Development of a mouse IgA monoclonal antibody-based enzyme-linked immunosorbent sandwich assay for the analyses of RBP4.

Elevated circulating Retinol-binding protein 4 (RBP4) has been associated with insulin resistance, dyslipidemia, and hypertension. However, many commonly used RBP4 ELISAs have limited dynamic range. We therefore developed an enzyme-linked immunosorbent sandwich assay (ELISA) employing a novel immunoglobulin A (IgA)-type capture mAb called AG102 instead of IgG subtypes, which was selected for its stability, capture efficiency, and specificity for human RBP 4. These features of RBP4 have hampered the development of quantitative immunological assays. Molecular analysis of AG102 revealed IgA heavy and light chains and a J chain, as expected. AG102 demonstrated notable detection of both bacterial- and HEK293-expressed RBP4 in Western blots. Serial and internal deletion experiments suggested that a putative epitope may be located in the first 35 amino acids of the mature RBP4. Compared with commercial ELISAs, the AG102-based system exhibited more significant recovery of RBP4 from serum or urine at any given dilution factor. To substantiate its quantitation capacity, comparison between RBP4 measurements from quantitative western blots and the AG102-based ELISA demonstrated a significant correlation (R2 = 0.859). After measurement for those analytes, our data suggested that IgA-based ELISA could be adapted for quantitative measurement of those analytes existing as major serum proteins or as multi-protein complexes like RBP4.

Defining the recognition elements of Lewis Y-reactive antibodies.

Antibody response to carbohydrate antigens is often independent of T cells and the process of affinity/specificity improvement is considered strictly dependent on the germinal centers. Antibodies induced during a T cell-independent type 2 (TI-2) response are less variable and less functionally versatile than those induced with T cell help. The antigen specificity consequences of accumulation of somatic mutations in antibodies during TI-2 responses of Marginal Zone (MZ) B cells is a fact that still needs explanation. Germline genes that define carbohydrate-reactive antibodies are known to sculpt antibody-combining sites containing innate, key side-chain contacts that define the antigen recognition step. However, substitutions associated with MZ B cell derived antibodies might affect the mobility and polyspecificity of the antibody. To examine this hypothesis, we analyzed antibodies reactive with the neolactoseries antigen Lewis Y (LeY) to define the residue subset required for the reactive repertoire for the LeY antigen. Our molecular simulation studies of crystallographically determined and modeled antibody-LeY complexes suggests that the heavy-chain germline gene VH7183.a13.20 and the light-chain Vκ cr1 germline gene are sufficient to account for the recognition of the trisaccharide-H determinant Types 1-4, while the specificity for LeY is driven by the CDR3 backbone conformation of the heavy chain and not the side chain interactions. These results confirm that these monoclonals use germline-encoded amino acids to recognize simple carbohydrate determinants like trisaccharide-H but relies on somatic mutations in the periphery of the combining site to modify affinity for LeY through electrostatic interactions that leads to their optimized binding. These observations bring further attention to the role of mutations in T-cell independent antibodies to distinguish self from non-self carbohydrate antigens.

Early postnatal B cell ontogeny and antibody repertoire maturation in the opossum, Monodelphis domestica.

Marsupials are a lineage of mammals noted for giving birth to highly altricial young, which complete much of their "fetal" development externally attached to a teat. Postnatal B cell ontogeny and diversity was investigated in a model marsupial species, the gray short-tailed opossum, Monodelphis domestica. The results support the initiation of B cell development late in gestation and progressing into the first two weeks of postnatal life. Transcription of CD79a and CD79b was detected in embryonic tissue prior to birth, while immunoglobulin heavy chain locus transcription was not detected until the first postnatal 24 hours. Transcription of the Ig light chains was not detected until postnatal day 7 at the earliest. The predicted timing of the earliest appearance of mature B cells and completion of gene rearrangements is consistent with previous analyses on the timing of endogenous antibody responses in newborn marsupials. The diversity of early B cell IgH chains is limited, as has been seen in fetal humans and mice, but lacks bias in the gene segments used to encode the variable domains. Newborn light chain diversity is, from the start, comparable to that of the adult, consistent with an earlier hypothesis that light chains contribute extensively to antibody diversity in this species.

