One antigen may form two precipitin lines and two spurs when tested with two monoclonal antibodies by gel diffusion assays

A monoclonal antibody reactive with human immunoglobulin (Ig)G4 and a monoclonal antibody reactive with IgG1, IgG2 and IgG4 were tested with an IgG4 myeloma protein by double diffusion in a polyethylene glycol-containing gel. In a three-well Ouchterlony pattern, the IgG4 myeloma protein formed lines of double partial identity (double spur) with the two monoclonal antibodies. The two spurs lengthened and thickened with decreasing concentrations of polyethylene glycol indicating that soluble immune complexes diffused past the precipitin lines and formed the spurs. In a two-well pattern, the myeloma protein formed two lines with mixtures of the two monoclonal antibodies indicating that the immune complexes formed by the two antibodies distributed bimodally in the gel as if two types of complexes were formed. These unpredicted findings indicate that the process of antigen-antibody precipitation in gels needs to be analyzed further by using monoclonal antibodies.     

Production and characterization of monoclonal antibodies to human growth hormone

Three BIOZZI-HR mice were immunized with human growth hormone (hGH). From the determination of the titer, the average equilibrium association constant and the heterogeneity index of the antisera, it was possible to select the most suitable mouse for production of monoclonal antibodies (Mabs). Resulting from a single fusion, eight Mabs were produced, purified and characterized. The equilibrium association constant of the Mabs ranged from 5.10(8) M-1 to 9.109 M-1 at physiological pH. Four areas on hGH are recognized by the Mabs (the topology of the Mabs was investigated by two-site immunoradiometric assays). The Mabs, which recognize a same area, show similar cross-reactivities between hGH and human Placental Lactogen (hPL). No selected Mabs bound human Prolactin (hPRL), equine Growth Hormone (eGH) and porcine Growth Hormone (pGH). Two complementary Mabs enable a two-site immunometric assay of pituitary and E. Coli derived hGH.     

Production and characterisation of monoclonal antibodies directed against different epitopes of human thyroid stimulating hormone.

We have produced monoclonal antibodies (mAbs) to human thyroid stimulating hormone (hTSH) and selected five that specifically recognize hTSH and do not cross-react with the other human glycoprotein hormones such as luteinizing hormone (LH), chorionic gonadotropin (CG), follicle stimulating hormone (FSH). All of the antibodies were of the IgG1 subclass with affinities ranging from 5.3 X 10(8) to 1.9 X 10(10) mol-1.l; they could be assigned to two subgroups on the basis of their epitope specificity.     

Antigenic sites of human growth hormone and related molecules detected by monoclonal antibodies to human growth hormone

Four major antigenic sites for human growth hormone (hGH) were identified by 27 mouse monoclonal antibodies to hGH. Sites 1 and 2 are spatially close whereas sites 3 and 4 are located in other parts of the molecule. There also appears to be a subdivision of antigenic sites. A panel of 10 monoclonal antibodies, which included representatives from each antigenic site group, were used to determine cross-reactivities between hGH and human placental lactogen (hPL), human prolactin (hPRL), the 20,000 mol. wt variant of hGH (hGH20K) and a disulfide-linked dimer of hGH (diS-dimer). The data suggest a high conformational dependence of antigenic sites in hGH. DiS-dimer retains all four antigenic sites of hGH, although all have been altered. hGH20K retains sites 2-4 but site 1 has been dramatically altered. hPL retains site 3, whereas sites 1 and 4 have been dramatically altered and site 2 may be lacking. The extremely low cross-reactivity observed for hPRL is consistent with the dissimilarity between hGH and hPRL. Antigenic site 3 is the most conserved of all sites. The lack of structural similarity compared with hGH of site 1 in hGH20K and of a portion of site 3 in diS-dimer suggests that it may be possible to develop specific radioimmunoassays for these structural variants of hGH.     

