Monoclonal antibodies and the transformation of blood typing

Today, when monoclonal antibodies (mAbs) have become one of the most important classes of therapeutic drugs, it is easy to forget how much they have transformed our healthcare in other ways. One of the first clinical areas, as this paper shows, where mAbs made their mark was in the field of blood typing. The adoption of mAbs for this purpose was done with little public fanfare or funding. Nonetheless, it radically transformed the accuracy and cost of blood typing and shifted the procedure away from a dependence on reagents made from human blood donated by volunteers. This paper argues that the development of mAbs as reagents for blood typing laid the foundation for the first large-scale production of mAbs thereby paving the way to the advent of mAb diagnostics and therapeutics.     

Trifunctional conjugation reagents. Reagents that contain a biotin and a radiometal chelation moiety for application to extracorporeal affinity adsorption of radiolabeled antibodies

A method of removing radiolabeled monoclonal antibodies (mAbs) from blood using a device external to the body, termed extracorporeal affinity-adsorption (EAA), is being evaluated as a means of decreasing irradiation of noncancerous tissues in therapy protocols. The EAA device uses an avidin column to capture biotinylated-radiolabeled mAbs from circulated blood. In this investigation, three trifunctional reagents have been developed to minimize the potential deleterious effect on antigen binding brought about by the combination of radiolabeling and biotinylation of mAbs required in the EAA approach. The studies focused on radiolabeling with (111)In and (90)Y, so the chelates CHX-A' '-DTPA and DOTA, which form stable attachments to these radionuclides, were incorporated in the trifunctional reagents. The first trifunctional reagent prepared did not incorporate a group to block the biotin cleaving enzyme biotinidase, but the two subsequent reagents coupled aspartic acid to the biotin carboxylate for that purpose. All three reagents used 4,7,10-trioxa-1,13-tridecanediamine as water-soluble spacers between an aminoisophthalate core and the biotin or chelation group. The mAb conjugates were radioiodinated to evaluate cell binding as a function of substitution. Radioiodination was used so that a direct comparison with unmodified mAb could be made. Evaluation of the number of conjugates per antibody versus cell binding immunoreactivities indicated that minimizing the number of conjugates was best. Interestingly, a decrease of radioiodination yield as a function of the number of isothiocyanate containing conjugates per mAb was noted. The decreased yields were presumably due to the presence of thiourea functionality formed in the conjugation reaction. Radiolabeling with (111)In and (90)Y was facile at room temperature for conjugates containing the CHX-A' ', but elevated temperature (e.g., 45 degrees C) was required to obtain good yields with the DOTA chelate. Stability of (90)Y labeled mAb in serum, and when challenged with 10 mM EDTA, was high. However, challenging the (90)Y labeled mAb with 10 mM DTPA demonstrated high stability for the DOTA containing conjugate, but low stability for the CHX-A' ' containing conjugate. Thus, the choice between these two chelating moieties might be made on requirements for facile and gentle labeling versus very high in vivo stability. Application of the trifunctional biotinylation reagents to the blood clearance of labeled antibodies in EAA is under investigation. The new reagents may also be useful for other applications.     

Blood typing South American camelids

Preliminary blood typing tests were made on New World camelids, guanacos, llamas, and two hybrids. Erythrocyte samples were tested against a battery of cattle blood typing reagents. Three different reagents were prepared from rabbit anti-erythrocyte sera. Transferrin variation and lectin polymorphism also were observed. No naturally occurring isoantibodies were found. Blood typing tests of New World camelids were shown to be feasible for studies of taxonomic relationships.     

Production of a diagnostic monoclonal antibody in perennial alfalfa plants.

The increasing use of monoclonal antibodies (mAbs) in diagnostic reagents necessitates efficient and cost-effective mAb production methods. In blood banks, one of the most routinely used reagents is the anti-human IgG reagent used for the detection of non-agglutinating antibodies. Here we report the production of a functional, purified anti-human IgG, through the expression of its encoding genes in perennial transgenic alfalfa. Transgenic plants expressing the light- and heavy-chain encoding mRNAs were obtained, and plants from crosses were found to express fully assembled C5-1. The purification procedure yielded mainly the H2L2 form with specificity and affinity identical to those of hybridoma-derived C5-1. The ability to accumulate the antibody was maintained both in parental F1 lines during repeated harvesting and in clonal material; the antibody was stable in the drying hay as in extracts made in pure water. Also, plant and hybridoma-derived C5-1 had similar in vivo half-lives in mice. These results indicate that plant C5-1 could be used in a diagnostic reagent as effectively as hybridoma-derived C5-1, and demonstrates the usefulness of perennial systems for the cost-effective, stable, and reliable production of large amounts of mAbs.     

