Passive immunization for the treatment and prevention of HIV infection.

Passive immunization using serum or immunoglobulin preparations has been used in the prophylaxis and treatment of many bacterial and viral diseases. Preliminary attempts to use these methods to prevent HIV infection in chimpanzees have been promising. With the identification of polyclonal and monoclonal antibodies with protective activity against HIV in in vitro systems, the possibility of using these reagents in vivo takes on new relevance. The potential and problems of using passively administered anti-HIV antibodies for HIV prophylaxis and treatment are discussed, as well as the relative merits of polyclonal versus monoclonal reagents.     

Monoclonal antibodies: methods and clinical laboratory applications

Kohler and Milstein have shown that individual clones of normal antibody-secreting lymphocytes could be immortalized by fusion with myeloma cells. These investigators initiated a new era of technology with the successful in vitro production of monoclonal antibodies via somatic cell hybridization. With the use of monoclonal antibodies, many major problems arising from the limited specificity and reproducibility of conventional antisera can be solved. Some of the commonly employed methods for the production of monoclonal antibody are: (1) fusion of sensitized lymphocytes and myelomas from different sources to produce continuous antibody-producing cell lines; (2) in vitro viral transformation of sensitized lymphocytes to form continuous antibody-producing cells; (3) hybrid fusion of sensitized lymphocytes and continuous B lymphocyte cell lines. During the past few years, monoclonal antibody methodology has been used in almost every area of biological research. Monoclonal antibodies have been used as structural probes for proteins and hormones, and as highly specific agents for histocompatibility testing, tumor localization, immunotherapy, purification of molecules, identification of new surface antigens on lymphocytes and tumor cells, and detection of drug levels and microbial and parasitic diseases. In addition, several investigators have developed alternative methods for the production of human as well as mouse and rat monoclonal antibodies. The new technology of in vitro production of animal and human monoclonal antibodies will have many future applications in diagnosis and therapy in laboratory and clinical medicine.     

A new type of anti-ganglioside antibodies present in neurological patients

High titers of anti-GA1 antibodies have been associated with neurological syndromes. In most cases, these antibodies cross-react with the structurally related glycolipids GM1 and GD1b, although specific anti-GA1 antibodies have also been reported. The role of specific anti-GA1 antibodies is uncertain since the presence of GA1 in the human nervous system has not been clarified. A rabbit was immunized with GD1a and its sera were screened for antibody reactivity by standard immunoassay methods (HPTLC-immunostaining and ELISA). Anti-GD1a antibodies were not detected but, unexpectedly, anti-GA1 IgG-antibodies were found. Antibody binding to GA1 was inhibited by soluble GA1 but also by GD1a. These results indicate that the rabbit produced antibodies that recognize epitopes present on the glycolipids, that are absent or not exposed on solid phase adsorbed GD1a. We investigated the presence of these unusual anti-ganglioside antibodies in normal and neurological patient sera. Approximately, 10% of normal human sera contained low titer of specific anti-GA1 IgG-antibodies but none of them recognized soluble GD1a. High titers of IgG-antibodies reacting only with GA1 were detected in 12 patient sera out of 325 analyzed. Of these, 6 sera showed binding that was inhibited by soluble GD1a and four of them also by GM1. This new type of anti-ganglioside antibodies should be considered important elements for understanding of the pathogenesis of these diseases as well as their diagnosis.     

A Method for the Parallel and Sequential Generation of Single-Domain Antibodies for the Proteomic Analysis of Human Blood Plasma

