Monoclonal Antibodies in Immunodiagnosis & Immunotherapy

Affinity-matured human antibodies have demonstrated efficacy as countermeasures for exposure to botulinum neurotoxin (BoNT), which is the cause of the disease botulism category A select bioterror agent. Little is known, however, about the potential role of natural (un-mutated) antibodies in the protective immune response to BoNT. Here we describe the cloning of two human IgM antibodies that bind serotype A BoNT. Both are un-mutated IgM antibodies, consistent with an origin in naive B cells. One of the antibodies is able to fully neutralize a lethal dose of serotype A BoNT in vivo. These results suggest that the natural human antibody repertoire may play a role in protection from exposure to biological toxins.

Monoclonal antibodies (MAbs) that bind neisserial lipooligosaccharides (LOS) have been widely used in structural studies of these glycolipids. MAb 2-1-L8 binds LOS with a lactosyl α chain (Gal β1-4 Glc β1-4 [GlcNAc α1-2 Hep2 α1-3] Hep1 α1-KDO) and at least one phosphoethanolamine (PEA) substitution of Hep2, but the requirement for PEA substitution and/or the exact position of this substitution, cyclic or exocyclic, remains unclear. In order to clarify the exact specificity of this MAb, we engineered an isogenic family of lpt mutants that each make LOS with a lactosyl α chain, but that lacked cyclic (-3Hep2), exocyclic (-6Hep2), or any PEA residues. Mass spectrometry showed that mutants that lack either Lpt3 or Lpt6 make small amounts of LOS with two PEA substitutions. Thus, each enzyme is able to phosphoethanolaminylate the alternate site, albeit with low efficiency. LOS made by the mutant that lacked both Lpt3 and Lpt6 was devoid of PEA. LOS made by the Δlpt3 mutant did not bind MAb 2-1-L8 on Western blot analysis, whereas Δ pt6 LOS did. Analysis of intact mutants by fluorescence-activated cell sorting confirmed that PEA substitution at 3Hep2, but not at 6Hep2, is needed for optimal binding of MAb 2-1-L8. These data confirm that the MAb 2-1-L8 epitope requires a -3Hep2 cyclic PEA substitution for optimal conformation and that this MAb specifies the PEA -3Hep2 lactosyl LOS structure.

Human (h) CD7 is a 40 kDa single chain Ig superfamily molecule that is expressed on thymocytes, a major subunit of peripheral T cells, and most natural killer cells. Ligands for hCD7 include the epithelial cell-produced molecule, K-12, and galectin. Mice deficient in CD7 have been shown to be resistant to LPS-induced endotoxic shock syndromes. However, monoclonal antibodies (MAb) to mouse (m) CD7 have yet to be produced, nor is the distribution of mCD7 protein in mice known. We have raised a panel of three rat MAbs to mCD7 by immunizing rats with recombinant mCD7 protein. However, using Western blot and immunoprecipitation of tissue extracts from mouse thymus, spleen, liver, brain, lymph node and skin, these anti-mouse CD7 MAbs bound only to murine heat shock protein 60 (HSP-60) present both in wild-type (CD7^+/+) and CD7-deficient (CD7^−/−) mice. Epitope mapping of the sites on HSP-60 and recombinant mCD7 recognized by mCD7 MAbs demonstrated non-homologous amino acid sequence epitopes recognized by anti-CD7 MAbs on both proteins. These data demonstrated molecular mimicry of mCD7 with HSP-60, and leave open the question of surface expression of mCD7.

Proliferating cell nuclear antigen (PCNA) is a homotrimeric ring-shaped protein that encircles the DNA and acts as a stationary loading platform for multiple, transiently interacting partners participating in various DNA transactions. This essential cellular component, originally characterized as a nuclear antigen of dividing cells, is evolutionary highly conserved from yeast to human. Within the eukaryotic cell, PCNA serves as a processivity factor for DNA polymerase δ and plays a key role in DNA replication, repair, cell cycle regulation, and post-replicative transactions like DNA methylation and chromatin remodelling. All these cellular processes are regulated by a complex network comprising cell cycle dependent changes in expression levels, dynamics, interactions, and localization of PCNA. Here we report the generation and characterization of the first rat monoclonal antibody (MAb) against human PCNA, designated as PCNA 16D10. We demonstrated that PCNA 16D10 is a highly affine and specific MAb suited for ELISA, immunoblotting, immunoprecipitation, and immunofluorescence. The characteristic punctate staining of S phase cells allows the identification of proliferating cells and the monitoring of cell cycle progression.

The natural selection of small organic molecules (osmolytes) that protect cellular proteins against denaturing stresses is characteristic of plants and animals that have adapted to environmental extremes. In this study the role of osmolytes on the structural stability of antibody in long-term storage was investigated. Native antibody preparations isolated from cell culture supernatant were formulated with different additives for storage stabilization. The additives studied were arginine, proline, and sorbitol. Structural stability of antibody investigated and effect of these osmolytes on retaining the native structure in long-term storage were established. CD spectra of native IgG showed the characteristics expected for β-proteins. After the passing of given time, the β-sheet structure of IgG was reduced but the addition of the osmolyte to antibody solution prevented loosing the ordered structures. These results can be effective for stabilization of proteins in commercial and industrial processes.