Bovine leukocyte antigens

Using bovine erythrocyte typing reagents in a leukocyte microcytotoxicity system, bovine peripheral blood leukocytes were found to have specific surface antigens. In this study, no obvious association between leukocyte.antigens and erythrocyte antigens of any individual animal was found. The leukocyte and erythrocyte antigens appeared to be distinct from each other.     

Monoclonal antibodies against Vibrio anguillarum O2 and Vibrio ordalii identify antigenic differences in lipopolysaccharide O-antigens

Abstract Monoclonal antibodies (mAbs) that recognize distinct species-specific antigenic epitopes in O-antigens from Vibrio anguillarum O2, O2a and certain O2b strains (mAb 7B4) and from Vibrio ordalii strains (mAbs A16 and 7D11) were generated. Western immunoblot analysis using these mAbs revealed that vibrio strains grown in the presence of fresh rainbow trout blood expressed lipopolysaccharide (LPS) with longer (high molecular mass) O-antigens and extracellular capsular layers when compared to strains grown without rainbow trout blood. We also generated mAbs that react with O-antigens from V. anguillarum serotype O1 (mAbs 7B8, 7B5 and 1C3) and serotype O3 (mAbs 13A1 and 14C5) strains. These mAbs provide rapid and accurate diagnostic reagents for serological differentiation of V. ordalii from serotype O2 strains of V. anguillarum , and for serotyping of these pathogenic vibrios.     

DNA typing of primate major histocompatibility complex (Mhc)-DQA1 locus by PCR and dot blot hybridization.

Non-human primates (NHPs) are increasingly utilized as models to investigate different aspects of immune responses against self (autoimmunity) and foreign antigens. These animals provide valuable models for testing the efficacy of candidate vaccines against pathogens such as human immunodeficiency virus (HIV) and also fertility regulating agents (immunocontraceptives). In order to fully understand the effects of vaccination, it may be necessary to elucidate the immunogenetic background of these animals. The major histocompatibility complex (Mhc) molecules play an important role in the generation of effective immune responses. Serological techniques have been used in the identification of human leukocyte antigens (HLA) necessary for cross-matching organs and tissues for transplantation. However, the application of this technique for typing monkey Mhc alleles has been hampered by unavailability of well characterized immunological reagents. Polymerase chain reaction (PCR)-based techniques such as restriction fragment length polymorphism (RFLP) and sequence-specific oligonucleotide probe hybridization (SSOP) have been extensively used for typing HLA-DP, DQ and DR alleles. A commercially available Kit (AmpliTypeR) designed for amplification and typing of HLA DQalpha alleles is routinely used in typing DNA samples for forensic casework. In the present study, we have evaluated this kit for possible application in routine typing of primate DQA1 alleles. Genomic DNA from ten African primate species (23 individuals) was isolated from peripheral blood lymphocytes and polymorphic second exon of DQA1 locus amplified using GH26 and GH27 PCR primers. The PCR products were hybridized on a nylon membrane containing immobilized sequence-specific oligonucleotide probes. Our results show seven of the nine probes hybridizing with primate DQA1 alleles, indicating that typing of equivalent primate alleles can be accomplished at lower stringency conditions. However, it may be necessary to design additional oligonucleotides probes (based on available primate DQA1 sequences) to improve the discriminating power of this kit for use in routine typing of Old World monkey DQA1 alleles.     

Immunogenetic studies of rhesus monkeys

Five alloimmune rhesus monkey blood typing reagents have been produced which define two new blood group loci inMacaca mulatto. Three of these reagents detect blood group factors at theM locus; the other two detect factors at theN locus. By typing over 1900 pedigreed monkeys we have established that these two loci are independent of each other and of any of our previously defined blood group systems.     

