used to immunize BALB/c mice. Monoclonal antibodies (MAbs) were raised and then characterized by enzyme-linked immunosorbent assay. Two MAbs of isotype immunoglobulin G2a, designated 2A5 and 6G7, reacted only with phages belonging to the c2 species and not with phages of the 936 and P335 species, with a Lactococcus lactis cell extract, or with phage DNA. Immunoelectron microscopy showed that both MAbs recognized only phage head proteins. They did not react with any denatured phage proteins in Western blot assays. However, when the nitrocellulose membranes were treated with a Triton-based buffer to assist in protein renaturation, MAbs 2A5 and 6G7 recognized the two major capsid proteins with molecular masses of 80 and 170 kDa. Competitive inhibition tests showed that the two MAbs bind to overlapping epitopes. These MAbs may be a useful tool for monitoring c2 bacteriophages during dairy fermentation and in genetic studies.     

Involvement of the Major Capsid Protein and Two Early-Expressed Phage Genes in the Activity of the Lactococcal Abortive Infection Mechanism AbiT

The dairy industry uses the mesophilic, Gram-positive, lactic acid bacterium (LAB) Lactococcus lactis to produce an array of fermented milk products. Milk fermentation processes are susceptible to contamination by virulent phages, but a plethora of phage control strategies are available. One of the most efficient is to use LAB strains carrying phage resistance systems such as abortive infection (Abi) mechanisms. Yet, the mode of action of most Abi systems remains poorly documented. Here, we shed further light on the antiviral activity of the lactococcal AbiT system. Twenty-eight AbiT-resistant phage mutants derived from the wild-type AbiT-sensitive lactococcal phages p2, bIL170, and P008 were isolated and characterized. Comparative genomic analyses identified three different genes that were mutated in these virulent AbiT-insensitive phage derivatives: e14 (bIL170 [e14(bIL170)]), orf41 (P008 [orf41(P008)]), and orf6 (p2 [orf6(p2)] and P008 [orf6(P008)]). The genes e14(bIL170) and orf41(P008) are part of the early-expressed genomic region, but bioinformatic analyses did not identify their putative function. orf6 is found in the phage morphogenesis module. Antibodies were raised against purified recombinant ORF6, and immunoelectron microscopy revealed that it is the major capsid protein (MCP). Coexpression in L. lactis of ORF6(p2) and ORF5(p2), a protease, led to the formation of procapsids. To our knowledge, AbiT is the first Abi system involving distinct phage genes.     

Detection by ELISA of Two Tobamoviruses in Orchids Using Monoclonal Antibodies

Five monoclonal antibodies (McAbs) were raised to the tobamovirus, odontoglossum ringspot virus (ORSV). All five McAbs reacted with the virus in double antibody sandwich (DAS) ELISA but not in an ELISA using virus-coated plates. All the McAbs recognized a panel of ORSV strains and isolates, although one of the antibodies reacted better with some isolates and another reacted less with certain isolates than with type ORSV. It was possible to use the same McAbs both as coating and as biotinylated antibody in DAS-ELISA. None of the five McAbs was able to bind to orchid strains of tobacco mosaic virus (TMV). In order to detect strains of both viruses, ORSV and TMV, in infected orchids it was necessary to include also McAbs raised against TMV in the immunoassays. The use of a mixed polyclonal-monoclonal antibody DAS-ELISA system is advocated for detecting both tobamoviruses in orchids.     

Uniform Nomenclature for Monoclonal Antibodies Directed Against Virus-Coded Proteins of Simian Virus 40 and Polyoma Virus

A uniform nomenclature has been agreed upon for monoclonal antibodies directed against virus-coded proteins of simian virus 40 and polyoma virus. Blocks of numbers from PAb1 to PAb999 have been allocated to workers involved in the isolation of monoclonal antibodies of this type. The correspondence between PAb numbers and previously used names is given.     

