Characterization of monoclonal antibodies against human leukocyte 5-lipoxygenase

Four mouse monoclonal IgG1 antibody-producing cell lines (5LO-1, 5LO-2, 5LO-3, 5LO-4), produced against highly purified human leukocyte 5-lipoxygenase have been characterized. The monoclonal antibodies produced by these cell lines exhibited differential reactivity against 5-lipoxygenase as determined by ELISA and immunoprecipitation analyses. Monoclonal antibodies 5LO-2 and 5LO-3 inhibited the activity of recombinant human leukocyte 5-lipoxygenase in a dose-dependent manner. This inhibition was selective for 5-lipoxygenase activity since these monoclonal antibodies did not inhibit human leukocyte 15-lipoxygenase or porcine leukocyte 12-lipoxygenase.     

Characterization and application of monoclonal antibodies against human endothelin B receptor expressed in insect cells

Endothelin B receptor (ET(B)R) is a G protein-coupled receptor that mediates a variety of signals by binding to vasoconstrictive peptides, endothelins. Monoclonal antibodies were prepared against human ET(B)R using the full-length protein expressed in Sf9 cells. Five typical monoclonal antibodies were characterized further for their recognition. The epitopes for the 2A5, 9A3 and 21A1 antibodies were mapped within the N-terminal extracellular sequences, V71-I85 and E27-Q41, respectively, which differ between the human and mouse ET(B)Rs. All of these antibodies labeled cell surface ET(B)R expressed in COS cells, suggesting that their recognition sites exist in the extracellular domain. In addition, the immobilized antibodies could purify ET(B)R expressed in Sf9 cells to the majority under mild conditions. Thus, immunization with the recombinant full-length membrane protein provides a strategy to produce monoclonal antibodies recognizing the native protein.     

Characterization of monoclonal antibodies against Gnathostoma nipponicum

Monoclonal antibodies (mAbs) were produced against the proteins of advanced third-stage larvae (AdL3) of Gnathostoma nipponicum. Six mAbs (Gn2C3, Gn2H3, Gn4C3, Gn4E9, GnSH1, and Gn10B7) were obtained as determined by enzyme-linked immunosorbent assay (ELISA). Gn4E9 and GnSH1 seemed to be genus-specific, as they did not cross-react with Anisakis sp., Dirofilaria immitis, Gongylonema pulchrum, Toxocara canis, Trichinella sp., Trichuris vulpis, Metagonimus sp., or Spirometra erinaceieuropaei by ELISA. Immunohistochemistry showed that Gn2C3, Gn4E9, and Gn5H1 reacted strongly with the central esophagus; Gn2H3 reacted with cuticle,muscle, intestine, and the cervical sac; and Gn4C3 and Gn10B7 reacted with cuticle, muscle, esophagus, intestine, and the cervical sac of AdL3. In Western blotting analysis, Gn2C3, Gn4E9, and Gn5H1 reacted to 60-, 53-, 46-, and 41-kDa proteins; Gn4C3 reacted to the AdL3 protein of G. nipponicum (>42 kDa). Moreover, proteins purified using a mAb Gn4E9 immunoprecipitation method (sizes 60-, 53-, 46-, and 41-kDa) were used as antigens in ELISAs. A significant difference (P < 0.01) was shown between mouse sera infected with G. nipponicum and sera infected with Trichnella sp. or not infected. These results provide a rationale for evaluating esophageal proteins for the development of diagnostic methods for detecting G. nipponicum or Gnathostoma sp. infections.     

Identification and mapping of neutralizing epitopes of human parvovirus B19 by using human antibodies.

We identified and mapped the regions responsible for neutralization in the human parvovirus B19 structural protein by using region-specific human antibodies derived from seropositive blood donors. The region-specific antibodies were purified by using affinity columns coupled with synthetic peptides of the hydrophilic regions including the beta-turn structure deduced by the predicted secondary structure of VP2. Fifteen highly specific antibodies against the synthetic peptides were obtained. Ten of them were able to precipitate the radiolabeled virus. Six of them proved to be able to protect the colony-forming unit erythroid cells in human bone marrow cell cultures from injury by the virus. The sequences recognized by the six neutralizing antibodies were sites corresponding to amino acids 253 to 272, 309 to 330, 325 to 346, 359 to 382, 449 to 468, and 491 to 515 from the amino-terminal portion of VP2. These observations suggest that the neutralizing epitopes were distributed in the region from amino acid 253 in the amino-terminal portion of VP2 to the carboxyl terminus of VP2.     

