Production of monoclonal antibodies to surface regions that are non-immunogenic in a protein using free synthetic peptide as immunogens: demonstration with sperm-whale myoglobin

Two synthetic peptides corresponding to surface regions of sperm whale myoglobin that are not antigenic in the native molecule were used in their free form (i.e. not coupled to a carrier) to immunize separate groups of Balb/cByJ mice. The synthetic peptides corresponded to regions 1-6 and 121-127. Serum samples obtained from each group of mice contained antibodies that bound specifically to myoglobin and exclusively to the immunizing peptide. Monoclonal antibodies to each of the two surface regions were subsequently obtained by hybridizing Fa/O mouse myeloma cells with spleen cells derived from each group of mice. These monoclonal antibodies were IgM (kappa). They expressed the same isotype as the antigen specific serum antibodies produced by the mice whose spleen cells were used for hybridization. Solid phase radioimmunoassay studies also indicated that each monoclonal antibody, like the immune serum of the parent animals, bound specifically to myoglobin and exclusively to the synthetic peptide used as an immunogen. These results suggested that the hybridoma antibodies expressed submolecular binding specificities that were the result of peptide immunization rather than hybrid selection and that monoclonal antibodies with preselected submolecular binding specificities to non-antigenic surface regions in a protein molecule can be produced by the techniques of somatic cell hybridization when the corresponding free synthetic peptides are used as immunogens.     

Production of monoclonal antibodies with preselected submolecular binding specificities to protein antigenic sites: antibodies to sperm whale myoglobin sites

The five synthetic antigenic sites of sperm whale myoglobin were used in their free form (i.e. not coupled to any carrier) to immunize separate groups of BALB/cByJ mice. The synthetic peptides corresponded to: site 1, residues 15-22; site 2, residues 56-62; site 3, residues 94-99; site 4, residues 113-119; site 5, residues 145-151. Serum samples obtained from each group of mice contained antibodies that bound specifically to myoglobin and exclusively to the immunizing antigenic site. Monoclonal antibodies to each of the five antigenic sites were subsequently obtained by hybridizing Fa/O mouse myeloma cells with spleen cells derived from each group of mice. These monoclonal antibodies were either IgM(kappa) or IgGl(kappa). They expressed the same isotypes as the antigen specific serum antibodies produced by the mice whose spleen cells were used for hybridization. Solid phase radioimmunoassay studies also indicated that each monoclonal antibody, like the immune serum of the parent animals, bound specifically to myoglobin and exclusively to the synthetic peptide used as an immunogen. These results suggested that the hybridoma antibodies expressed submolecular binding specificities that were the result of peptide immunization rather than hybrid selection. This strongly supports our previous findings that it is possible to produce monoclonal antibodies with preselected submolecular binding specificities to continuous protein determinants by the techniques of somatic cell hybridization when the corresponding free synthetic determinants are used as immunogens.     

Immunogenicity of a free synthetic peptide: carrier-conjugation enhances antibody affinity for the native protein

The synthetic peptide 166-174 of hCS sequence, corresponding to an antigenic determinant of the protein, was used to elicit MAbs to the native hCS molecule. The synthetic peptide was administered according to different immunization protocols to BALB/c mice, both in the free form or conjugated to carrier proteins. Spleens and popliteal lymph nodes from primed mice were fused with a myeloma cell line to produce MAbs, and selected clones were characterized for isotype and affinity. Spleen fusions gave rise to IgM MAbs, whereas lymph node fusions gave preferentially IgG MAbs. No correlation was found between antibody class and affinity since affinity is highly increased by carrier-conjugation, while it did not enhance IgG production. The free synthetic peptide showed a low immunogenicity: affinities of MAbs produced ranged from 10(5) to 10(7) l/mole, an average 1000-fold lower than the values obtained with carrier-conjugated peptide. In one case, however, carrier conjugation did not give rise to anti-hCS MAbs. Overall, these studies provide a rational approach to the production of anti-protein MAbs by synthetic peptide immunization and offer the opportunity to obtain MAbs of the desired isotype and affinity.     

Identification of a mixture of two different monoclonal antibodies by nonlinear regression analysis after solid phase radioimmunoassay with labeled antigen

The results of competitive solid phase radioimmunoassay with labeled antigen, when analyzed by nonlinear regression, may reveal the presence of two different monoclonal antibodies, occasionally produced in a hybridoma culture. In fact, a hybridoma-derived antihuman chorionic somatomammotropin antibody prepared in our laboratories showed an unsatisfactory curve fitting when the experimental data were elaborated on the basis of a monoclonal model. The antibody was subjected to recloning, and gave two separate homogeneous monoclonal antibodies with different affinity constants.     