Inhibition of Bordetella pertussis filamentous hemagglutinin-mediated cell adherence with monoclonal antibodies

Abstract Filamentous hemagglutinin (FHA), a 220-kDa protein located on the surface of Bordetella pertussis , is one of the major cell adhesins of this bacterium. We have produced three hybridoma cell lines that express monoclonal antibodies (mAbs) against FHA: X3C, X3E and X4B. The anti-FHA mAbs X3C and X3E reacted with 220-kDa FHA protein bands on Western blots. The mAb X4B, which reacted with FHA in ELISA, did not bind to FHA in a Western blot assay. All three mAbs seemed to be directed to the same epitope or to epitopes in close proximity as suggested by competition ELISAs. All three mAbs were able to inhibit the adherence of Chinese hamster ovary cells to purified FHA, and they could also inhibit the FHA-mediated agglutination of goose red blood cells. The attachment of B. pertussis to epithelial cell monolayers was inhibited by the mAb X3C. These antibodies are very useful probes to identify the presence of FHA in bordetellae species and in clinical reagents such as pertussis vaccines, and to characterize the functional domains of this important bacterial adhesin.     

Immunogenetic studies of rhesus monkeys

Five alloimmune blood typing reagents have been produced which define five new blood group systems in rhesus monkeys. Each of the five independent blood group loci is comprised of a detectable allele and a null allele. Using these new reagents and those previously described, we can potentially identify close to a million phenotypes in rhesus monkeys.     

Efficient isolation of novel human monoclonal antibodies with neutralizing activity against HIV-1 from transgenic mice expressing human Ig loci

Despite considerable interest in the isolation of mAbs with potent neutralization activity against primary HIV-1 isolates, both for identifying useful targets for vaccine development and for the development of therapeutically useful reagents against HIV-1 infection, a relatively limited number of such reagents have been isolated to date. Human mAbs (hu-mAbs) are preferable to rodent mAbs for treatment of humans, but isolation of hu-mAbs from HIV-infected subjects by standard methods of EBV transformation of B cells or phage display of Ig libraries is inefficient and limited by the inability to control or define the original immunogen. An alternative approach for the isolation of hu-mAbs has been provided by the development of transgenic mice that produce fully hu-mAbs. In this report, we show that immunizing the XenoMouse G2 strain with native recombinant gp120 derived from HIV(SF162) resulted in robust humoral Ab responses against gp120 and allowed the efficient isolation of hybridomas producing specific hu-mAbs directed against multiple regions and epitopes of gp120. hu-mAbs possessing strong neutralizing activity against the autologous HIV(SF162) strain were obtained. The epitopes recognized were located in three previously described neutralization domains, the V2-, V3- and CD4-binding domains, and in a novel neutralization domain, the highly variable C-terminal region of the V1 loop. This is the first report of neutralizing mAbs directed at targets in the V1 region. Furthermore, the V2 and V3 epitopes recognized by neutralizing hu-mAbs were distinct from those of previously described human and rodent mAbs and included an epitope requiring a full length V3 loop peptide for effective presentation. These results further our understanding of neutralization targets for primary, R5 HIV-1 viruses and demonstrate the utility of the XenoMouse system for identifying new and interesting epitopes on HIV-1.     

Engineered antibody fragments and the rise of single domains

With 18 monoclonal antibody (mAb) products currently on the market and more than 100 in clinical trials, it is clear that engineered antibodies have come of age as biopharmaceuticals. In fact, by 2008, engineered antibodies are predicted to account for >30% of all revenues in the biotechnology market. Smaller recombinant antibody fragments (for example, classic monovalent antibody fragments (Fab, scFv)) and engineered variants (diabodies, triabodies, minibodies and single-domain antibodies) are now emerging as credible alternatives. These fragments retain the targeting specificity of whole mAbs but can be produced more economically and possess other unique and superior properties for a range of diagnostic and therapeutic applications. Antibody fragments have been forged into multivalent and multi-specific reagents, linked to therapeutic payloads (such as radionuclides, toxins, enzymes, liposomes and viruses) and engineered for enhanced therapeutic efficacy. Recently, single antibody domains have been engineered and selected as targeting reagents against hitherto immunosilent cavities in enzymes, receptors and infectious agents. Single-domain antibodies are anticipated to significantly expand the repertoire of antibody-based reagents against the vast range of novel biomarkers being discovered through proteomics. As this review aims to show, there is tremendous potential for all antibody fragments either as robust diagnostic reagents (for example in biosensors), or as nonimmunogenic in vivo biopharmaceuticals with superior biodistribution and blood clearance properties.     

