Quantification of a major bovine allergen by a two-site immunometric assay based on monoclonal antibodies

A two-site immunometric assay based on monoclonal antibodies (mAb) was developed for the measurement of a 20-kDa major allergen of cow. The sensitivity of this assay was (BDA20) 1 ng/ml. It was used to measure airborne allergen concentrations in 10 Finnish cowsheds. The mean concentration of the BDA20 measured at two stationary sites was 280 ng/m³. Concentrations varied more than 10-fold among cowsheds (54–804 ng/m³). The mean intertest coefficient of variation was 8.2%, and the mean intratest variation 4.1 %.     

Identification and characterization of a 30 kd major allergen from Parapenaeus fissurus

The allergenic components of the shrimp (Parapenaeus fissurus) were identified by immunoblotting, with sera from 10 allergic patients. Six components, ranging in molecular weight from about 86 to 39 kd, showed IgE-binding activity and were identified as allergens of the shrimp. The component with a molecular weight of about 39 kd showed the highest frequency of IgE binding (70%) and was considered to be one of its major allergens. Two monoclonal antibodies against this 39 kd component were generated, and their antigenic cross-reactivity with five different kinds of seafood, shrimp, crab, cuttlefish, oyster, and pomfret was analyzed. Monoclonal antibody 1-6-10B reacted with the 39 kd component from shrimp only, but monoclonal antibody 2-7-IE also reacted with the 39 kd component from crab. By extraction with 0.5% sodium dodecylsulfate and ethanol precipitation, a highly purified shrimp 39 kd component was obtained. In two-dimensional gel electrophoresis six isoforms of this purified 39 kd component, with isoelectric point values from purified 39 kd component, with isoloelectric point values from 5.1 to 5.6, were identified. No marked difference was observed when the amino acid composition of this purified 39 kd allergen was compared with those of serum albumin from different animals. They all contain a high proportion of acidic amino acids. There was also a 62% to 83% sequence homology among three different pairs of peptide fragments of purified 39 kd components of shrimp and crab. In conclusion, a 39 kd major allergen from the shrimp has been identified and characterized in the present study. According to the suggestions of the International Union of Immunological Societies, this allergen is designated as Par f I.     

Identification by monoclonal antibodies of a new epitope in the glycoprotein complex of Sindbis virus

Monoclonal antibodies against a deletion mutant of Sindbis virus were produced and characterized in order to determine the fine mapping and functional activities of single viral epitopes. All monoclonal antibodies so far tested showed a certain degree of reciprocal competition and were directed against an antigenic determinant which was present only on the undissociated complex of the E1 and E2 glycoproteins. A biological assay measuring viral haemagglutination showed no decrease in the titre of viral samples preincubated with monoclonal antibodies. Conversely, a reduction in viral infectivity was demonstrated, particularly with two of these antibodies. The results suggest that the antibodies which we characterized seem to recognize a new epitope which is represented on both glycoproteins on the surface of this mutant of Sindbis virus.     

平榛主要过敏原Corh1单克隆抗体的制备与鉴定

目的制备和鉴定鼠抗平榛主要变应原Cor h 1的单克隆抗体(Monoclonal Antibody,McAb)。方法用重组Cor h 1蛋白为免疫原,免疫Balb/c小鼠,取免疫小鼠脾细胞与小鼠骨髓瘤NS-1细胞融合。通过间接ELISA法筛选分泌特异性McAb的杂交瘤细胞。用杂交瘤细胞株诱导小鼠产生腹水,再用蛋白A亲和层析法纯化抗体。采用Ig类与亚类鉴定试剂盒鉴定该单克隆抗体的Ig亚型;通过间接ELISA、Western Blotting鉴定该McAbs的特性和交叉性。结果获得4株可稳定分泌鼠抗平榛主要变应原Cor h 1的McAbs,其Ig亚型均为IgG1,均具有良好的效价;ELISA和Western Blotting分析表明该4株单抗均能识别重组Cor h 1蛋白,其中3株单抗能够识别天然平榛提取物。结论成功制备了4株鼠抗平榛主要变应原Cor h 1的单克隆抗体,为建立平榛主要变应原Cor h 1检测及纯化方法奠定了基础。     