Production of monoclonal antibodies to thyroglobulin by in vitro immunization with a free synthetic peptide

A synthetic peptide corresponding to amino acids 1-19 of thyroglobulin was used to test the possibility of generating protein-reactive monoclonal antibodies by immunization in vitro with a synthetic peptide as antigen. Splenocytes from non-immunized Balb/c mice were cultured in serum-free medium for 3 days in the presence of thymocyte-conditioned medium and the synthetic peptide prior to fusion with SP2/0 murine myeloma cells. The synthetic peptide was used in its free form, i.e. not coupled to a protein carrier. Hybridomas secreting monoclonal antibodies reactive with the synthetic peptide were obtained after immunization in vitro with as little as 10 ng/ml of the synthetic peptide. Between 50 and 70% of the primary clones obtained in different experiments produced monoclonal antibodies also reactive with the intact protein. Six stable hybridomas were isolated; all produced antibodies of the IgM class. We conclude that immunization in vitro with a free synthetic peptide is an efficient method for the generation of monoclonal antibodies reactive with the intact protein.     

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.     

Antigenic structure of human chorionic somatomammotropin: four epitopes revealed by monoclonal antibodies

The characterization of epitope specificity of a panel of 15 monoclonal antibodies against human chorionic somatomammotropin (hCS), previously prepared in our laboratory, allowed us to distinguish 4 antigenic determinants on the hCS molecule. Experiments were carried out with competition and sandwich assays. The results allowed us to divide our MAbs into 4 groups, recognizing 4 different epitopes, and to select 4 MAbs each distinguishing a different epitope in order to pursue further studies on the antigenic structure of hCS.     

Production of monoclonal antibodies with pre-selected submolecular binding specificities to protein determinants: Demonstration with sperm whale myoglobin

Two monoclonal antibodies with pre-selected submolecular binding specificities to sperm whale myoglobin (Mb) were obtained by hybridizing Fa/0 mouse myeloma cells with spleen cells derived from mice which had been immunized with free (not coupled to any carrier) Mb synthetic peptides 132–153 or 145–151 (antigenic site 5). Both monoclonal antibodies were IgG₁ (_k). Their homogeneity was confirmed by analytical isoelectric focusing electrophoresis. According to competitive inhibition studies in which Mb, the five synthetic antigenic sites of Mb, Mb peptide 132–153, bovine serum albumin (BSA), and lysozyme were used as inhibitors, the binding specificities of both monoclonal antibodies were restricted to determinants present in the peptides used for immunizations. The results of direct binding studies confirmed this conclusion and suggested that monoclonal antibodies with pre-selected submolecular binding specificities can be readily obtained by the techniques of somatic cell hybridization when the corresponding free synthetic determinants are used as immunogens.     

Affinity purification of IgG monoclonal antibodies using the D-PAM synthetic ligand: chromatographic comparison with protein A and thermodynamic investigation of the D-PAM/IgG interaction

This study investigates the applicability of D-PAM, the inverso form of the Protein A Mimetic synthetic peptide affinity ligand (PAM) obtained from the screening of a multimeric combinatorial peptide library, in monoclonal IgG isolation from ascitic fluids and cellular supernatants. D-PAM affinity columns, prepared by immobilizing the all-D peptide on the commercially available support Emphaze, were able to capture monoclonal antibodies in a single chromatographic step, with a recovery yield and purity degree above 90% and full recovery of antibody activity. D-PAM/Emphaze resin showed a host cell protein (HCP) and DNA reduction similar to protein A sorbent. Indeed, column capacity, determined by applying a large excess of purified antibodies to 1 mL of column bed volume, was always higher than 50 mg/mL. D-PAM/IgG interaction was characterized by isothermal titration calorimetry (ITC) and an analysis of binding isotherms, obtained for titration of ST2146, ST1485 and 7H3 IgG monoclonal antibodies, suggested that two peptides bind simultaneously to the IgG molecule, with a K(A) (equilibrium association constant) of 3.4, 6.2 and 3.4 x 10(4) M(-1), and a DeltaH (change in enthalpy) of -1.3, -4.2 and -4.1 kcal mol(-1), respectively.     