Characterization of monoclonal antibodies raised against the latent nuclear antigen of human herpesvirus 8

Human herpesvirus 8 (HHV-8; also designated Kaposi's sarcoma-associated herpesvirus) is the likely etiological agent of Kaposi's sarcoma (KS). HHV-8 encodes a latent nuclear antigen (LNA) which is the product of the viral gene orf 73. LNA is recognized by most infected patient sera and is the basis of current immunofluorescence assays used in epidemiological studies of HHV-8 infection. Here we describe the characterization of four monoclonal antibodies raised to the C-terminal third of LNA-glutathione S-transferase fusion proteins. These monoclonal antibodies recognized discrete linear epitopes within the C terminus and repetitive region of LNA, detected antigen in primary effusion lymphoma (PEL) cells, and precipitated a 220- to 230-kDa protein doublet corresponding to LNA from HHV-8-infected PEL cell lines. In situ immunocytochemistry of KS lesions with these antibodies show that LNA is extensively expressed in KS spindle cells.     

Characterization of monoclonal antibodies against human red blood group cells antigens by laser backscattering

A problem in immunohematology is to define the antibody quality which is related to its affinity expressed by the equilibrium constant. The activity of an antibody can be measured by the strength of its interaction, related to the adhesive energy exchanged during RBC agglutination which depends on the antigen-antibody liaison strength. To estimate this adhesive energy, two methods are used in this paper. Firstly, the dissociation behaviour of suspended RBC agglutinates was analysed by laser backscattering intensity (r) in a Couette flow. Backscattered intensity issued from shear-induced mechanical dissociation is recorded and submitted to a numerical process to obtain the energy parameter (ED). Secondly, a modification of this technique is proposed for measuring specific binding energy. Samples were exposed to increasing shear stress, and backscattered intensity was recorded. A constant increase of this intensity with raising shear stress was observed, pointed to a progressive dissociation of RBC agglutinates into smaller ones. Considering that complete dissociation of agglutinates is only approached asymptotically it is assumed that the final break-up of doublets (two-cell agglutinates) is produced at a critical shear stress (tauC) reflecting the work done to breaking-up the molecular bridges between both adjacent cells. This shear stress is defined by the extrapolation of the linear part of the curves [r-log tau] to the backscattered signal (r0) corresponding to the complete dispersion of RBCs. These approaches permit to define the specific surface adhesive energy (Gamma) by using the Derjaguin relation and to assess the functional characterization of specific immunoglobulins. In conclusion, two parameters characterizing monoclonal antibody agglutination properties, ED and Gamma, were estimated by laser backscattering methods, which could be very useful for antibodies quality control.     

Characterization of mouse monoclonal antibodies to human interferon-gamma

Mouse monoclonal antibodies (mAb) to human interferon-gamma (HuIFN-gamma) were characterized. The mAbs studied--E4-18, G4-15, and SAT-1--which are all IgGl-type, reacted to all HuIFN-gamma molecular species, both glycosylated and non-glycosylated. Affinity constants calculated of E4-18 and G4-15 didn't have considerable differences for both kinds of HuIFN-gamma (1-3 x 10(8) liter/mol), but SAT-1 had a difference--a higher value (10(10) liter/mol) for the former than for the latter (8 x 10(8) liter/mol). In epitope specificity, the results suggested that E4-18 and G4-15 recognized an overlapped region remote from the region of SAT-1. Competition experiment using synthetic peptides suggested that epitope of G4-15 is around N9-26 of the HuIFN-gamma sequence. Those mAbs could be used for sandwich radioimmunoassay of HuIFN-gamma using double mAbs in two combinations, one (G4-15/E4-18) based on dimer forms of HuIFN-gamma and the other (SAT-1/E4-18) based on epitope difference. The mAbs are all neutralizing antibodies in which SAT-1 neutralized at a lower concentration than did G4-15, and at a much lower one than did E4-18. The receptor binding of HuIFN-gamma was inhibited by mAbs G4-15 and SAT-1. Efficacy of G4-15 and SAT-1 for the inhibition correspond with that for neutralization.     