Characterization of the allergen Der f 7 from house dust mite extracts by species-specific and crossreactive monoclonal antibodies

Background The group 7 mite allergens react with IgE in 50% of sera from allergic patients. Objective To determine the molecular and antigenic characteristics and heterogeneity of Der f 7 in mite extracts. Methods Monoclonal antibodies (MoAbs) produced from mice immunized with recombinant Der f 7 were examined for crossreactivity to Der p 7 and then used for immunoblotting of 1 and 2-D gel electrophoresis. Deglycosylation was studied with N-glycosidase-F and N-terminal sequencing by Edman degradation. The epitopes of the monoclonal antibodies were compared by cross-inhibitory immunoassays. Results Immunoblotting of D. farinae extracts with all the anti Der f 7 MoAbs showed major reactivities at 31, 30 and 25 kDa. The strongest immunostaining was at 25 kDa which contrasted with Der p 7 where the 31 and 30 kDa bands were strongest. The relative strength of staining however varied between extracts. The 31 and 30 kDa components were glycosylation products of the 25 kDa form which had the N-terminal sequence predicted from cDNA analysis. Two MoAbs stained an 18 kDa band consistent with a degradation product. The 2-D gels showed that different components with pls from 5.6–6.4. Both species-specific and Der p 7 crossreactive MoAbs were produced and a two-site ELISA assay for detecting group 7 allergen was developed with MoAbs recognizing different epitopes. Conclusions Der f 7 has been defined by its natural N-terminal sequence and MoAbs. It apparently exists as different glycosylation and degradation products in mite extracts, the relative abundance of which differs with different preparations. A two-site ELISA to measure the allergen was developed.     

Identification of a novel cofilin-related molecule (Der f 31) as an allergen from Dermatophagoides farinae

House dust mite (HDM) allergen is a major cause of allergic disease. In this study, two-dimensional immunoblot and Matrix-Assisted Laser Desorption Ionization tandem Time-of-flight mass spectrometry (MALDI-TOF-MS) were used to identify Der f 31. After Der f 31 was cloned, expressed and purified, skin prick test (SPT), Immune inhibitory assays, Western blot, ELISA and asthmatic mouse model were employed to examine the allergenicity of recombinant Der f 31. The gene of Der f 31 includes 447 bps, and encoded 148 amino acids. Positive responses of SPT to r-Der f 31 were 32.5% in 43 HDM-allergic patients. r-Der f 31 can induce allergic pulmonary inflammation in the mouse model. In conclusion, Der f 31 is a novel subtype of dust mite allergens.     

IgE and monoclonal antibody binding by the mite allergen Der p 7

Background The recently characterized group 7 house dust mite allergens give positive skin-test reactions in 53% of allergie patients. Objective This study was performed to compare the IgE bindinig activity of natural and recombinant Der p 7, to measure the binding in allergic sera in comparison to major allergen Der p 2 and characterize the response by competitive inhibition with monoclonal antibodies. Methods IgE anti Der p 2 and Der p 7 antibodies against the recombinant allergens and monoclonal binding activities were measured by a solid phase radioimmune assay. Reslts A competitive binding assay showed that rDer p 7 inhibited 91% of IgE-binding to natural Der p 7 in 2 sera and 73% in a further two. The IgE binding of rDer p 2 and Der p 7 from 41 sera was then compared. Of the sera 88% and 46% respectively showed positive binding. All of the 19 sera which bound Der p 7 also bound Der p 2 but 11 (58%) had bound IgE to Der p 7 as high or higher than the binding to Der p 2. These sera were mostly high responders to both allergens. A panel of six monoclonal antibodies produced against either rDer p 7 or rDer f 7 was used for epitope analysis. All of these reaeted with eaeh allergen by enzyme linked immunosorbent assay (ELISA) and immunoblotting. Two patterns of cross inhibition of monoclonal antibody binding were observed and of five monoclonal antibodies tested, lour could inhibit the binding of IgE (WH9, WH22. WPS and HD19) while one (WH14) could not. Conclusions Although Der p 7 only reacts with 50% of allergic sera it often has a high IgE binding activity and may be more important than the major Der p 2 allergen in a high percentage of subjects. The combined competitive inhibition experiments show the IgE response is directed at several specilieities.     