Two Monoclonal Antibodies Recognize Alzheimer''s Neurofibrillary Tangles, Neurofilament, and Microtubule-Associated Proteins

Two monoclonal antibodies that recognize Alzheimer's neurofibrillary tangles (ANTs), AD10 and AB18, have been characterized by immunoblotting against human and calf spinal cord neurofilament (NF) and calf brain microtubule preparations. Both antibodies bind to the 200-kilodalton (kd) (NF-H) and 160-kd (NF-M) but not to the 68-kd (NF-L) NF triplet proteins. They also bind to high-molecular-weight microtubule-associated proteins (MAPs) and tau. AD10 immunostains MAP2 and MAP1 families, whereas AB18 stains mainly MAP1 bands. Preincubation of intact filament preparation or nitrocellulose strips containing electroblotted NF proteins with Escherichia coli alkaline phosphatase completely blocks AD10 binding and partially blocks binding of AB18. These results suggest that the determinants recognized by these antibodies are phosphorylated. Immunoblotting of peptide fragments generated by limited proteolysis of NF proteins with alpha-chymotrypsin and Staphylococcus aureus V8 protease shows that the localization of the antigenic determinants to AD10 and AB18 in NF-H is approximately 100 and 60 kd, respectively, away from the carboxy terminal, a region previously shown to form the NF projection side arm. In NF-M, the antigenic determinants to both antibodies are located also in the projection side arm, in a 60-kd polypeptide adjacent to the alpha-helical filament core. The results show that ANTs contain at least two phosphorylated antigenic sites that are present in NF and MAPs, a finding suggesting that ANTs may be composed of proteins or their fragments with epitopes shared by cytoskeletal proteins.     

Monoclonal Antibodies for Detection of 4-Hydroxynonenal Modified Proteins

A promising approach to study lipid peroxidation pathology is antibodies recognizing aldehydes which react with and became bound to amino acid side chains of proteins. We present in this study the characterization of several monoclonal antibodies which recognize 4-hydroxynonenal (HNE) modified proteins. Six out of 20 antibodies recognizing HNE modified BSA were able to detect HNE-protein adducts in peroxidized liver microsomes. Two of these antibodies were selected and characterized. Both antibodies could also detect HNE-protein adducts in oxidized low density lipoprotein. They exhibit no detectable cross reaction with proteins modified by malonaldehyde, nonanal, nonenal and 4-hydroxyhexenal. Protein bound 4-hydroxyoctenal and 4-hydroxydecenal were recognized to some extent. Further characterization revealed that the two antibodies are highly selective for HNE bound to histidine with only some cross reaction to HNE bound to lysine and cysteine. Preliminary quantitative ELISA-analysis showed that oxidized microsomes and oxidized LDL contain 12 nmol and 3 nmol HNE-histidine per mg protein respectively.     

用多抗原肽(MAP)免疫制备单克隆抗体的研究

目前制备单克隆抗体杂交瘤细胞株的传统方法是用纯度很高的天然蛋白作为抗原来免疫动物。此方法虽然成功率很高,但提取和纯化作为抗原用的高纯蛋白确非常困难,在某些情况下甚至是不可能的。为了克服以上不足,近年来发展起用合成多肽作为抗原制备单克隆抗体^[1,2]。这个技术的难点是用合成多肽作为抗原来免疫动物不易获得与天然蛋白呈特异反应的抗体^[3-5]。近年来国外科学家发明了一种称为“多抗原肽”(Multiple antigenic peptide, MAP)物质用作抗原^[6,7]。它是一个由赖氨酸核和多个由同一多肽分子构成的具有免疫原性的大分子。这个大分子的多肽部分组成丁造成动物免疫应答的多个相同抗原决定簇．而赖氨酸核部分只起连接多肽的作用,它本身没有免疫原性。实验证明多抗原肽和佐剂配合使用会引起动物强烈的免疫应答。本文介绍的是从Calpain [EC 3.4.22.17] 28kDa 小亚基上选取的一段氪基酸序列 AAQYNPEPPP-PRTH(氨基酸从73到86)与赖氨酸核偶联形成多抗原肽来制备单克隆抗体。Calpain的水解蛋白活性依附于钙离子浓度。它有两种异构酶：在μmol/L钙离子浓度下被激活的称为“μ-calpain．而在mmol/L钙离子浓度下被激活的称为m-calpain。这两种异构酶都由两个亚基组成。其大亚基的分子量为80kDa,它包括酶的活性区和与钙离子结合区。两种酶的大亚基氨基酸序列各不相同。其小亚基的分子量为28 kDa,这两种酶具有完全相同的小亚基氨基酸序列。有关Calpain的详细资料可参阅文献[8]。     