The effect of mild hypothermic (32°C) conditions on cell growth, cell-cycle progress, and antibody production of hybridoma C₂E₇ cells was investigated in the present study. The growth of hybridoma cells was slower during the mild hypothermic condition compared to that at 37°C; this led to about 10% decrease in maximum viable cell density and volumetric antibody productivity. However, under mild hypothermic growth conditions, the culture viability was substantially improved and the specific antibody productivity was enhanced compared to that at 37°C. The average specific productivity for the entire batch culture at 32°C is about 5% higher than that at 37°C. Cell-cycle analysis data showed that there was no growth arrestment during the mild hypothermic growth of hybridoma cells. The G1-phase cells were increased, while the S-phase cells were decreased gradually as the culture time progressed. Further analysis showed that the specific antibody productivity of hybridoma cells was correlated to the fraction of S-phase cells.

Monoclonal antibodies (MAbs) were produced by fusing SP2/0 myeloma cells with spleen cells of BALB/c mice that were immunized with live whole cells of the four most prevalent Leptospira serovars—namely, autumnalis, australis, grippotyphosa, and icterohaemorrhagiae. A total of 26 MAbs (10 autumnalis, 5 australis, 4 grippotyphosa, and 7 icterohaemorrhagiae) were produced that showed specific, restricted, or broad cross-reactivity when tested with 19 standard pathogenic and 3 standard saprophytic serovars by MAT and dot-ELISA. Monoclonal antibodies like AT4 and AT5 against serovar autumnalis; AS1 and AS2 against serovar australis; GR1, GR3, and GR4 raised against serovar grippotyphosa; and also the MAbs IC3 to IC7 against serovar icterohaemorrhagiae were all usable as typing reagents in a rapid dot-ELISA. Selected MAbs were subsequently utilized in a rapid sandwich dot-ELISA for identification of Leptospira serovars as well as for antigen detection in experimentally infected mice and guinea pigs. Results of rapid sandwich dot-ELISA were compared with dark field microscopy, culture, and PCR in experimentally infected animals and sandwich dot-ELISA detected the presence of Leptospira antigen during the bacteremia stage in all experimental animals. Besides detecting antigens in animals infected with homologous serovars, the sandwich dot-ELISA employing pooled capture and revealing antibodies also detected Leptospira in the group of animals infected separately with the serovars australis and icterohaemorrhagiae. Results showed PCR to be a reliable and rapid test for demonstration of Leptospira in the plasma samples. The rapid sandwich dot-ELISA appeared more advantageous over PCR in being simple, rapid, field based, and economical. This method shows better promise of being used as a bedside test for routine diagnostic purposes.

Conformation, heat-induced conformational changes, and the stability of mouse monoclonal antibody against digoxin were analyzed by circular dichroism (CD), fluorescence, UV-Vis spectroscopy, and ELISA techniques. Since both biological function and aggregation depend on the conformational properties of mouse IgG molecules, a series of sensitive and rapid physical methods were introduced to assess conformational changes and structural stability as a function of temperature. CD spectra of IgG show the characteristics expected for β-proteins. Denaturation experiments performed through optical spectroscopic methods and affinity measurement indicated that the antibody unfolds cooperatively in a single transition. Thus, unfolding/refolding equilibrium proceeds via a simple two-state mechanism (N ⇆ U), where only the native and the unfolded states are significantly populated. Thermal-induced denaturation, however, is not completely reversible, and the partial loss of binding capacity might be due, at least in part, to incorrect refolding of the long loops (CDRs), which are responsible for antigen recognition. The results show that purified IgG is temperature stable. Of course, affinity assessment as measured by ELISA indicated that increasing the temperature decreases the affinity of IgG toward digoxin.

Disulfide oxidoreductase A (also known as disulfide bond formation protein A, or DsbA) is produced in Escherichia coli (E. coli) periplasm, which plays an important role in correct formulation of disulfide bonds during protein exportation in vivo. DsbA prokaryotic expression vectors are designed for periplasmic co-expression of recombinant proteins with an improving secretion. In the present study, the first domain of human CD226 (CD226D1) was expressed as a His-tagged fusion protein with DsbA and purified by Ni-NTA resin. Three monoclonal antibodies (MAbs) against DsbA were raised by using the recombinant fusion protein as immunogen. We have demonstrated that one of them can detect DsbA via Western blotting, in addition to its ability to immunoprecipitate DsbA fusion protein. Furthermore, anti-DsbA MAb can be employed to prepare antibody-coupled affinity column for purification of DsbA fusion protein from E. coli lysate.