Recent advances in the generation of human monoclonal antibody

The use of monoclonal antibodies (mAbs) has now gained a niche as an epochal breakthrough in medicine. Engineered antibodies (Abs) currently account for over 30% of biopharmaceuticals in clinical trials. Several methods to generate human mAbs have evolved, such as (1) immortalization of antigen-specific human B cell hybridoma technology, (2) generation of chimeric and humanized antibody (Ab) from mouse Ab by genetic engineering, (3) acquisition of antigen-specific human B cells by the phage display method, and (4) development of transgenic mice for producing human mAbs. Besides these technologies, we have independently developed a method to generate human mAbs by combining the method of in vitro immunization using peripheral blood mononuclear cells and the phage display method. In this paper, we review the developments in these technologies for generating human mAbs.     

Characterization of monoclonal antibody's binding kinetics using oblique-incidence reflectivity difference approach

Monoclonal antibodies (mAbs) against human proteins are the primary protein capture reagents for basic research, diagnosis, and molecular therapeutics. The 2 most important attributes of mAbs used in all of these applications are their specificity and avidity. While specificity of a mAb raised against a human protein can be readily defined based on its binding profile on a human proteome microarray, it has been a challenge to determine avidity values for mAbs in a high-throughput and cost-effective fashion. To undertake this challenge, we employed the oblique-incidence reflectivity difference (OIRD) platform to characterize mAbs in a protein microarray format. We first systematically determined the K_on and K_off values of 50 mAbs measured with the OIRD method and deduced the avidity values. Second, we established a multiplexed approach that simultaneously measured avidity values of a mixture of 9 mono-specific mAbs that do not cross-react to the antigens. Third, we demonstrated that avidity values of a group of mAbs could be sequentially determined using a flow-cell device. Finally, we implemented a sequential competition assay that allowed us to bin multiple mAbs that recognize the same antigens. Our study demonstrated that OIRD offers a high-throughput and cost-effective platform for characterization of the binding kinetics of mAbs.     

The relationship between the S system of blood groups and potassium levels in red blood cells of cattle

Red cell potassium concentrations were determined in 5 breeds of cattle. Most cattle had red cells with less than 50 mmol K/litre and although a few Hereford and Friesian cows had levels approaching 70 mmol K/litre, it was only in the Jersey breed that values above this were found. Attempts were made to correlate potassium levels with serological specificity using cattle S system and sheep M system blood typing reagents. Red cells negative for cattle factors S, H', and S were never positive for sheep L; cells positive for H' were usually positive for M, and those positive for S were always positive for L. No obvious relationship was found between potassium levels and serological type, although some consistent trends were noted in Friesians and Herefords. Limited family data indicated that a major difference in potassium levels of red cells in cattle may be controlled by two codomi-nant alleles.     

Epitope mapping analysis of apolipoprotein B-100 using a surface plasmon resonance-based biosensor

Using a surface plasmon resonance (SPR)-based biosensor (BIA-technology), we have studied the interaction of ten different murine monoclonal antibodies (mAbs, all IgG₁), raised against the main protein constituent of human low density lipoprotein (LDL), i.e. the apolipoprotein B-100 (apoB-100). These mAbs identify distinct domains on apoB-100, relevant to LDL-receptor interaction: epitopes in the amino-terminal region (mAbs L7, L9, L10 and L11: aa 1–1297) and in the middle region (mAb 6B: aa 1480–1693; mAbs 2A, 3B: aa 2152–2377; mAbs 9A, L2 and L4: aa 2657–3248) of native apoB-100. A multisite binding analysis was performed to further characterize the epitopes recognized by all these mAbs. A rabbit anti-mouse IgG₁-Fc antibody (RAM.Fc) was first coupled to the gold surface in order to capture one anti-human apoB-100 mAb. ApoB-100 protein was subsequently injected and allowed to react with this immobilized, oriented antibody. Multisite binding assays were then performed, by sequentially flowing other mAbs, in different orders, over the sensing surface. The capacity of each mAb to interact with the entrapped apoB-100 in a multimolecular complex was monitored in real time by SPR. The results achieved were comparable to those obtained by western immunoblotting using the same reagents. However, SPR ensures a more detailed epitope identification, demonstrating that BIA-technology can be successfully used for mapping distinct epitopes on apoB-100 protein in solution dispensing with labels and secondary tracers; moreover, compared with conventional immunoassays, it is significantly time saving (CNR-P.F. MADESS 2).     