Identification of feline MAGE-1 gene product by monoclonal antibodies

Melanoma antigens (MAGE) are thought to induce a tumor-specific immune response and to be potential therapeutical targets for cancer immunotherapy. We have earlier identified the cDNA of feline melanoma antigen 1 (fMAGE-1), but its product was not characterized in detail. We have expressed the recombinant fMAGE-1 protein and have generated monoclonal antibodies (mAbs) against it, to identify the native fMAGE-1 protein in feline lymphoma cell lines and tumor tissues. The fMAGE-1 protein was found to be approximately 39 kDa in molecular mass on sodium dodecyl-sulphate-polycrylamide gel electrophoresis (SDS-PAGE), and it was found to be located in the cytoplasm of the cells by immunofluorescence. Immunoblotting analysis detected the fMAGE-1 gene product in the fMAGE-1-mRNA-positive cells, but not in the fMAGE-1-mRNA-negative cells. An interesting finding of the present study was the distribution of the fMAGE-1 protein, which was found to have a spindle-like distribution, with filaments twining around the nucleus, suggesting that the fMAGE-1 protein may be associated with or form some cytoplasmic filaments. This type of finding is so far the first report of its kind, and to the best of our knowledge it has not been reported in either human or mouse MAGE proteins until now. It most probably implies the major diversity of the MAGE family genes.     

Polyreactivity of human monoclonal antibodies: Human IgM anti-rhesus monoclonal antibodies are frequently polyreactive

The aim of this study was to characterize human anti-Rhesus monoclonal antibodies cross-reacting with tissue antigens. Of the 155 monoclonal alloantibodies tested, 49 also reacted with intracellular antigens, as demonstrated by immunofluorescence assay on cryostat sections of animal and human tissues. This cross-reactivity was mainly a property of monoclonal alloantibodies belonging to the IgM isotype (among the 49 cross-reacting Mabs, 37 were IgM). The results confirm that during an immune response against a foreign antigen (alloantigen), B cells that produce polyreactive antibodies are not excluded from the pool of responding cells.     

Purification of murine IgM monoclonal antibodies by hydroxylapatite chromatography

Summary A simple method is described for the isolation of murine monoclonal immunoglobulin M using hydroxylapatite chromatography.     

Epitope mapping of the cat (Felis domesticus) major allergen Fel d I by overlapping synthetic peptides and monoclonal antibodies against native and denatured Fel d I

The major cat allergen Fel d I is a homodimer of which each monomer consists of two disulfide-linked polypeptide chains: chain 1 (70 amino acid residues) and chain 2 (92 amino acid residues). Twenty-one synthetic peptides of 14 amino acid residues length, overlapping by seven residues and spanning the entire sequence of both chains, were synthesized. These peptides were coupled to CNBr-activated Sepharose-4B and used as solid-phase antigens in epitope-mapping studies with monoclonal antibodies against native and reduced/alkylated Fel d I.
Two monoclonal antibodies directed against reduced/alkylated chain I bound to the overlapping peptides 53–66 and 60–70 of chain 1. The monoclonal antibody directed against reduced/alkylated chain 2 bound to the overlapping peptides 36–49 and 43–56 of chain 2. Binding specificity was demonstrated by inhibition by reduced/alkylated Fel d I for all three monoclonal antibodies.
Another monoclonal antibody against reduced/alkylated Fel d I had been found to bind predominantly to reduced/alkylated chain 2 on immunoblot in previous studies (27). It bound to peptides 1–16 and 60–70 of chain 1 and peptides 1–14 and 50–63 of chain 2; it is therefore probably directed against a conformational epitope formed by these four regions. Possibly because of low affinity of this monoclonal antibody, specificity of its binding could not be verified by inhibition studies.
A panel of monoclonal antibodies directed against native Fel d I bound to peptides 1-16 and 60–70 of chain 1 and peptides 1–14 and 43–56 of chain 2. For two monoclonal antibodies, binding to each peptide was investigated and shown to be inhibitable by native Fel d I. These antibodies are therefore probably directed against a conformational epitope formed by these four regions.
These studies give us substantial information about the quaternary structure of Fel d I.     