Computer-aided antibody design.

Recent clinical trials using antibodies with low toxicity and high efficiency have raised expectations for the development of next-generation protein therapeutics. However, the process of obtaining therapeutic antibodies remains time consuming and empirical. This review summarizes recent progresses in the field of computer-aided antibody development mainly focusing on antibody modeling, which is divided essentially into two parts: (i) modeling the antigen-binding site, also called the complementarity determining regions (CDRs), and (ii) predicting the relative orientations of the variable heavy (V(H)) and light (V(L)) chains. Among the six CDR loops, the greatest challenge is predicting the conformation of CDR-H3, which is the most important in antigen recognition. Further computational methods could be used in drug development based on crystal structures or homology models, including antibody-antigen dockings and energy calculations with approximate potential functions. These methods should guide experimental studies to improve the affinities and physicochemical properties of antibodies. Finally, several successful examples of in silico structure-based antibody designs are reviewed. We also briefly review structure-based antigen or immunogen design, with application to rational vaccine development.

Hexameric immunoglobulin M in humans: desired or unwanted?

Immunoglobulin M (IgM) is the first antibody produced upon infection, and is often suggested as the first line of defense of human immune system. In addition to being present on the surface of naïve B cells as a monomeric molecule, IgM is always secreted as a polymer. The most abundant IgM polymer in humans is pentamer, composed of five monomeric units, joined together by so-called joining or J chain. On the other hand, it is well known that hexameric IgM can be also found in human sera. Its presence is often related to different dissorders (Waldenström's macroglobulinemia, cold agglutinin, and recurrent urinary bacterial infections), although it is believed that small amounts of hexamer are present in normal human sera as well. Unlike pentamer, IgM hexamer contains six monomeric blocks and completely lacks J chain. Although it has been decades since its discovery, the precise function of IgM hexamer is still unknown. Since it was documented that hexamer is very potent in activating complement, it is suggested that its production in humans must be under strict control, and that it is produced in special conditions, when strong activation of complement is absolutely needed. However, the question is whether hexameric IgM is really a secret weapon or just an undesirable molecule in humans. According to structural and known functional characteristics of both pentamers and hexamers of IgM, it can be concluded that hexamers are, in addition to being maybe too reactive to be around, probably not that efficient in protecting us from bacterial and viral infections.

How antibodies fold.

B cells use unconventional strategies for the production of a seemingly unlimited number of antibodies from a very limited amount of DNA. These methods dramatically increase the likelihood of producing proteins that cannot fold or assemble appropriately. B cells are therefore particularly dependent on 'quality control' mechanisms to oversee antibody production. Recent in vitro experiments demonstrate that Ig domains have evolved diverse folding strategies ranging from robust spontaneous folding to intrinsically disordered domains that require assembly with their partner domains to fold; in vivo experiments reveal that these different folding characteristics form the basis for cellular checkpoints in Ig transport. Taken together, these reports provide a detailed understanding of how B cells monitor and ensure the functional fidelity of Ig proteins.

Exclusion of natural autoantibody-producing B cells from IgG memory B cell compartment during T cell-dependent immune responses.