Differentiation of Yam Virus Isolates Using Symptomatology, Western Blot Assay, and Monoclonal Antibodies

Monoclonal antibodies (mAbs) were prepared against a yam mosaic virus (YMV) isolate from the Côte d'Ivoire. Symptomatology, Western immunoblotting, and ELISA were used to discriminate 69 isolates of YMV originating from different Dioscorea species and from various yam producing areas. These isolates induced two types of symptoms, were of four different electrophoretic mobilities and formed two serogroups. These results suggest that at least six differetit groups of isolates exist, three of which infect the main cultivated species in various geographical areas while two others were from unusual samples in our collection. An isolate from 'Pilimpikou Yam' from Central Burkina Faso was serologically distinct. It is concluded that there is a significant variability among yam virus isolates which is unrelated to the origin of the isolate (geographic or host species). It is suggested that precautions should be taken in order to avoid international exchange of infected material.     

Subcellular Distribution of Distinct Nucleolin Subfractions Recognized by Two Monoclonal Antibodies

Monoclonal antibodies binding to different domains of nucleolin have been used to localize nucleolin in tissue culture cells ofXenopus laevis.The monoclonal antibody b6-6E7 binds to an epitope in the N-terminal domain, which contains arrays of phosphorylation consensus sites. This monoclonal antibody binds to nucleolin of oocytes and of eggs with high affinity. In contrast, the monoclonal antibody Nu-1H6 binds poorly to the modified forms of nucleolin arising during meiosis and mitosis. In interphase cells, monoclonal antibody b6-6E7 preferentially stains the periphery of the nucleoli, where most of the rRNA accumulates. Staining by monoclonal antibody Nu-1H6 complements this pattern by staining mainly the center of the nucleoli. The epitope of monoclonal antibody Nu-1H6 is within the central domain of nucleolin, which contains the first two RNA binding domains. RNase treatment of cells results in loss of nucleolin from nucleoli. In mitotic cells, both monoclonal antibodies decorate the surface of condensing chromosomes in prophase. The periphery of the condensed chromosomes in metaphase and anaphase is preferentially stained by monoclonal antibody b6-6E7.     

Functional Activity of Antibodies Directed towards Flagellin Proteins of Non-Typhoidal Salmonella

Non-typhoidal Salmonella (NTS) serovars Typhimurium and Enteritidis are major causes of invasive bacterial infections in children under 5 years old in sub-Saharan Africa, with case fatality rates of ~20%. There are no licensed NTS vaccines for humans. Vaccines that induce antibodies against a Salmonella Typhi surface antigen, Vi polysaccharide, significantly protect humans against typhoid fever, establishing that immune responses to Salmonella surface antigens can be protective. Flagella proteins, abundant surface antigens in Salmonella serovars that cause human disease, are also powerful immunogens, but the functional capacity of elicited anti-flagellar antibodies and their role in facilitating bacterial clearance has been unclear. We examined the ability of anti-flagellar antibodies to mediate microbial killing by immune system components in-vitro and assessed their role in protecting mice against invasive Salmonella infection. Polyclonal (hyperimmune sera) and monoclonal antibodies raised against phase 1 flagellin proteins of S. Enteritidis and S. Typhimurium facilitated bacterial uptake and killing of the homologous serovar pathogen by phagocytes. Polyclonal anti-flagellar antibodies accompanied by complement also achieved direct bacterial killing. Serum bactericidal activity was restricted to Salmonella serovars expressing the same flagellin used as immunogen. Notably, individual anti-flagellin monoclonal antibodies with complement were not bactericidal, but this biological activity was restored when different monoclonal anti-flagellin antibodies were combined. Passive transfer immunization with a monoclonal IgG antibody specific for phase 1 flagellin from S. Typhimurium protected mice against lethal challenge with a representative African invasive S. Typhimurium strain. These findings have relevance for the use of flagellin proteins in NTS vaccines, and confirm the role of anti-flagellin antibodies as mediators of protective immunity.     