Human sera with precipitating antibodies to human soluble immune complexes. A brief communication

INTRODUCTION: Previous numerous papers by the senior author dealt with the human serum factor referred to as anti-antibody which is specifically directed against IgG antibodies that underwent molecular transformation in the course of the reactions with their corresponding antigens. The reactions of this serum factor could be conveniently detected by means of agglutination of Rh-positive erythrocytes sensitized by anti Rh antibodies. No precipitation tests could be developed. MATERIAL AND METHODS: Most studies were conducted by means of double diffusion in gel precipitation. RESULTS: A rheumatoid arthritis serum, G, was noted that produced a strong reaction of double diffusion in gel precipitation with serum samples of a renal graft recipient, T. Further screening detected one more rheumatoid arthritis serum reacting with T; of 28 sera from renal graft recipients, 6 reacted in a similar way to T, but the reactions were considerably weaker and poorly reproducible. Evidence was presented that the precipitin in the two rheumatoid arthritis sera under study had properties of previously described anti-antibody. CONCLUSIONS: Sera with precipitating anti-antibodies may serve as exquisite reagents for detection of soluble immune complexes in human sera.     

Characterization of two monoclonal antibodies to Epstein-Barr virus diffuse early antigen which react to two different epitopes and have different biological function.

Five monoclonal antibodies (mAbs) were identified using immunofluorescence that were specific for the Epstein-Barr virus (EBV) encoded 52/50 kDa early antigen (EA-D) protein complex. Evidence to suggest that these mAbs react with the same 52/50 kDa EA-D protein was obtained by Western blotting, immunoprecipitation and ELISA. Two of the mAbs, 90E2 and 214A9, neutralized EBV DNA polymerase activity. The 214A9 mAb also inhibited the activity of bacteriophage T4 DNA polymerase while the 90E2 mAb did not. These data suggest that the 90E2 and 214A9 mAbs recognize two different epitopes on the 52/50 kDa EA-D protein. The high frequency of recovery of hybridomas producing anti 52/50 kDa EA-D mAbs suggest that this protein may have an important role in EBV pathogenesis/replication.     

Comparative reactivity of different HLA-G monoclonal antibodies to soluble HLA-G molecules

Different HLA-G monoclonal antibodies (mAbs) were first evaluated for their capability to identify soluble HLA-G (sHLA-G) in ELISA. Three of them, namely 87G, BFL.1 and MEM-G/9, when used as coating mAbs together with W6/32 capture mAb, identified beta2-microglobulin (beta2m)-associated-sHLA-G but not soluble HLA-B7 (sHLA-B7) in cell culture supernatants from transfected cells. By comparison, the anti-HLA class I mAb 90 did recognize both sHLA-G and sHLA-B7. By using these HLA-G mAbs, sHLA-G was identified in amniotic fluids as well as in culture supernatants of first trimester and term placental explants but not in cord blood. Intron 4-retaining sHLA-G isoforms were identified in some amniotic fluids by the use of an intron 4-specific mAb (16G1). Reactivity of these different HLA-G mAbs was then compared to determine their respective binding sites on soluble and membrane-bound HLA-G. Using both ELISA and flow cytometry analysis, we showed that they did not compete with each other, which suggested that they did not recognize the same determinants. Finally, we report that two mAbs directed against the alpha1 domain of HLA class I heavy chain (mAb 90 and YTH 862) did compete with 87G, therefore demonstrating that this latter mAb recognized an epitope localized on this external domain of HLA-G.     

Structural analysis of the CD69 early activation antigen by two monoclonal antibodies directed to different epitopes.

The biochemical structure of CD69 early activation antigen has been characterized by means of two newly isolated mAb, namely C1.18 and E16.5. Upon analysis by SDS-PAGE, C1.18-reactive molecules immunoprecipitated from 125I-surface labeled PMA activated PBL consisted of a 32 + 32 kD dimer, a 32 + 26 kD dimer, a 26 + 26 kD dimer and a 21 + 21 kD dimer. E16.5-reactive molecules consisted of a 26 + 26 kD dimer and a 21 + 21 kD dimer. Cross absorption experiments showed that E16.5 mAb reacts with an epitope of the CD69 molecule distinct from the one recognized by C1.18 mAb and present only on a subpopulation of the CD69 molecular pool. The patterns of migration of C1.18- and E16.5-reactive molecules in two-dimensional gel-electrophoresis, under reducing conditions before and after treatment with Endoglycosidase F enzyme suggest that the two mAb recognize the same glycoprotein structure, but in two distinct glycosylation forms, both expressed on the cell surface membrane. Finally, p32, p26 and p21 of CD69 complex obtained from three distinct normal donors did not show appreciable structural polymorphism, by two-dimensional peptide mapping, not only among single subunits within the same individual, but also among homologous subunits in distinct individuals. Further, it was found that CD69 complex is expressed at the cell surface of resting PBL, although at a very reduced level in comparison to PMA activated cells. C1.18 and E16.5 mAb induced comparable cell proliferation and IL-2 production in PBL in the presence of PMA. C1.18 mAb increased intracellular free calcium concn in PMA activated PBL after cross-linking with goat anti mouse Ig, while the effect induced by E16.5 mAb after cross-linking was consistently lower. Finally, it was found that Sepharose-linked C1.18 mAb, in the presence of rIL-2 or PMA, did not induce TNF release from 6 NK cell clones.     