In healthy individuals, a substantial proportion of circulating Abs exhibit polyreactivity and self-reactivity. These Abs are referred to as natural autoantibodies (NAAs). As part of the innate immunity, NAAs play an important role in eliminating pathogens. However, inherent to their poly/autoreactivity is the potential for NAAs to differentiate to high-affinity autoantibodies during an immune response. We recently generated site-directed transgenic mice that express a prototypic NAA, ppc1-5, which binds a variety of self- and non-self-Ags including DNA and phosphocholine. We have shown previously that B cells expressing the ppc1-5 NAA are positively selected during their primary development. In this study, we demonstrate that following immunization with the T-dependent Ag, phosphocholine conjugated to keyhole limpet hemocyanin, ppc1-5 NAA B cells mounted a quick IgM Ab response and entered germinal centers, but they failed to differentiate to IgG-producing cells during late primary and memory responses. Hybridomas and cDNA clones derived from the immunized mice included many IgM NAA-producing cells, but IgG NAA clones were extremely rare. Instead, many of the IgG B cells replaced their IgH transgene with an endogenous V(H) gene and produced non-autoreactive Abs. These results indicate that although NAA B cells are positively selected in the preimmune repertoire and can participate in early IgM Ab response, they are subjected to regulatory mechanisms that prevent them from developing to high-affinity IgG autoantibody production. This would explain, at least in part, why NAAs do not cause autoimmunity in most individuals.

Human single chain monoclonal antibody that recognizes matrix protein of heterologous influenza A virus subtypes.

Matrix protein (M1) is predominant and has pivotal role in the influenza A virus replication and assembly. It is therefore an attractive target for antiviral drugs, siRNA studies, and therapeutic antibodies. Nevertheless, therapeutic antibody that interferes with the M1 multiplex function has never been developed. In this study, human single monoclonal antibody fragments (HuScFvs) to M1 were generated. Full length recombinant M1 (rM1) was produced from cDNA prepared from genome of highly pathogenic avian influenza virus, A/H5N1. The rM1 was used as an antigen in phage bio-panning to select phage clones displaying HuScFv from a human antibody phage display library. Several phage clones displaying HuScFv bound to the rM1 and harboring the respective huscfv gene inserts were isolated. RFLP experiments revealed multiple DNA banding patterns which indicated epitope/affinity diversity of the HuScFv. The HuScFv were tested for their binding to native M1 of homologous and heterologous influenza A viruses using ELISA as well as incorporating immunostaining and immunofluorescence studies with infected MDCK cells. One such protein produced from a selected phage clone blocked binding of M1 to viral RNA. The HuScFv in their in vivo functional format, e.g. cell-penetrating molecules, should be developed and tested as a broad spectrum anti-A/influenza.

Development of ALS-like disease in SOD-1 mice deficient of B lymphocytes.

Several recent studies proposed a role for innate immunity and inflammation in the pathogenesis of amyotrophic lateral sclerosis (ALS). However, possible links, if any, between disease and adaptive immunity are poorly understood. The present study probed for the role of B cells in ALS disease using the G93A-SOD-1 transgenic mouse model. In agreement with other studies, we show here that autoantibodies are detectable in SOD-1 mice. However, SOD-1 B cells did not express any altered phenotype and exhibited indistinguishable responsiveness to immunogenic stimuli relative to wild-type B cells. This was obtained for B cells isolated before, during and after the onset of ALS-like disease. Finally, to obtain an in vivo conclusion, we generated SOD-1 mice that are deficient of B cells, by crossing SOD-1 mice with Igmu-deficient mice (muMT), where B cell development is blocked at the proB stage. The meteoric assays performed on a rota-rod clearly showed the development of ALS-like disease in SOD-1 mice that are deficient of B cells not differently than in control SOD-1 mice. Our results propose that B lymphocytes do not have a major role in the pathogenesis of ALS-like disease in SOD-1 mice.

[Development of the monoclonal antibody to deoxynivalenol].