A new efficient method for the parallel and sequential stepwise generation of single-domain antibodies to various high-abundance human-plasma proteins has been described. Single-domain antibodies have a number of features that favorably distinguish them from classical antibodies. In particular, they are able to recognize unusual unique conformational epitopes of native target proteins, small in size, and relatively easily produced and modified; have enhanced stability; and rapidly renature after denaturation. As a consequence, the immunosorbents that utilize these antibodies can be reused without any significant loss of activity. The principal novelty and universality of the described method is that it enables the sequential generation of antibodies to a number of high-abundance and yet unknown antigens of a complex protein mixture without the need for purified antigens. The effectiveness of the method is demonstrated by the example of generation of single-domain antibodies to a number of high-abundance proteins of the human blood plasma. The produced antibodies are promising biotechnological tools that can be used to develop prototypes for new diagnostic and therapeutic agents, as well as appropriate immunoaffinity-based methods for removal, enrichment, analysis, and/or targeting of specified proteins and their complexes from (in) the human blood. As we show, the generated single-domain antibodies can be efficiently used in designing new immunosorbents. As a rule, commercially available analogous immunosorbents that utilize classical antibodies remove many major proteins from the blood plasma immediately, while immunosorbents for many individual proteins are difficult to find and rather expensive. Single-domain antibodies generated by our method are unique new materials that allow for the development of more efficient and delicate approaches to pretreatment of plasma and the analysis of various blood plasma biomarkers.     

Nucleic acid capture assay,a new method for direct quantitation of nucleic acids

Technologies allowing direct detection of specific RNA/DNA sequences occasionally serve as an alternative to amplification methods for gene expression studies. In these direct methods the hybridization of probes takes place in complex mixtures, thus specificity and sensitivity still limit the use of current technologies. To address these challenges, we developed a new technique called the nucleic acid capture assay, involving a direct multi-capture system. This approach combines a 3′-ethylene glycol scaffolding with the incorporation of 2′-methoxy deoxyribonucleotides in the capture sequences. In our design, all nucleotides other than those complementary to the target mRNA have been replaced by an inert linker, resulting in significant reductions in non-specific binding. We also provide a versatile method to detect the presence of captured targets by using specific labeled probes with alkaline phosphatase-conjugated anti-label antibodies. This direct, flexible and reliable technique for gene expression analysis is well suited for high-throughput screening and has potential for DNA microarray applications.     

Candida endophthalmitis after heroin abuse

Three cases of ocular candidosis involving heroin abusers have been observed in 1983 in Toulouse department of ophthalmology. These three patients had used iranian brown heroin. Twenty similar cases have been published in these last years. This new pathology can be explained on two reasons. The first is that the drug abusers have some immunity pertubation; however, immunity exploration in these patients does not reveal any immunodeficiency. The second reason, certainly more important, is the method of using heroin. The diagnosis of Candida endophthalmitis of course based on clinical context must be proved by biological tests. Candida albicans is never identified in aqueous humor. For this reason, it seems very interesting to detect anti-candida antibodies in aqueous humor. It has been used as methods of dosage laser Nephelemetry for IgG and immunofluorescence for candidosis antibodies. The criterion used is similar to the toxoplasmosis coefficient established by Desmonts (3). In two cases, this test was the only way that permits us to have certitude of candidosis ocular diagnosis. Otherwise the observations show that anterior chamber punction is more significant when there is an anterior uveitis.     

The flow cytometric analysis of cytokines using multi-analyte fluorescence microarray technology

Cytokines, which are small peptides that act as hormones of the immune system, affect cells throughout the body in a variety of different ways. These cellular signaling molecules often have synergistic or opposing effects on various cell types and often different cytokines have overlapping activities. There is great advantage, therefore, to be able to assess a pattern of cytokine responses in certain inflammatory, autoimmune, transplant or immunodeficiency states. This is one of the major advantages of the new particle-based flow cytometric assays, which have become available. We have employed such assays to analyze up to 10 different cytokines in cultured supernatants of stimulated mononuclear cells and in as little as 75 microL of serum from patients with a variety of different disorders. In developing these assays and validating them for use in our esoteric reference laboratory (ARUP Laboratories), we have found that a variety of heterophile antibodies can lead to both false positive and false negative results. This review will describe the development of our multi-analyte cytokine assays and document the interference derived from heterophile antibodies. Lastly, we will point out various procedures that we have utilized to include internal controls directly in the assays, which allow one to routinely detect these interfering antibodies, as well as methods we have developed to circumvent the interference posed by these antibodies.     