Specificity and association constants of 33 monoclonal antibodies to human IgA epitopes

We used an immunofluorescent sequential-saturation-of-antibody assay and an interactive computer program for Scatchard analysis to determine association constants (Ka) of 33 murine monoclonal antibodies (Mabs) specific for human IgA epitopes. Ka ranged from 0.37 to 690 x 10(7) liters per mole (an approximate 1900-fold difference). Specificity was validated with a panel of 18 highly purified IgA1 and IgA2 myeloma proteins and secretory IgA using an immunofluorometric assay. Western blots of bacterial IgA protease digests were used to locate the epitopes of IgA specific Mabs in either the Fab, Fc, or hinge region. Mabs specific for unique epitopes on secretory IgA or free secretory component (FSC) were produced and evaluated.     

Quantitative measurement of human immunoglobulin E using monoclonal antibodies to distinct epitopes

Monoclonal antibodies (MAb) which recognize distinct epitopes on human immunoglobulin E have been used to develop two-site sandwich radio- and enzyme-linked immunoassays for the quantitation of human IgE. In the first step, a purified anti-IgE MAb coated to polyvinyl or polystyrene microtiter plates specifically bound the IgE contained in the samples. In the second step, another anti-IgE MAb (either iodinated or conjugated to beta-galactosidase) directed to a different antigenic determinant was used to estimate the amount of bound IgE. This simple method permitted the determination of IgE concentrations of 10 ng/ml and greater in about 3 h. Coefficients of variation on a single day did not exceed 7.5% for IgE levels, covering a wide range of the standard curve. The values obtained on serum samples showed a good correlation with those obtained using the paper radioimmunosorbent test (PRIST).     

Comparative study of pituitary and bacteria-derived human growth hormone by monoclonal antibodies

We have established hybridoma lines which secrete mouse monoclonal antibodies (Mabs) to human pituitary growth hormone, hGH. Using indirect competitive ELISA and indirect passive hemagglutination inhibition twelve different Mabs were characterized with regard to cross-reactivity with the hGH-related hormones, human chorionic somatomammotropin, hCS, and human prolactin, hPRL. The reactivity of these Mabs with pituitary hGH was compared to that with either bacterially-produced methionyl-hGH or to that of reduced and S-carboxymethylated hGH, which has an altered conformation. None of the Mabs reacted with hPRL. Four did not react with hCS whereas the others showed varying degree of cross-reactivity with hCS. All Mabs reacted more weakly with reduced and S-carboxymethylated hGH than with the native form of the hormone, which was not seen with conventional rabbit antisera to hGH. Thus in the case of hGH the Mabs are superior to conventional antisera in revealing small conformational differences. However the pituitary and bacterially-derived methionyl-hGH were indistinguishable as determined by the 12 Mabs.     

抗人血小板/T细胞活化抗原1(PTA1)不同表位单克隆抗体的制备及鉴定

目的 研制一套可识别PTA1分子不同功能性表位的单克隆抗体（mAb)。方法 采用亲和层析法从血小板裂解液中纯化的天然PTA1分子免疫Balb/c小鼠,以间接ELISA筛选阳性杂交瘤,并以PTA1 cDNA转染COS7细胞,进行间接免疫荧光染色和流式细胞术鉴定mAb的特异性。竞争结合试验确定mAb识别PTA1分子的表位。纯化的PTA1经SDS－PAGE后,转移到PVDF膜,确定mAb是否可用于Western blot。结果 共获得7株可稳定分泌mAb的杂交瘤细胞株,分别命名为FMU1,FMU2,FMU3,FMU4,FMU5,FMU6和FMU7。所有mAb与纯化天然PTA1和PTA1／Ig融合蛋白均有良好的免疫反应性,均可识别PTA1 cDNA转染的COS7细胞。流式细胞仪检测阳性荧光峰型与PTA1阳性对照Leo A1 mAb的荧光峰型相似;7株mAb识别PTA1分子的5个不同表位;FMU1,FMU2,FMU4,FMU5,FMU6和FMU7可应用于Westernblot。结论 成功地制备7株抗PTA1分子的特异性mAb。这些mAb可分别识别PTA1的5个表位,为PTA1分子结构与功能的研究提供了新的手段。     