Characterization of monoclonal antibodies directed against trail or trail receptors

A subset of tumour necrosis factor receptor family members is involved in death transducing signals and is, therefore, referred as the "death receptors." Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in many tumour cells but only rarely in normal cells. Five distinct receptors have been described for TRAIL: TRAIL R1 (DR4), TRAIL R2 (DR5, TRICK), TRAIL R3 (TRID, DcR1), TRAIL R4 (TRUNDD, DcR2), and osteoprotegerin. In the Eighth International Workshop on Human Leukocyte Differentiation Antigens, 10 monoclonal antibodies (mAbs) reported to be specific for TRAIL or for TRAIL receptors were submitted. In the present study, the mAb specificity was determined by ELISA. Using these mAbs, investigation on the expression of TRAIL and TRAIL receptors was performed. Some of them were able to modulate TRAIL induced programmed cell death.     

Characterization of monoclonal antibodies against waterfowl parvoviruses VP3 protein

Background

Ebola viruses (EBOVs) cause severe hemorrhagic fever with a high mortality rate. At present, there are no licensed vaccines or efficient therapies to combat EBOV infection. Previous studies have shown that both humoral and cellular immune responses are crucial for controlling Ebola infection. CD8⁺ T cells play an important role in mediating vaccine-induced protective immunity. The objective of this study was to identify H-2^d-specific T cell epitopes in EBOV glycoproteins (GPs).

Results

Computer-assisted algorithms were used to predict H-2^d-specific T cell epitopes in two species of EBOV (Sudan and Zaire) GP. The predicted peptides were synthesized and identified in BALB/c mice immunized with replication-deficient adenovirus vectors expressing the EBOV GP. Enzyme-linked immunospot assays and intracellular cytokine staining showed that the peptides RPHTPQFLF (Sudan EBOV), GPCAGDFAF and LYDRLASTV (Zaire EBOV) could stimulate splenoctyes in immunized mice to produce large amounts of interferon-gamma.

Conclusion

Three peptides within the GPs of two EBOV strains were identified as T cell epitopes. The identification of these epitopes should facilitate the evaluation of vaccines based on the Ebola virus glycoprotein in a BALB/c mouse model.     

Characterization of monoclonal antibodies against human prorenin profragment and identification of active prorenins in plasma

Monoclonal antibodies were raised against a synthetic peptide (43 amino acid residues) that corresponds to the complete profragment of human prorenin. Seven monoclonal antibodies were chosen for further characterization. Two antibodies, 2-X-C1 and 4-X-E1, reacted with the middle region and C-terminus of the profragment and were isotyped IgG1. The affinity constants of these antibodies against the human profragment were 7.6 x 10(8) and 3.0 x 10(7) M-1, respectively. Immunoaffinity columns containing the antibodies 2-X-C1 and 4-X-E1, respectively, were used for the characterization of active prorenin in human plasma. This active prorenin strongly bound to the 4-X-E1 column and eluted as two separate peaks which corresponded to fully and partially active prorenin, respectively. The partially active prorenin had higher activity with a small substrate, tridecapeptide, than with a large one, angiotensinogen, although the fully active prorenin had the same renin activity irrespective of the size of the substrate. These data suggest that new forms of prorenin, active prorenin, exist in human plasma and that their active sites are completely or partially exposed to the substrates. Moreover, the active prorenin in plasma was found not only in human but also in all tested mammalians. Cross-reactivity among the profragments of mammalian plasma prorenins can be explained by conservation of the amino acid sequence (epitope) of the combining site.     