Production and characterization of monoclonal antibodies to rabbit lymphocyte subpopulations. I. Tissue immunofluorescence and flow cytometric analysis

Nine monoclonal antibodies to rabbit T cells and B subpopulations have been generated from three separate fusions of spleen cells from mice immunized with fractionated populations of rabbit lymphocytes. These monoclonal antibodies, as well as a previously described rabbit T cell monoclonal antibody, 9AE10, have been analyzed by immunofluorescence staining on frozen tissue sections of rabbit thymus, spleen, and appendix. This screening method permits rapid identification of the lymphocyte subdomains in each tissue which is not possible by other screening methods. Each monoclonal antibody selected has a unique tissue staining pattern. Flow cytometric analysis of these monoclonal antibodies, using indirect immunofluorescence techniques on thymocytes, splenocytes, and PBL, revealed varying percentages of positive cells and individual mean fluorescence intensities indicating different epitope densities for each antigen. These monoclonal antibodies are now being used to characterize normal lymphocyte function and the role of specific lymphocyte subpopulations in experimental disease models in the rabbit.     

Applications of monoclonal antibodies in aquaculture

Monoclonal antibodies (MAbs) currently are being applied to the study of fish immunology and fish infectious diseases. MAbs to fish immunoglobulins (Igs) have helped isolate fish Igs, identify heavy and light chain variants in fish Ig, study the ontogeny of B lymphocytes, and improve techniques for the measurement of fish Ig and specific antibodies (Abs). MAbs have been obtained against several leucocyte surface antigens and are being used as markers for different subsets of fish leukocytes: neutrophils, non-specific cytotoxic cells and cells responsible for the mixed leucocyte reaction. The sensitivity and specificity of many immunoassays for identifying fish pathogens have been improved by the use of MAbs. Further improvement of these tests is currently being attempted by the use of MAbs together with the polymerase chain reaction (PCR). Epizootiological studies of fish diseases are beginning to emerge from the use of these reagents and techniques. In the near future these new methods should detect low levels of pathogens in adult carriers and perhaps in eggs, thus helping to prevent the dissemination of fish diseases. MAbs to fish pathogens are also being used for passive immunization studies as well as for conformational probes in the development of genetically engineered vaccines.     

Manufacture of recombinant polyclonal antibodies

Polyclonal antibody therapy in the form of hyper-immune serum has for more than a century been used for treatment of many infectious diseases. However, with the emergence of first antibiotics and later recombinant monoclonal antibody therapy, the use of hyper-immune serum has declined. The main reason for this is that methods for consistent manufacturing of safe hyper immune immunoglobulin products have been lacking. In contrast, manufacturing processes of recombinant monoclonal antibodies follow a well established schedule and it appears obvious to use similar methods to produce recombinant polyclonal products. However, the methods for monoclonal antibody manufacturing are, for several reasons, not directly applicable to generation and manufacture of polyclonal recombinant antibodies. A new production strategy based on recombinant mammalian producer cells has recently been developed to support consistent generation of recombinant polyclonal antibodies for therapeutic use. This review describes aspects of this novel technology with emphasis on the generation, production and characterization procedures employed, and provides comparison with alternative polyclonal and monoclonal antibody manufacturing strategies.     

Biotechnology techniques for the development of new tumor specific peptides

Peptides, proteins and antibodies are promising candidates as carriers for radionuclides in endoradiotherapy. This novel class of pharmaceuticals offers a great potential for the targeted therapy of cancer. The fact that some receptors are overexpressed in several tumor types and can be targeted by small peptides, proteins or antibodies conjugated to radionuclides has been used in the past for the development of peptide endoradiotherapeutic agents such as ⁹⁰Y-DOTATOC or radioimmunotherapy of lymphomas with Zevalin. These procedures have been shown to be powerful options for the treatment of cancer patients.Design of new peptide libraries and scaffolds combined with biopanning techniques like phage and ribosome display may lead to the discovery of new specific ligands for target structures overexpressed in malignant tumors. Display methods are high throughput systems which select for high affinity binders. These methods allow the screening of a vast amount of potential binding motifs which may be exposed to either cells overexpressing the target structures or in a cell-free system to the protein itself. Labelling these binders with radionuclides creates new potential tracers for application in diagnosis and endoradiotherapy. This review highlights the advantages and problems of phage and ribosome display for the identification and evaluation of new tumor specific peptides.     