Non-complement-dependent adsorption of soluble immune complexes to human red cells

Heat aggregated IgG and soluble immune complexes (IC) prepared by combining human serum albumin with rabbit anti-serum albumin and tetanus toxoid with rabbit antiserum to tetanus toxoid were shown to bind to human O+ RBC. The binding was greater for soluble IC prepared at antigen excess, and although it was usually maximal when IC were pre-opsonized, it could also be demonstrated using non-opsonized heat aggregated IgG or soluble IC prepared in the absence of complement. These observations suggest that two types of receptors may be involved in binding of soluble IC to human RBC: the classical C3b receptor, and a non-complement-dependent receptor, perhaps recognizing the Fc region of the immunoglobulin molecule exposed after heat aggregation or antigen-antibody reaction.     

Difference in cell binding patterns of two monoclonal antibodies recognizing distinct epitopes on a human melanoma-associated oncofetal antigen

Two monoclonal antibodies (MAbs), 140.240 and 96.5, generated independently in different laboratories, have been shown to detect the target structures of 87,000 (gp87) and 97,000 (p97) glycoproteins, respectively, both strongly expressed by melanoma cells and fetal small intestine. To determine whether MAb 140.240 and MAb 696.5 recognized a same target structure, they were tested in immunoprecipitation/SDS-PAGE using NP-40 lysates of melanoma cells labelled with [35S]methionine for 18 hr. Both antibodies precipitated a single band with Mr = 87,000. Reciprocal immunodepletion studies showed that neither of the two antibodies detected the 87,000 band in the lysate immuno depleted by either antibody, suggesting that these two antibodies recognize the same or extremely similar molecules. Two-dimensional tryptic peptide mapping analysis showed that the two identified molecules shared the same finger-printing pattern. A 40,000 fragment of the 87,000 molecule produced by protease digestion was precipitated by MAb 96.5 but not MAb 140.240, indicating that the epitopes recognized by the two antibodies are localized at discrete sites on the molecule. Serological studies on these two antibodies revealed slightly different binding patterns in the MAb 140.240 exhibited a more melanoma-restricted specificity, while MAb 96.5 had a specificity to melanoma and to some other cell types. The observed difference in epitope specificity may be important in the clinical applications of these antibodies.     

Immunization with immune complexes: Characterization of monoclonal antibodies against a TSH-antibody complex

Monoclonal antibodies (MAb) to human thyrotropin (hTSH) were prepared by immunization of mice and rats according to different procedures. We have previously demonstrated that a specific antigenic region on the surface of the hTSH molecule was highly immunogenic; in order to produce specific MAb to weakly immunogenic regions of hTSH, we immunized mice and rats with a complex composed of hTSH and an anti-β hTSH MAb 27 directed against the highly immunogenic region. Monoclonal antibodies elicited by this immunization procedure were highly specific and a high percentage was found complementary to the MAb 27 used in the immunogen. We did not search for anti-MAb 27 antibodies, however one hybridoma produced antibody that preferentially reacted with the immune complex. This antibody, called 515, is an IgG₁ that binds the complex with 100-fold greater affinity than it does to the anti-β hTSH MAb 27 alone. This enhancement was also observed with the Fab fragment of the MAb suggesting that the epitope recognized by this anti-complex MAb is displayed in a very different way when hTSH is bound to the first MAb.     