Identification of allergen components of the opportunistic yeast Pityrosporum orbiculare by monoclonal antibodies

The yeast Pityrosporum orbiculare (P. orbiculare) is a member of the normal human cutaneous flora, but it is also associated with several clinical manifestations of the skin. We have previously observed IgE-binding components in P. orbiculare extracts, using sera from patients with atopic dermatitis. In the present study, we raised several monoclonal antibodies (MoAbs) against P. orbiculare to characterize some of its antigens, and used Candida albicans (C. albicans) as a control. We obtained several IgGI MoAbs which specifically recognized P. orbiculare in ELISA. Two of these were selected for immunoblotting studies on P. orbiculare and two patterns of reactivity emerged. Firstly, one MoAb showed a distinct band at a molecular mass of 67 kDa. In the second pattern, a sharp band at about 37 kDa appeared. In contrast, the IgM antibodies raised reacted with a 14-kDa component; but they reacted with C. albicans in addition to P. orbiculare The IgGI antibodies seemed to react with proteins, as their ability to react in ELISA with extract pretreated with protease was greatly reduced. In contrast, IgM MoAbs were much less affected, suggesting that they recognized nonprotein components. To determine whether these MoAbs-binding components were also recognized by human IgE, we adopted a radioimmunoassay (RIA) using the MoAbs as catcher antibodies. Both the 67-kDa and the 37-kDa components were IgE-binding proteins. P. orbicular RAST positive sera were scored as positive in the RIA, whereas the control serum was not.     

IgE antibodies to recombinant forms of Fel d I: Dichotomy between fluid-phase and solid-phase binding studies

Background: The major cat allergen Fel d I consists of two polypeptide chains linked by disulfide bonds, each of which has been expressed in bacteria. To investigate the antigenic structure of Fel d I, antibody binding to the native molecule and to each recombinant chain were compared. Methods: Polyclonal human IgE and IgG antibodies and monoclonal antibodies (mAbs) to Fel d I were compared for binding to Fel d I, chain 1, or chain 2 by fluid-phase inhibition radioimmunoassay, RAST, and immunoabsorption. Results: In the fluid-phase assay, neither recombinant chain significantly inhibited the binding of antibody to native Fel d I at concentrations of up to 10 μg/ml. Partial inhibition was observed when chain 1 was used, which inhibited the binding of two mAbs by 40% and 75%. In contrast, when the solid-phase RAST assay was used, IgE antibodies bound both chains with high specificity, and there was a good quantitative correlation between IgE antibody binding to Fel d I and both chain 1 (r = 0.58, p < 0.01) and chain 2 (r = 0.47, p < 0.01). Up to 70% of IgG or IgE anti-Fel d I antibodies could be absorbed by either chain 1 or chain 2, and both chains in combination produced similar absorption values in response to native Fel d I. Four mAbs were fully absorbed by chain 1, but not chain 2, and three mAbs were not absorbed by either chain. Conclusions: The results demonstrate a dichotomy between antibody binding to recombinant Fel d I chains, which may be explained by confirmational differences between the chains in the fluid phase or on solid supports. The results also suggest that chain 1 is an important site for mAb-defined B-cell epitopes on Fel d I. (J ALLERGY CLIN IMMUNOL 1995;95:1221-8.)     

Identification by monoclonal antibodies of T-cell subpopulations activated by 12-O-tetradecanoylphorbol-13-acetate (TPA)

Two major T-cell subpopulations obtained from human peripheral blood were studied their capacity to be activated by the tumor promoting agent, 12-O-tetradecanoylphorbol-13-acetate (TPA). The subpopulations were identified and separated from each other by their reactivity either to the monoclonal antibody, OKT4 or OKT8, followed by complement-dependent lysis. The fraction enriched in T8+ cells showed approximately a 1.2-2.0-fold greater proliferative response to TPA (20 ng/ml) than did the T4+ cell subset. Optimal activation required the presence of monocytes. Comparison of the mitotic activity showed that the sum of each T-cell subset was less than a mixture of the two. This indicates either a unilateral or cooperative interaction of mitogenesis in the presence of TPA.     