Designing fast converging phylogenetic methods

Absolute fast converging phylogenetic reconstruction methods are provably guaranteed to recover the true tree with high probability from sequences that grow only polynomially in the number of leaves, once the edge lengths are bounded arbitrarily from above and below. Only a few methods have been determined to be absolute fast converging; these have all been developed in just the last few years, and most are polynomial time. In this paper, we compare pre-existing fast converging methods as well as some new polynomial time methods that we have developed. Our study, based upon simulating evolution under a wide range of model conditions, establishes that our new methods outperform both neighbor joining and the previous fast converging methods, returning very accurate large trees, when these other methods do poorly.     

Monoclonal antibodies, 30 years of success

The hybridoma fusion technology, proposed in 1975, gave for the first time an access to murine monoclonal antibodies. The high potential of these new molecules, as laboratory tools, was exploited during the two following decades. Nowadays, antibodies, still omnipresent in both diagnostic and research domains, have progressively invaded the therapeutic field. New technologies, such as phage display and transgenic mice, have been implemented, allowing for the isolation of fully human antibodies. The natural complexity of the antibody molecules and the development of engineering methodologies helped making them ideal candidates for new applications and immunotherapeutic challenges. The present review is a temporary update of the different antibody-derived molecules as well as a walk-through among the techniques recently applied to antibody engineering. In addition it also address an important issue, such as the development of expression systems suitable large-scale production of recombinant antibodies.     

Non-infectious aggregates of the prion protein react with several PrPSc-directed antibodies

The key event in the pathogenesis of prion diseases is the conformational conversion of the normal prion protein (PrP) (PrP^C) into an infectious, aggregated isoform (PrP^Sc) that has a high content of β-sheet. Historically, a great deal of effort has been devoted to developing antibodies that specifically recognize PrP^Sc but not PrP^C, as such antibodies would have enormous diagnostic and experimental value. A mouse monoclonal IgM antibody (designated 15B3) and three PrP motif-grafted monoclonal antibodies (referred to as IgG 19–33, 89–112, and 136–158) have been previously reported to react specifically with infectious PrP^Sc but not PrP^C. In this study, we extend the characterization of these four antibodies by testing their ability to immunoprecipitate and immunostain infectious and non-infectious aggregates of wild-type, mutant, and recombinant PrP. We find that 15B3 as well as the motif-grafted antibodies recognize multiple types of aggregated PrP, both infectious and non-infectious, including forms found in brain, in transfected cells, and induced in vitro from purified recombinant protein. These antibodies are exquisitely selective for aggregated PrP, and do not react with soluble PrP even when present in vast excess. Our results suggest that 15B3 and the motif-grafted antibodies recognize structural features common to both infectious and non-infectious aggregates of PrP. Our study extends the utility of these antibodies for diagnostic and experimental purposes, and it provides new insight into the structural changes that accompany PrP oligomerization and prion propagation.     

Development of rat anti-mouse interleukin 3 monoclonal antibodies which neutralize bioactivity in vitro

Several rat anti-mouse interleukin 3 (IL-3) monoclonal antibodies have been developed which inhibit the biologic activity of mouse IL-3. These antibodies were produced in rats immunized with preparations of purified, recombinant mouse IL-3, obtained from transiently transfected COS7 cell supernatant. Hybridomas secreting anti-IL-3 were selected initially either on the basis of their giving a positive signal in an indirect enzyme-linked immunosorbent assay, or for their ability to inhibit the proliferation of the IL-3-dependent mouse mast cell line, MC/9. Neutralizing rat monoclonal IgG1, IgG2a, and IgG2b antibodies have been identified; these also block IL-3-induced proliferation of the NFS-60 and IC2 cell lines. These antibodies also blocked the IL-3-induced proliferation of mouse bone marrow-derived colony-forming units-culture suggesting that the same epitopes on IL-3 influence receptor recognition for both the proliferation of factor-dependent cell lines as well as normal bone marrow cells. Fab fragments produced from certain of the IgG2a-neutralizing antibodies blocked as well as the parent IgG. Antibody cross-blocking studies identified one neutralizing antibody apparently recognizing an epitope that was spatially distinct from those recognized by the other blocking antibodies tested. The development of these neutralizing rat monoclonal antibodies to mouse IL-3 should facilitate further investigation on the role of this factor in hemopoietic regulation.     