Abortive Infection of Shigella dysenteriae P2 by T2 Bacteriophage

We have investigated some of the biochemical events that accompany the abortive infection by T2 of Shigella dysenteriae lysogenized with the temperate phage P2. After infection with T2, protein and RNA synthesis continued for 3 to 5 min. The virus-induced enzyme, deoxycytidylate hydroxymethylase was produced in reduced amounts (15% of normal), and the extent of deoxyribonucleic acid (DNA) synthesis was 0.1% of that found with a nonlysogenic strain. Measurements of the production of acid-soluble fragments and sedimentation analyses failed to detect enzymatic degradation of the infecting viral DNA which could be specifically related to the presence of the prophage P2. Each interaction between T2 and a bacterium resulted in the death of the cell. This observation is consistent with results obtained with other types of bacteria which show that only when a nucleolytic attack occurs on T2 DNA does the cell have an increased capacity to survive after adsorption of T2.     

Characterization of Monoclonal Antibodies Directed Against the Rat Neurotensin Receptor NTS1

G protein-coupled receptors (GPCRs) are integral membrane proteins that mediate cellular responses to a variety of ligands and represent major drug targets. Despite their medical importance, detailed structural information is limited because only one GPCR has been crystallized and its structure determined. To develop tools to aid in the formation of well-ordered crystals, we generated monoclonal antibodies with high affinity to the rat neurotensin receptor. All antibodies bound to the C-terminus of the receptor, which may reflect the selection strategy used to identify high-affinity binders. Further characterization revealed that some antibodies bound to the receptor in a sodium chloride sensitive manner, but others did not. Epitope mapping revealed distinct antigenic regions within the receptor C-terminus. Tight binding of Fab fragments to the receptor was verified by size exclusion chromatography.     

Determination of Tetanus Toxin and Toxoid by ELISA Using Monoclonal Antibodies

The procedure for obtaining monoclonal antibodies TT-1, TT-2, and TT-3 against tetanus toxin/toxoid is described. It is shown that the commercial DTP vaccine and tetanus toxoid conjugated with a low-molecular-weight hapten can both be used as immunogens. Monoclonal antibodies TT-1 and TT-2 neutralized tetanus toxin in vivo. The monoclonal antibodies obtained were used to design and compare several schemes of quantitative determination of tetanus toxoid and toxin by ELISA. A more sensitive competitive ELISA allowed the detection of as much as 0.01 EC/ml toxoid and 50 LD₅₀/ml toxin.     

High Throughput Identification of Monoclonal Antibodies to Membrane Bound and Secreted Proteins Using Yeast and Phage Display

Antibodies are ubiquitous and essential reagents for biomedical research. Uses of antibodies include quantifying proteins, identifying the temporal and spatial pattern of expression in cells and tissue, and determining how proteins function under normal or pathological conditions. Specific antibodies are only available for a small portion of the proteome, limiting study of those proteins for which antibodies do not exist. The technologies to generate target-specific antibodies need to be improved to obtain high quality antibodies to the proteome at reasonable cost. Here we show that renewable, validated, and standardized monoclonal antibodies can be generated at high throughput, without the need for antigen production or animal immunizations. In this study, 60 protein domains from 24 selected secreted proteins were expressed on the surface of yeast and used for selection of phage antibodies, over 400 monoclonal antibodies were identified within 3 weeks. A subset of these antibodies was validated for binding to cancer cells that overexpress the target protein by flow cytometry or immunohistochemistry. This approach will be applicable to many of the membrane-bound and the secreted proteins, 20–40% of the proteome, accelerating the timeline for Ab generation while reducing the cost.     