Dissection of an antibody paratope into peptides discloses the idiotope recognized by the cognate anti-idiotypic antibody

Using methods of parallel synthesis, the complete amino acid sequence of an Ab 1 antibody (Tg 10, an anti-human thyroglobulin monoclonal antibody) was made in the form of a set of 100 synthetic overlapping peptides. This set of immobilized peptides was allowed to react with the cognate Ab2 (AI 10, a highly purified rabbit anti-idiotypic polyclonal antibody to Tg 10). A dominant peptide idiotope, INTFSGVPTYA, was thus mapped, which corresponds mainly to the CDR2 region from the V(H) domain of the Tg 10 mAb. A synthetic peptide replica of this idiotope was found to bind to AI 10 with an affinity (K(D) in the 10(-8) M range, as measured using BIACORE technology) which represents a significant part of the affinity of the complete Tg 10 antibody (K(D) in the 10(-9) M range). The synthetic peptide also elicited anti-idiotypic antibodies in rabbits that recognized specifically the Ab1 antibody in an Ab1- and antigen-inhibitable manner. The peptide idiotope was further characterized chemically by the identification of residues important for binding to the Ab2 and by modelization of its structure. Our approach makes it readily possible to map and characterize functional, continuous-type idiotopes that could be further used to manipulate the immune response by peptide technologies.     

Peptide mimics of hapten DNP: the effect of affinity of anti-DNP monoclonal antibodies for the selection of phage-displayed mimotopes

Biopanning of two linear (6- and 15-mer) and two constrained (10- and 17-mer) phage-displayed peptide libraries with two anti-DNP monoclonal antibodies (mAbs) selected seven unique peptide sequences using only the low affinity anti-DNP monoclonal antibody. The selected peptides contained two of 6, one of 10, two of 15 and two of 17 amino acids in length. They were all rich in hydrophobic residues. Both 15-mer peptides had antigenic regions of eight amino acids as revealed by a spot scan assay. Two of the 17-mer and one of the 10-mer peptides displayed on phage competed with free DNP for the low affinity anti-DNP mAb. These findings highlight (i) the selective power of phage displayed peptide libraries to identify peptides that mimic the shape of a small hapten molecule such as DNP, (ii) the possible preferential bias of phage libraries towards low affinity antibodies, (iii) the importance of using a panel of phage libraries for selecting peptide mimics.     

Monoclonal antibodies to human alpha-foetoprotein: analysis of the behaviour of three different antibodies

Three mouse monoclonal antibodies directed to different epitopes on human alpha-foetoprotein have been produced. Two are of IgG1 subclass and the third is IgA. The polyethylene glycol-dependent immunoprecipitation system, designed for conventional antisera, had to be adapted before reproducible results could be obtained with the reagents. In this adapted system, as well as in a radioimmunoassay using solid-phase second antibody, a mixture of the 3 monoclonal antibodies exhibits cooperativity. However, the sensitivity of the radioimmunoassay with pooled monoclonals is not good as that of conventional antiserum. Low affinity monoclonal antibodies have been used for immunopurification of the antigen, whilst the high affinity one is useful for antigen quantitation in a labelled antibody-dependent system which requires absolute antibody specificity.     

A simple enzyme-linked immunosorbent assay (ELISA) for the neuron-specific gamma isozyme of human enolase (NSE) using monoclonal antibodies raised against synthetic peptides corresponding to isozyme sequence differences.

Monoclonal antibodies specific for the gamma isozyme of human enolase (known as neuron-specific enolase or NSE) have been raised against synthetic peptides after coupling to carrier protein: the selected peptides were those corresponding to regions of amino acid sequence difference between the alpha and gamma subunits of these closely similar isozymes. This technique gave monoclonal antibodies of high specificity and affinity. Two monoclonal antibodies raised against different peptides were used to develop a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA), using one as the solid-phase antibody and the other conjugated to horseradish peroxidase to detect the bound NSE. This assay provides a simple and routine method of detecting NSE in serum samples from patients with small-cell carcinoma of the lung and related tumours.     

A two-site sandwich radioimmunoassay of human gamma interferon with monoclonal antibodies

Two monoclonal antibodies (nos. 6008 and 6016) were raised against human gamma interferon (IFN-gamma) derived from E. coli harboring the recombinant cDNA for IFN-gamma, and one (3710) against a synthetic peptide representing its C-terminus amino acid sequence of 20 residues. The monoclonal antibody against the synthetic peptide (3710) reacted either with IFN-gamma or the synthetic peptide. One monoclonal anti-IFN-gamma (6008) did not react with the synthetic peptide, while the other (6016) showed a weak binding with the peptide. The binding of the monoclonal antibody against the synthetic peptide (3710) with IFN-gamma was not inhibited by 6008, but to a certain extent by 6016. A 2-site '1-step' radioimmunoassay was developed in which 6008 was fixed on a solid-phase support, and the test sample together with radiolabeled 3710 was added for the binding with it. The assay was rapid with a sensitivity capable of detecting a few ng/ml of IFN-gamma.     