Preparation of monoclonal antibodies against duck hepatitis B core antigen and analysis of the monoclonal antibodies

Mice were immunized against duck hepatitis B virus core (DHBc) particles isolated from the liver of asymptomatic carrier ducks of duck hepatitis B virus (DHBV) by ultracentrifugation. Their spleen cells were fused with mouse myeloma (NS-1) cells, and 12 clones of hybridoma cells secreting antibodies against DHBc (anti-DHBc) were isolated. According to the reactivity to core particles and core peptide obtained from DHBc particles treated with SDS-2ME, the 12 antibodies were classified into two groups. Two monoclonal antibodies reacted against both core particles and core peptide (B-type), the other ten monoclonal antibodies reacted against core particles but did not react against core peptide obtained from DHBc particles treated with SDS-2 ME. (A-type). Solid phase enzyme immuno assay (EIA) using these two types of antibodies could detect core antigenisity not only in the liver homogenate but also in the DHBV infected serum. Sucrose gradient analysis and gel filtration analysis revealed this DHBc antigenisity in the serum is not carried by core particles but carried by core peptide, equivalent to HBe antigen in the serum of Hepatitis B virus (HBV) carrier. This EIA may provide sensitive test monitoring both serum DHBe antigen levels and DHBc antigen levels in the liver during DHBV infection.     

Impaired gamma interferon responses against parvovirus B19 by recently infected children

Parvovirus B19 is the causative agent of fifth disease of childhood. It has been implicated in a variety of conditions, including unsuccessful pregnancy and rheumatoid arthritis, and is a potential contaminant of blood products. There has been little study of immunity to parvovirus B19, and the exact nature of the protective humoral and cell-mediated immune response is unclear. Immune responses to purified virus capsid proteins, VP1 and VP2, were examined from a cohort of recently infected children and compared with responses from long-term convalescent volunteers. The results demonstrate that antibody reactivity is primarily maintained against conformational epitopes in VP1 and VP2. The unique region of VP1 appears to be a major target for cell-mediated immune responses, particularly in recently infected individuals. We confirm that antibody reactivity against linear epitopes of VP2 is lost shortly after infection but find no evidence of the proposed phenotypic switch in either the subclass of parvovirus B19-specific antibody or the pattern of cytokine production by antigen-specific T cells. The dominant subclass of specific antibody detected from both children and adults was immunoglobulin G1. No evidence was found for interleukin 4 (IL-4) or IL-5 production by isolated lymphocytes from children or adults. In contrast, lymphocytes from convalescent adults produced a typical type 1 response associated with high levels of IL-2 and gamma interferon (IFN-gamma). However, we observed a significant (P<0.001) deficit in the production of IFN-gamma in response to VP1 or VP2 from lymphocytes isolated from children. Taken together, these results imply that future parvovirus B19 vaccines designed for children will require the use of conformationally preserved capsid proteins incorporating Th1 driving adjuvants. Furthermore, these data suggest novel mechanisms whereby parvovirus B19 infection may contribute to rheumatoid arthritis and unsuccessful pregnancy.     

Identification of the region including the epitope for a monoclonal antibody which can neutralize human parvovirus B19.

In this study, we identified a region in the human parvovirus structural protein which involves the neutralization of the virus by a monoclonal antibody and site-specific synthetic peptides. A newly established monoclonal antibody reacted with both viral capsid proteins VP1 and VP2. The epitope was found in six strains of independently isolated human parvovirus B19. The monoclonal antibody could protect colony-forming unit erythroid in human bone marrow cell culture from injury by the virus. The monoclonal antibody reacted with only 1 of 12 peptides that were synthesized according to a predicted amino acid sequence based on nucleotide sequences of the coding region for the structural protein of B19 virus. The sequence recognized by the antibody was a site corresponding to amino acids 328 to 344 from the amino-terminal portion of VP2. This evidence suggests that the epitope of the viral capsid protein is located on the surface of the virus and may be recognized by virus-neutralizing antibodies.     

抗细小病毒B19-VP2单克隆抗体的建立

制备抗细小病毒B19－VP2单克隆抗体,用于检测人血清中的B19抗原,辅助诊断相关疾病;也可用于制备人类细小病毒基因工程疫苗。用纯化的基因工程表达的B19－VP2蛋白免疫BALB/c小鼠,取免疫小鼠的脾细胞和小鼠骨髓瘤Sp2/0细胞融合,有限稀释法克隆细胞。ELISA及IF证明抗体特异性。克隆筛选出4株细胞,并初步建立了检测B19－VP2抗原的双抗体夹心酶联免疫吸附试验,为双抗体夹心法检测B19抗原为临床相关疾病诊断提供了检测手段。