OBJECTIVE: Deoxynivalenol (DON) is a trichothecene mycotoxin produced by Fusarium graminearum, a pathogen causing Fusarium Head Blight of wheat. It is necessary to establish a rapid and simple assay to detect DON. METHODS: High affinity monoclonal antibodies (Mab) against DON were produced by cell fusion with 500 mg/mL Polyethylene Glycol 400, and cell sub-cloning in HAT (H: hypoxanthine, A: aminopterin; T: thymidine) culture medium for screening and limiting dilution. Hybridoma lines were screened for specificity to DON by Enzyme-linked Immunosorbent Assay (ELISA). One hybridoma cell line (3B2) secreting monoclonal antibody (MAb) against DON was produced by fusing mouse myeloma cells (SP 2/0) with spleen cells from BAL B/C mice which were immunized by the artificial antigen conjugated with Ovalbumin (OVA). RESULTS: The MAb obtained in this experiment could specifically react with DON without cross-reactivity to DON related compounds except 3-acetyldeoxynivalenol (3-Ac-DON), with the titres of ascitic fluids up to 1 x 10(-7) by indirect ELISA. Isotype and subclass of the monoclonal cell line (3B2) showed that it belonged to IgG1. The light chain of the MAb was identified to be kappa. Ascites antibodies generated by hybridoma of 3B2 cells were purified. Inhibition rate studies showed that the detection limit of the ELISA was 8 microg/L, with the regression equation of indirect ELISA Y = 0.7331g(x)-0.572, R2=0.9652, IC50 value being 29 ug/L. CONCLUSION: The MAb can be used to prepare the reagents for analyzing DON residue.

A focused antibody library for selecting scFvs expressed at high levels in the cytoplasm.

BACKGROUND: Intrabodies are defined as antibody molecules which are ectopically expressed inside the cell. Such intrabodies can be used to visualize or inhibit the targeted antigen in living cells. However, most antibody fragments cannot be used as intrabodies because they do not fold under the reducing conditions of the cell cytosol and nucleus. RESULTS: We describe the construction and validation of a large synthetic human single chain antibody fragment library based on a unique framework and optimized for cytoplasmic expression. Focusing the library by mimicking the natural diversity of CDR3 loops ensured that the scFvs were fully human and functional. We show that the library is highly diverse and functional since it has been possible to isolate by phage-display several strong binders against the five proteins tested in this study, the Syk and Aurora-A protein kinases, the alphabeta tubulin dimer, the papillomavirus E6 protein and the core histones. Some of the selected scFvs are expressed at an exceptional high level in the bacterial cytoplasm, allowing the purification of 1 mg of active scFv from only 20 ml of culture. Finally, we show that after three rounds of selection against core histones, more than half of the selected scFvs were active when expressed in vivo in human cells since they were essentially localized in the nucleus. CONCLUSION: This new library is a promising tool not only for an easy and large-scale selection of functional intrabodies but also for the isolation of highly expressed scFvs that could be used in numerous biotechnological and therapeutic applications.

[Development and characterization of monoclonal antibodies against VP1 protein of AsiaI type foot-and-mouth virus].

AIM: To prepare the monoclonal antibodies against VP1 protein of type AsiaI foot-and mouth disease virus (FMDV) and identify the characterization. METHODS: Three cell of hybridization that strains secreted monoclonal antibody(mAb) against type AsiaI FMDV were produced by fusing mouse myeloma cells (Sp2/0) with spleen cells from BALB/c immunized with the purified recombinant VP1 protein. RESULTS: The three hybridized cell lines reacted with cattle type AsiaI FMDV, the titres of ascetic fluids of the three mAbs ranged from 1:10(5) to 1:10(6) indirect ELISA showed. Among the three mAbs,1B8 belonged to IgG1, 5E1 and 5E2 pertained to IgG2a. Besides, the three strain antibodies stably excreted antibodies and reacted with type AsiaI FMDV. CONCLUSION: VP1 protein of FMDV could replace FMDV can be use to replace FMDV to prepare mAbs.

[Development and characterization of monoclonal antibodies against H7 hemagglutinin of avian influenza virus].