Analysis of the Epitopes Recognized by Mouse Monoclonal Antibodies Directed to Yersinia enterocolitica Heat-Shock Protein 60

To determine amino acid sequences of the epitopes recognized by monoclonal antibodies (mAbs) 3C8 and 5C3 directed against Yersinia enterocolitica heat-shock protein (HSP60), a dot blot analysis was perfomed using synthesized peptides of Y. enterocolitica HSP60 such as peptides p316-342, p327-359, p340-366, p316-326, p316-321, p319-323, and p321-326 which represent positions of amino acids in Y. enterocolitica HSP60. The dot blot analysis revealed that 5C3 mAb reacted with p316-342, p316-326 and p321-326, and 3C8 mAb p316-342 and p316-326. These results indicate that the epitopes recognized by the mAbs were associated with eleven amino acids, Asp Leu Gly Gln Ala Lys Arg Val Val Ile Asn, of p316-326. The sequence homology between p316-326 of Y. enterocolitica HSP60 and the rest of the HSP60 family suggests that the five amino acids of Lys, Arg, Val, Ile and Asn, which are highly conserved in the HSP60 family, might be related with the epitope recognized by 3C8. In contrast, it was also demonstrated that three amino acids of Leu, Gly and Val, which are not well conserved in the HSP60 family, might be related to the epitope recognized by 5C3.     

Generation of Neutralizing Human Monoclonal Antibodies against Parvovirus B19 Proteins

Infections caused by human parvovirus B19 are known to be controlled mainly by neutralizing antibodies. To analyze the immune reaction against parvovirus B19 proteins, four cell lines secreting human immunoglobulin G monoclonal antibodies (MAbs) were generated from two healthy donors and one human immunodeficiency virus type 1-seropositive individual with high serum titers against parvovirus. One MAb is specific for nonstructural protein NS1 (MAb 1424), two MAbs are specific for the unique region of minor capsid protein VP1 (MAbs 1418-1 and 1418-16), and one MAb is directed to major capsid protein VP2 (MAb 860-55D). Two MAbs, 1418-1 and 1418-16, which were generated from the same individual have identity in the cDNA sequences encoding the variable domains, with the exception of four base pairs resulting in only one amino acid change in the light chain. The NS1- and VP1-specific MAbs interact with linear epitopes, whereas the recognized epitope in VP2 is conformational. The MAbs specific for the structural proteins display strong virus-neutralizing activity. The VP1- and VP2-specific MAbs have the capacity to neutralize 50% of infectious parvovirus B19 in vitro at 0.08 and 0.73 μg/ml, respectively, demonstrating the importance of such antibodies in the clearance of B19 viremia. The NS1-specific MAb mediated weak neutralizing activity and required 47.7 μg/ml for 50% neutralization. The human MAbs with potent neutralizing activity could be used for immunotherapy of chronically B19 virus-infected individuals and acutely infected pregnant women. Furthermore, the knowledge gained regarding epitopes which induce strongly neutralizing antibodies may be important for vaccine development.     

Mechanisms Mediating Enhanced Neutralization Efficacy of Staphylococcal Enterotoxin B by Combinations of Monoclonal Antibodies

Staphylococcal enterotoxin B (SEB) is a superantigen that cross-links the major histocompatibility complex class II and specific V-β chains of the T-cell receptor, thus forming a ternary complex. Developing neutralizing mAb to disrupt the ternary complex and abrogate the resulting toxicity is a major therapeutic challenge because SEB is effective at very low concentrations. We show that combining two SEB-specific mAbs enhances their efficacy, even though one of the two mAbs by itself has no effect on neutralization. Crystallography was employed for fine-mapping conformational epitopes in binary and ternary complexes between SEB and Fab fragments. NMR spectroscopy was used to validate and identify subtle allosteric changes induced by mAbs binding to SEB. The mapping of epitopes established that a combination of different mAbs can enhance efficacy of mAb-mediated protection from SEB induced lethal shock by two different mechanisms: one mAb mixture promoted clearance of the toxin both in vitro and in vivo by FcR-mediated cross-linking and clearance, whereas the other mAb mixture induced subtle allosteric conformational changes in SEB that perturbed formation of the SEB·T-cell receptor·major histocompatibility complex class II trimer. Finally structural information accurately predicted mAb binding to other superantigens that share conformational epitopes with SEB. Fine mapping of conformational epitopes is a powerful tool to establish the mechanism and optimize the action of synergistic mAb combinations.