DARPins and other repeat protein scaffolds: advances in engineering and applications

Antibodies have long been regarded as the only class of binding proteins. With the emergence of protein engineering techniques, new binding proteins based on alternative scaffolds have been designed. Additionally, modern technologies for selection and evolution from libraries are independent of the antibody scaffold and could thus be readily used for obtaining specific binding proteins. One important group of alternative scaffolds is based on repeat proteins. Nature is widely using these proteins to modulate protein-protein interactions, and even in the adaptive immune system of jawless vertebrates; the step to their application as an alternative to antibodies seems therefore logical. In this review, progress on DARPins and other repeat protein scaffolds will be discussed. Advances in their design as well as novel applications will be highlighted.     

A challenge to the ultrasensitive chemical method for the analysis of oligo- and polysialic acids at a nanogram level of colominic acid and a milligram level of brain tissues

Polysialic acid (polySia) is a functional epitope and is known: 1) to regulate normal fertilization of lower vertebrates and invertebrates; 2) to be expressed on neural cell adhesion molecule (NCAM) when the formation or re-arrangement of nervous tissues takes place during embryonic stages as well as in adults of higher vertebrates; and 3) to be re-expressed in several human tumors. Thus, polySia serves as oncodevelopmental antigen. To date sensitive biochemical diagnostic probes (antibodies and endo-N-acylneuraminidase) to detect polySia are known. However, these reagents are not commercially available yet and they are only reactive to specific types of polySia structure. Moreover, precise information not only on diversity but also on the length or degree of polymerization (DP) of extended polySia chains is considered important in understanding the molecular mechanism of biosynthesis of polySia chains and fine-tuning of NCAM-NCAM adhesive interaction by polySia chain but cannot be obtained with these biochemical probes. We have been continuously making efforts to develop and improve the sensitivity of chemical methods for polySia analysis toward these challenging problems. This article presents our most recently developed chemical method for polySia analysis and its use in obtaining new information on DP of colominic acid samples and polySia chains present in rat brain tissues with the highest sensitivity that has ever been attained.     

Antibodies to the alpha-subunit of insulin receptor from eggs of immunized hens

Simple methods for the generation, purification, and assay of antibodies to the alpha-subunit of insulin receptor from eggs of immunized hens have been described. Chicken antibodies against the alpha-subunit inhibit insulin binding to the receptor and stimulate glucose oxidation as well as autophosphorylation of the beta-subunit. Thus the properties of chicken antibodies are very similar to those of antibodies found in human autoimmune diseases and different from rabbit antibodies obtained against the same antigen.     

Frontier of therapeutic antibody discovery:The challenges and how to face them

Therapeutic monoclonal antibodies have become an important class of modern medicines.The established technologies for therapeutic antibody discovery such as humanization of mouse antibodies,phage display of human antibody libraries and transgenic animals harboring human IgG genes have been practiced successfully so far,and many incremental improvements are being made constantly.These methodologies are responsible for currently marketed therapeutic antibodies and for the biopharma industry pipeline which are concentrated on only a few dozen targets.A key challenge for wider application of biotherapeutic approaches is the paucity of truly validated targets for biotherapeutic intervention.The efforts to expand the target space include taking the pathway approach to study the disease correlation.Since many new targets are multi-spanning and multimeric membrane proteins there is a need to develop more effective methods to generate antibodies against these difficult targets.The pharmaceutical properties of therapeutic antibodies are an active area for study concentrating on biophysical characteristics such as thermal stability and aggregation propensity.The immunogenicity of biotherapeutics in humans is a very complex issue and there are no truly predictive animal models to rely on.The in silico and T-cell response approaches identify the potential for immunogenicity;however,one needs contingency plans for emergence of antiproduct antibody response for clinical trials.