AIM: To prepare monoclonal antibodies (mAbs) against the hemagglutinin protein of H7 subtype of avian influenza virus (AIV). METHODS: (6-8 weeks old) BALB/c mice of were immunized endermicly with H7 subtype of AIV. The splenocytes from the immunized mice were fused with Sp2/0-Ag-14 myeloma cells after the last immunization. Hybridoma cells were screened by hemagglutination (HA) and hemagglutination inhibition (HI) tests. The reactivity and specificity of mAbs were evaluated by HI test and Western blot assay. RESULTS: Four hybridoma cell lines secreting specific mAbs named 2E2, 2A4, 5F5 and 7G5 were developed. The HI titers of these mAbs were 5 x 2(7)-5 x 2(11), and the immunoglobulin subclass of 2E2 was IgM, that of 2A4 was IgG1, and that of 5F5 and 7G5 was IgG2a. Western blot analysis confirmed that the mAbs only reacted with M(r) 75 000 HA protein of H7 subtype of AIV but did not react with the proteins of Newcastle disease virus (NDV). The results of HI reactivity assay suggested that 2E2, 5F5 and 7G5 only reacted with H7 subtype of AIV but did not react with other subtypes of AIV, NDV and infectious bronchitis virus (IBV). However 2A4 reacted not only with H7 subtype of AIV but also with H15N8 reference strain of AIV at low HI level. CONCLUSION: These mAbs can be used as a useful tool to analyze the HA structure of AIV. They also provide the effective reagents for the rapid detection of H7 subtype of AIV.

[Preparation of three functional monoclonal antibodies against human FL molecule and analysis of their bioactivity].

AIM: To Prepare three functional monoclonal antibodies(mAbs) against human FL molecule and analyze their bioactivity. METHODS: The cell line L929-FL transfected with human FL gene was used as immunogen. The hybridomas secreting the antibodies against human FL were obtained by fusing splenoecytes from the immunized mice with murine myeloma cells(Sp2/0). Their subclasses were analyzed using fast-strip method. The monoclonal antibodies were produced in mouse peritoneal cavity and purified by Protein G affinity chromatography. The inhibitory effect of mAbs against FL on leukemia cell lines U937 and HL-60 was detected by MTT. The apoptosis of U937 and HL-60 cells stained by annexin-V/PI was determined by FCM. RESULTS: Three hybridomas named 3C2, 3C6 and 8D10 were successfully obtained, which secreted monoclonal antibodies against human FL molecule stably. Their subclasses were the mouse IgG2a with kappa light chains. The three monoclonal antibodies recognized the FL molecule on U937 and HL-60 cells that also coexpressed Flt3 molecule. When U937 and HL-60 cells were cultured in presence of 3C2, 3C6 and 8D10, their proliferation was reduced as compared to that in control in MTT assay(P < 0.05). The analysis of annexin-V/PI binding to U937 and HL-60 cells by FCM showed the mAbs had the apoptotogenic activity of the monoclonal antibodies against human FL molecule. CONCLUSION: 3C2, 3C6 and 8D10 are three funtional monoclonal antibodies against human FL molecule. They may be of some value in the study of the roles of FL/Flt3 interaction in leukemia pathogenesis.

Immunoglobulin crystal-storing histiocytosis in a pleural effusion from a woman with IgA kapa multiple myeloma: a case report.

BACKGROUND: Crystal-storing histiocytosis (CSH) is a rare disorder occurring in patients with lymphoproliferative diseases, predominantly multiple myeloma and low grade B-cell lymphoma. This report presents the first case of CSH diagnosed on pleural fluid from a patient with multiple myeloma (MM). CASE: A 79-year-old women with IgA kappa MM underwent thoracocenthesis and thoracic drainage because of a pleural effusion. Cytologic and immunocytochemical examination of pleural fluid revealed abundant histiocytic, CD68-positive cells with prominent intracytoplasmic, needlelike, crystalloid inclusions showing strong immunopositivity for IgA heavy and kappa light chains. Identical crystals were observed on an extracellular background. No myeloma infiltration was detected. Two weeks later, examination of new pleural fluid from the patient showed a similar cytologic picture, but, in addition, isolated plasma cell features were identified. They were too few for a meaningful determination of clonality. The patient died I month after the CSH diagnosis. CONCLUSION: This case illustrates the value of cytologic examination of serous fluids from patients with plasma cell dyscrasias, not only to evaluate possible infectious or neoplastic causes but also to diagnose CSH.

[Immune response induced by combinant immunization of different HBsAg vaccines in mice].