T cell activation by monoclonal antibodies directed to different epitopes on the human T cell receptor/CD3 complex: evidence for two different modes of activation

The mouse monoclonal antibody (mAb) BMA031 (IgG2b) has recently been described to be directed against a monomorphic part of the human T cell receptor (TcR) alpha/beta. In vitro analysis of its stimulatory potential for mononuclear cells revealed two patterns of responsiveness. Out of 35 tested individuals only 2 generated a proliferative response to low antibody concentrations (15 ng/ml; "high responders"), the others ("low responders") responded only to high antibody concentrations (1.5 micrograms/ml); the anti-CD3 mAb UCHT1 (IgG2b) stimulated only the two high responders. This response pattern to BMA031 was determined by the accessory cell compartment in the culture. Stimulation by BMA031 in low responders demonstrated some unusual features: (a) high antibody concentrations were required, (b) addition of autologous serum had no inhibitory effect and (c) vigorous depletion of macrophages reduced but did not abolish the proliferative response. These characteristics were shared by two other mAb, BMA032 and BW239/347, presumably directed against the TcR alpha/beta but not by several other antibodies to the TcR/CD3 complex. Thus, the results demonstrate unusual stimulatory properties of three anti-TcR alpha/beta mAb, inducing a proliferative response without antibody cross-linking. This suggests that the stimulatory effect of anti-TcR/CD3 complex mAb is not only determined by their isotype, but also strongly depends on their epitope specificity.     

Two monoclonal antibodies raised against a Burkitt lymphoma cell line recognise different cell types within lymphoid follicles.

Monoclonal antibodies were raised against a laboratory derived variant of the Raji Burkitt lymphoma cell line. We have characterized two of these antibodies by screening on haematopoietic cell lines, and frozen sections of both reactive and neoplastic lymphoid tissue, and found reactivity with separate cellular components of lymphoid follicles. Monoclonal FW37.4.D5 reacts in section specifically with follicular dendritic reticular cells. Monoclonal 35.1C5 reacts with a subpopulation of splenic marginal zone, and lymph node mantle zone, B lymphocytes, but stains only rare cells within germinal centres. This monoclonal is operationally B lymphocyte specific, distinguishing an 'intermediate' B cell subset, represented in lymphoma by B chronic lymphocytic leukaemia, B hairy cell leukaemia and centrocytic lymphoma.     

Estimation of association constants of 42 monoclonal antibodies to human IgG epitopes using a fluorescent sequential-saturation assay

We measured the association constant (Ka) for 42 murine monoclonal antibodies (MAbs) to human IgG epitopes. Included are antibodies, previously evaluated in an IUIS/WHO collaborative study, to various epitopes on the four subclasses of human IgG - IgG Fc, IgG Fab, kappa, and lambda - and to selected IgG allotopes. We used a sequential-saturation immunofluorescent assay and interactive computer program to determine the Ka by Scatchard analysis. Kas ranged from unmeasurably low by this method (approximately 10(6) L/M) to 3.8 X 10(9) L/M. Some class specific MAbs had large Ka differences for different subclasses and some subclass specific MAbs had large Ka differences for molecules of the same subclass but of different light-chain types.     

Multiplicity of cross-reactive epitopes on Bet v I as detected with monoclonal antibodies and human IgE

Six monoclonal antibodies against Bet v I, the major cross-reactive allergen of birch pollen ( Betula verrucosa ), were obtained. Four did not react with fruits, but two monoclonal antibodies (mAbs) (5H8 and 9C11) were reactive with apple and other fruits. These two cross-reactive antibodies reacted with identical or overlapping sites, but differed in their relative degree of cross-reactivity toward various fruits and hazelnut. Cross-reactive human IgE antibodies reacted with a nonoverlapping epitope, as indicated by results of a two-site radioimmunoassay (RIA) with the fruit-reactive mAb 9C11. By isoelectric focusing (IEF) in conjunction with immunoblotting, a maximum of seven isoforms could be distinguished. Depletion of birch-pollen extract for Bet v I with the most reactive mAb (7F7) removed approximately 95% of the IgE cross-reactivity between birch pollen and apple extract. The remaining 5% cross-reactive material was still capable of inhibiting the binding of IgE to apple allergen completely, and was reactive with mAbs 5H8 and 3C4. By means of IEF/immunoblot, it was shown that these mAbs recognize an isoform of Bet v I that is poorly, if at all, recognized by mAb 7F7. These results illustrate the heterogeneity of Bet v I, both with respect to the cross-reactive sites as well as to the backbone structure. This type of heterogeneity has possible implications for the use of monoclonal antibodies in allergen standardization.