The recognition by monoclonal antibodies of various portions of a major antigenic site of human growth hormone

The reactivities of five mouse monoclonal antibodies against human growth hormone (hGH) were defined by either a competitive radioimmunoassay with insolubilized antibodies or by an agglutination-inhibition method with hGH-coated polystyrene particles. The five antibodies reacted significantly but to various degrees with human placental lactogen and at least three antibodies reacted with human prolactin and three synthetic peptides extending from residues 19 to 128, 73 to 128 and 98 and 128 of hGH. Four tested monoclonal antibodies failed to react with bovine growth hormone and with hGH oxidized by performic acid. The antibodies were further distinguished by their different reactions with hGH modified by reduction and alkylation or by adsorption on a polystyrene surface. The unique specificity of each antibody was confirmed for most of them by an agglutination method in which the agglutinating activity of hGH was tested on latex particles coated with various paired combinations of the monoclonal antibodies. The lack of agglutination with certain combinations suggested that the specificities of such a pair of antibodies overlapped each other. These results suggest that the sequences corresponding to the synthetic peptides participate in the structure of a major antigenic site of which various portions are recognized by the monoclonal antibodies.     

Monoclonal antibodies to human immune interferon and their application for affinity chromatography

Two IgG1/kappa class monoclonal antibodies specific for human immune interferon (IFN-gamma), designated B1 and B3, were developed. Specific binding of both monoclonal antibodies to natural or Escherichia coli-derived recombinant human IFN-gamma was demonstrated in a solid-phase radioimmunoassay or by immunoprecipitation. Antibody B3 showed potent neutralizing activity against both natural and recombinant IFN-gamma. Antibody B1, which showed neutralizing activity only when very high concentrations were employed, was used for preparing immunosorbents for affinity chromatography of IFN-gamma. When a highly purified preparation of 125I-labeled natural IFN-gamma was loaded onto the affinity column, all of the biological activity was retained on the column. The bulk of 125I-labeled IFN-gamma bound to the affinity column be eluted in biologically active form, suggesting that antibody B1 could be used for the purification of human IFN-gamma. Analysis of IFN-gamma eluted from the column by NaDodSO4/polyacrylamide gel electrophoresis (SDS-PAGE) indicated that both of the known molecular weight subspecies of IFN-gamma (25,000 and 20,000 MW), as well as the presumed dimer of 45,000 MW, were retained by the B1 antibody affinity column.     

A murine monoclonal anti-metallothionein autoantibody recognizes a chemically synthesized amino-terminal heptapeptide common to various animal metallothioneins

A murine hybridoma clone (MT 189-14-7) producing an IgM-class monoclonal antibody specific for metallothionein (MT) was produced with rat Zn-MT 2-keyhole limpet hemocyanin conjugate as an immunogen. The reactivity of the monoclonal antibody with MTs obtained from various animal species was determined by the competitive radioimmunoassay. The MT 189-14-7 antibody bound equally to various MTs 1 and 2 including mouse MTs 1 and 2. This indicates that the antibody is a murine autoantibody reactive with various animal MTs and recognizes a common structure of the various MTs. Subsequently, the location of the antigenic determinant recognized by the MT 189-14-7 antibody was determined by using various synthetic human MT 2 peptides. Among the synthetic peptides examined, both the amino-terminal beta-domain, Ac-(hMT 2 1-29)-OH, and an amino-terminal heptapeptide, Ac-(hMT 2 1-7)-OH, but not the carboxy-terminal alpha-domain peptides, H-(hMT 2 29-35)-OH and H-(hMT 2 30-61)-OH, showed inhibitory activities slightly higher than the native MTs in the competitive radioimmunoassay. These results demonstrate that the MT 189-14-7 autoantibody recognizes the epitope located within the amino-terminal heptapeptide common to various MTs.     