AIM: To investigate the specific humoral and cellular immune response induced by prime-boost immunization of HBsAg protein vaccine (P), recombinant vaccinia virus vaccine (V) and DNA vaccine (D) in mice. METHODS: Groups of BALB/c mice were primed by one of the three vaccines P, V or D and boosted by another vaccine at 2, 5, 8 and 11 week later, thus 9 immune combinations were made: PP, PV, PD, VP, VV, VD, DP, DV and DD. Serum samples were collected at week 2, 5, 8 and 11 and levels of anti-HBsAg IgG antibodies and their sub-isotypes were determined. Seven days after every boost, spleen cells of vaccinated mice were separated and the specific CTL lysis ratio of P815S cells were determined. RESULTS: Among the three HBsAg vaccines P, V and D, V could induce the quickest humoral immune response. The memory humoral immune response induced by P was the strongest. D induced the weakest antibody titer. The ratio of specific IgG1/IgG2a indicated that antibody induced by PP was more polarized to Th2. The other groups induced balanced Th1/Th2 immune response. Among all the groups, VD and DV induced the strongest CTL response, and the specific lysis ratio of P815S cells was 71% and 64%, respectively. CONCLUSION: The experimental results suggested that among all the immune combinations, PV, PD, VP and VD can induce better humoral immune response while DV and VD can induce stronger CTL response in BALB/c mice.

[Preparation and characterization of the monoclonal antibody against human sperm protein 17].

AIM: To prepare and characterize the monoclonal antibody (mAb) against sperm protein 17 (Sp17). METHODS: Human Sp17 cDNA was cloned and recombinant Sp17 protein was expressed as a fusion protein with an N-terminal 6-His tag. The purified recombinant Sp17 protein was used to immunize BALB/c mice for preparing mAb. mAb-produced hybridoma cells were screened by ELISA and the specificity of mAb was analyzed by immunohistochemical staining and blocking test. RESULTS: Two hybridoma cells (3C12 and 3D6) secreting mAb against Sp17 were developed. The isotypes of these two mAbs, 3C12 and 3D6, were IgG(1) and IgM, respectively. ELISA detection showed that titers of mAbs, 3C12 and 3D6, were 1:64, 1:32 in cultured supernatant and 1:1 x 10(5), 1:5 x 10(4) in ascites, respectively. The results of immunohistochemical staining and blocking test indicated that 2 mAb specifically bound to Sp17 in human and rat testis and human ejaculated spermatozoa. Anti-Sp17 mAb also detected Sp17 in ovarian cancer. CONCLUSION: The successful preparation of anti-Sp17 mAb will be useful for assessing the native distribution and aberrant expression of Sp17 protein.

Selection of anti-double-stranded DNA B cells in autoimmune MRL-lpr/lpr mice.

Abs to DNA and nucleoproteins are expressed in systemic autoimmune diseases, whereas B cells producing such Abs are edited, deleted, or inactivated in healthy individuals. Why autoimmune individuals fail to regulate is not well understood. In this study, we investigate the sources of anti-dsDNA B cells in autoimmune transgenic MRL-lpr/lpr mice. These mice are particularly susceptible to lupus because they carry a site-directed transgene, H76R that codes for an anti-DNA H chain. Over 90% of the B cells are eliminated in the bone marrow of these mice, and the few surviving B cells are associated with one of two Vkappa editors, Vkappa38c and Vkappa21D. Thus, it appears that negative selection by deletion and editing are intact in MRL-lpr/lpr mice. However, a population of splenic B cells in the H76R MRL-lpr/lpr mice produces IgG anti-nuclear Abs, and these mice have severe autoimmune organ damage. These IgG Abs are not associated with editors but instead use a unique Vkappa gene, Vkappa23. The H76R/Vkappa23 combination has a relatively high affinity for dsDNA and an anti-nuclear Ab pattern characteristic of lupus. Therefore, this Vkappa gene may confer a selective advantage to anti-DNA Abs in diseased mice.