Activation of human complement by mouse and mouse/human chimeric monoclonal antibodies

The complement (C)-activating capabilities in human serum of 32 mouse and 10 mouse/human chimeric MoAbs of different isotypes, and their fragments, were tested in vitro. Activation of C via the classical pathway (CP) was performed in 1% factor D-deficient serum in gelatin containing Veronal buffer in the presence of calcium and magnesium (GVB⁺⁺), while activation of the alternative pathway of C (AP) was assessed in 10% Clq-depleted serum in the presence of 5 mm MgCl₂ in GVB⁺⁺. The C-activating ability of MoAbs was expressed relative to the degree of activation of complement by aggregated IgG for the CP and relative to mouse IgG 1 for the AP. All of seven mouse IgG2a MoAbs were potent activators of the CP. The results of CP activation by IgG1, IgG2b and IgG3 isotypes were different for individual MoAbs. Only three (two IgG 1 and one IgG3) of 32 mouse MoAbs were potent activators of the AP. IgG2a and IgG2b were relatively poor AP activators. There were a few MoAbs which activated both the AP and CP. Of 10 chimeric MoAbs, two IgG1, one IgG2 and one IgG4 were poor or non-activators of the CP. On the other hand, IgG2 and IgG4 were good AP activators. IgG3 was the most potent AP activator. Most of the F(ab')₂ fragments were activators of the AP and displayed no activation of the CP. Fc fragments only activated the CP. whereas Fab'did not activate the CP or the AP. These studies suggest that the route of complement activation by class and subclass MoAbs can not always be predicted in advance and based only on their subclass identity.     

Biosynthetic radiolabeling and immunoprecipitation of monoclonal antibodies

Summary A simple method is described for the radiolabeling in vitro and immunoprecipitation of monoclonal antibodies without altering their native structures.     

Measurement of serum IgG4 levels by a competitive immunoenzymatic assay with monoclonal antibodies

A competitive indirect ELISA is described for the measurement of IgG4 levels. It uses a monoclonal anti-subclass antibody and purified monoclonal IgG4 as standards. This method is sensitive and reproducible and more accurate than hemagglutination inhibition and radial immunodiffusion. Serum IgG4 levels in 173 normal adults were < 0.01–2.1 mg/ml (mean 0.30 mg/ml) in women and < 0.01–1.87 mg/ml (mean 0.465 mg/ml) in men.     

Identification of cells of the human mononuclear phagocyte system with rat monoclonal antibodies

Five rat monoclonal antibodies (McAbs) to human macrophages are described: YTH 8.18, YTH 25.7, YTH 51.1, YTH 85.12.1, and YHB 65.5. These McAbs are divided into three groups, since YTH 8.18, YTH 51.1, and YHB 65.5 are thought to identify the same antigen. These McAbs react with some bone marrow blast cells, granulocytes, and different percentages of peripheral blood monocytes. When studied on different body tissues, they were found to identify all members of the mononuclear phagocyte system (MPS), except Langerhans cells of skin and epithelium and in the case of one group (YTH 8.18/YTH 51.1/YHB 65.5) osteoclasts. In nine reactive lymph nodes the anti macrophage McAbs identified germinal centre macrophages, sinus macrophages, and interdigitating cells, but not dendritic reticulum cells. They also identified epithelioid macrophages and Langhans-type multi-nucleated giant cells in lymph nodes involved in granulomatous lesions (sarcoidosis and toxoplasmosis). In 24 cases of non-Hodgkin's lymphoma, the antimacrophage McAbs identified reactive macrophages in cases of B- or T-lymphocyte origin, whereas in three selected cases of true histiocytic lymphoma all the McAbs were found to be reactive with the vast majority of neoplastic macrophages as they were with the cells of a neoplastic macrophage line (U937). The possible use of these McAbs in the identification of benign and malignant macrophages in different systems is discussed.