Real Time Analysis of Antibody-Antigen Reaction Kinetics

Surface plasmon resonance, i.e. detection of changes in refractive index on a surface, was used in a biosensor to evaluate the dissociation/association rate and affinity constants of human monoclonal IgG and IgM antibodies and Fab fragments. The results showed that an observed difference in affinity constants between intact and fragmented IgG anti-tetanus antibody was related to approximately 10-fold differences in dissociation rate constants, since the association rate constants were in the same range, i.e. 2-3 x 10(5) (M-1 s-1). Affinity constants, as determined by conventional solid phase enzyme immunoassay, were substantially higher than the constants produced by the biosensor. Human monoclonal IgM anti-Tn alpha antibodies showed, furthermore, one order of magnitude higher association rate constants, as compared with the IgG antibodies, but since the dissociation rate constants were more than ten times higher, the resulting affinity constants of the anti-carbohydrate IgM antibodies were still somewhat lower than those of the IgG antibodies.     

A crossreactive antipeptide monoclonal antibody with specificity for lysyl-lysine

Synthetic peptides meeting certain guidelines have been used as immunogens to generate antibodies with predefined specificity. We have raised and characterized using established methods a monoclonal antibody against a synthetic peptide corresponding to the 18-amino acid carboxyterminal sequence (A194-211) of the platelet-derived growth factor (PDGF) A chain expressed by the U343 human glioma cell line. This antibody was generated in order to carry out structure-function studies on this region of PDGF whose biological significance is not yet clear. Anti-PDGF-A194-211 was found to be a low titre, IgMκ molecule, with K_{rmd of} 2.8 × 10⁻⁷M. When antibody reactivity was tested with parent PDGF-AA_L (A chain homodimer containing a carboxyterminal extension) significant binding was observed. Suprisingly, ¹²⁵I-PDGF-AA_S, consisting of truncated A chains but lacking the extension was also bound. Moreover, poly- -lysine, β-thromboglobulin, PDGF-A194-211, and myoglobin competed dose-dependently with ¹²⁵I-PDGF-AA_L for antibody, ¹²⁵I-bovine serum albumin was also bound. Examination of the primary sequence of proteins and peptides bound by the antibody revealed only one shared structural motif: a lysyl-lysine moiety. Selected small synthetic peptides containing this and other sequences were used as potential competitors of ¹²⁵I-PDGF-A194-211 in antibody binding. Lysyl-lysyl-glycyl-glutamine and lysyl-lysine competed, whereas lysyl-leucine did not. These results suggest that as few as two amino acid residues constitute a functional antigenic determinant and contrast with most previous estimates of the minimum number of residues required. Furthermore, we show that guidelines governing the design of synthetic peptides for their use as antigens to produce monoclonal antibodies of predetermined specificity may be unreliable.     

Monoclonal antibodies against isotypic and isoallotypic determinants of human IgA1 and IgA2: fine specificities and binding properties

We have analysed and compared the fine specificity and behavior in various immunoassays of 10 mouse monoclonal antibodies, from three independent laboratories, directed against IgA1, IgA2 or non-IgA2m(2). The following observations were made. (1) Although all of the monoclonal antibodies were specific for a particular IgA subclass or isoallotype in a radioimmunoassay, three of them were not specific when tested in indirect immunofluorescence on plasma cells derived from pokeweed-activated peripheral blood lymphocytes. In this highly sensitive system, contrary to direct immunofluorescence previously performed using formalin-fixed lymphoid tissue, the anti-IgA1 69.114 reacted with some of the IgA2 plasma cells, the anti-IgA2 DLDB7 reacted with some of the IgA1 plasma cells and the anti-IgA2 16.512 dimly reacted with all IgM plasma cells. (2) Among the eight anti-IgA subclass antibodies, seven were directed against the CH2 domain of IgA whereas the anti-IgA1 1-155-1 recognised an epitope destroyed by Streptococcus sanguis IgA1 protease and localised in the hinge region of IgA1. The two anti-isoallotype antibodies were directed against epitope(s) probably localised in the 65 C-terminal amino acid residues of the alpha-CH3 domain. All of the 10 antibodies were able to react with endogeneously produced surface IgA on B-cells. (3) Using monoclonal anti-IgA subclass antibodies in radioimmunoassay may be hazardous in the absence of knowledge of their affinity constants and of careful control experiments: some of the antibodies were not sensitive in radioimmunoassays designed to measure the serum titer of specific IgA1 and IgA2 antibodies. Moreover, major differences were observed between the different monoclonal reagents with respect to the influence of the size of IgA on a solid-phase sandwich radioimmunoassay. While three of the anti-IgA1 underestimated dimeric IgA relative to monomeric IgA, the fourth anti-IgA1 and all the anti-IgA2 overestimated dimeric IgA relative to monomeric IgA, by a factor sometimes close to 7.