Characterization of monoclonal antibody's binding kinetics using oblique-incidence reflectivity difference approach

Monoclonal antibodies (mAbs) against human proteins are the primary protein capture reagents for basic research, diagnosis, and molecular therapeutics. The 2 most important attributes of mAbs used in all of these applications are their specificity and avidity. While specificity of a mAb raised against a human protein can be readily defined based on its binding profile on a human proteome microarray, it has been a challenge to determine avidity values for mAbs in a high-throughput and cost-effective fashion. To undertake this challenge, we employed the oblique-incidence reflectivity difference (OIRD) platform to characterize mAbs in a protein microarray format. We first systematically determined the K_on and K_off values of 50 mAbs measured with the OIRD method and deduced the avidity values. Second, we established a multiplexed approach that simultaneously measured avidity values of a mixture of 9 mono-specific mAbs that do not cross-react to the antigens. Third, we demonstrated that avidity values of a group of mAbs could be sequentially determined using a flow-cell device. Finally, we implemented a sequential competition assay that allowed us to bin multiple mAbs that recognize the same antigens. Our study demonstrated that OIRD offers a high-throughput and cost-effective platform for characterization of the binding kinetics of mAbs.     

Oligoclonal Enzyme-linked Immunosorbent Assay Capable of Determining the Major Food Allergen, Ovomucoid, Irrespective of the Degree of Heat Denaturation

We have recently established an enzyme-linked immunosorbent assay (ELISA) for total ovomucoid determination, irrespective of the degree of its heat denaturation, by using a monoclonal antibody (mAb) 7D specific to the carbohydrate moiety of ovomucoid (Biosci Biothechnol Biochem, 68, 2490–2497, 2004). Two novel methods have been developed to improve the ELISA. First, its sensitivity was enhanced 100 times by using an oligoclonal cocktail of mAb 7D and two other mAbs with different epitopes as a second antibody. Second, it was shown that usage of denaturing reagents such as SDS and β-mercaptoethanol for extraction was acceptable for ELISA within a range of stability of a first antibody on a solid phase. Properties of the oligoclonal sandwich ELISA system thus constructed were discussed in connection with allergen labeling.     

Bovine monoclonal alloantibodies to blood group antigens prepared by murine x bovine or (murine x bovine) x bovine interspecies fusions.

In order to obtain monoclonal alloantibodies against bovine blood group antigens, lymph node cells from calves immunized with bovine red blood cells (RBC) were fused with either murine NSO/1 myeloma cells or a HAT sensitive murine x bovine heterohybridoma cell line. Both fusion partners resulted in heterohybridoma cell lines, producing monoclonal alloantibodies against bovine red blood cell antigens. Several clones produced antibodies against identical antigens and some of these clones have been further analysed. The antibodies produced by these selected cell lines have been compared with conventional polyclonal antisera used in bovine blood typing service. Thus extensive tests--including the ISAG Comparison Tests 1989/90 and 1991/92--have proved that monoclonal alloantibodies specific for the internationally recognized bovine red cell antigens A2, I1, O1, Q, A', B', Q', C1, R1, X1, S and Z have been produced. The Q, A', B', and C1 antibodies react weakly with certain phenogroups, whereas the A2, I1, O1, Q', R1, X1, S and Z antibodies have proved to be excellent blood typing reagents and have now substituted the polyclonal antisera in routine bovine blood typing in our laboratory.     

Canine blood groups: description of 20 specificities

Twenty blood typing reagents, four agglutinins and 16 operable in the antiglobulin test, were prepared from 54 antisera which were produced in 24 dogs. Two of the reagents were identified as anti-B and Nf6. Two of the antigens were shown by absorption and family studies to be linear subtypes. In most cases, detailed family studies demonstrated a Mendelian dominant inheritance for the genes controlling the canine red cell antigens. Gene frequencies were determined in various breeds of dogs and in the dingo.