Human monoclonal antibodies to synthetic viral peptides]

Cells producing human antibodies to synthetic viral peptides MH-24 (corresponding to 302-325 residues of HIV-1 gp120) and p107 (main peptide of EBV antigen EBNA-1) were fused with human x mouse heteromyeloma B6B11. The efficacy of specific hybridization was 40%. Hybrid cells H1F2 (MN-specific) and HF4 (p107-specific) were cloned by the limiting dilutions method. Monoclonal antibodies (MAb) were tested in enzyme immunoassay on a panel of synthetic peptides. All resultant MAbs were polyspecific for tested synthetic peptides. The data permit a conclusion that previously detected polyspecificity of human cultural antibodies to virus peptides is due to the true polyspecificity of antigen-binding centers of MAb, but not to the presence of strange cells in the culture.     

Use of monoclonal antibodies to quantify subclasses of human IgG. I. Development of two-site immunoenzymometric assays for total IgG subclass determinations

Dissection of the IgG antibody response into its subclass components has been difficult largely because of the lack of adequate supplies of specific reagents. The development of monoclonal antibodies (Mcab) promises to overcome this problem, but the use of such antibodies has certain inherent problems. It has been shown recently that Mcabs which were avid, potent and specific for well defined epitopes may partially or completely lose their activity depending on the assay system in which they were used. In order to identify Mcabs that would be specific and useful as capture antibodies in a simple two-site enzymometric assay, a panel of 18 Mcabs was screened and one Mcab to each of the four IgG subclasses was identified for quantitation of subclass levels in human serum.     

A highly specific two-site ELISA for pneumococcal C-polysaccharide using monoclonal and affinity-purified polyclonal antibodies

A two-site ELISA for the detection of pneumococcal C-polysaccharide (PnC) has been developed. A monoclonal antibody directed against the phosphorylcholine residue of the PnC was used as catcher and an affinity-purified polyclonal anti-PnC rabbit antiserum for detection. Polyclonal antibodies against the PnC as well as capsular antigens were obtained by immunizing rabbits with type 1 pneumococci. Antibodies against the phosphorylcholine determinant of PnC could be removed by affinity purification. Remaining antibodies reacted in an ELISA with type 1 capsular polysaccharide as well as with PnC. Only in the fraction with the highest antibody activity against PnC, phosphorylcholine exhibited a slight inhibitory action. It is concluded that the purified antibody preparation reacted with an antigenic determinant shared by the two polysaccharides, in all probability a determinant associated with 2-acetamido-4-amino-2,4,6-trideoxygalactose which is the only monosaccharide component in common between PnC and the type 1 capsular polysaccharide. By the use of this affinity-purified antibody preparation, reactions with alpha-streptococci, occurring with non-purified serum, were abolished. The sensitivity and specificity of the test was determined using capsulated and non-capsulated pneumococci and alpha-streptococci known to cross-react with unpurified serum against the pneumococcal C-polysaccharide.     

Analysis of immune responses in the sheep to synthetic peptides of foot-and-mouth disease virus using ovine polyclonal and monoclonal antibodies.

A 40-residue peptide incorporating residues 200-213 and 141-158 of foot-and-mouth disease virus VP1 capsid protein strain O1 Kaufbeuren was injected uncoupled into sheep, and the immune responses analysed. Direct-binding and inhibition experiments showed that the polyclonal antibody response was directed mainly against epitopes unique to the 40-residue peptide but absent from the constituent peptides containing residues 200-213 or 141-158, respectively. Further confirmation of the presence of unique epitopes on the 40-residue peptide was obtained from similar experiments performed with sheep monoclonal antibodies generated through the use of an aminopterin-sensitive sheep/mouse heterohybridoma cell line as a fusion partner. The sheep polyclonal antisera to the 40-residue peptide had high neutralization titres and were fully active in a mouse protection assay, whereas none of the sheep monoclonal antibodies conferred protection. The results suggest that the conformation of the 40-residue peptide is important for its ability to induce neutralizing antibodies.     

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.     

Structural probing of human lutropin using antibodies raised against synthetic peptides constructed by classical and multiple antigen peptide system approaches.

Antibodies were elicited against a synthetic peptide which encompassed two different regions of the human lutropin beta-subunit (hLH-beta). These antibodies were raised against either the peptide which was assembled using a conventional approach and conjugated to the tetanus toxoid, or with the peptide assembled using the multiple antigen peptide system approach. Automated simultaneous synthesis of the two forms of the immunizing peptide was successfully achieved. Animal injected with the peptide conjugated to tetanus toxoid produced high titers of antibodies to the synthetic peptide, but did not bind to the native hLH-beta subunit. In contrast, antisera induced by the peptide in its MAP form displayed reactivity with both the peptide and the native hLH-beta subunit; these latter antisera appeared to preferentially recognize the beta 47-55 portion of the molecule and were able to bind to the beta-subunit of human choriogonadotropin. Present results demonstrate that the beta 47-55 region is accessible to antibody binding and appears to be located at the surface of both hLH-beta and hLH. Moreover, this study confirms that the MAP approach provides a chemically unambiguous method for obtaining antibodies of predetermined specificity, capable of recognizing cognate sequences of various native proteins.     

Selection and optimisation of monoclonal antibodies for a two-site immunoradiometric assay for ACTH

Four monoclonal antibodies with predominant specificities towards different sequences within the ACTH molecule were investigated in a 2-site immunoradiometric assay (IRMA) for human ACTH. Antibody 3H9 recognises the extreme N-terminal sequence, antibodies 1A12 and 1D1 are specific for the mid N-terminal sequence but differ in that the former cross-reacts with alpha MSH whereas the latter does not, and antibody 2A3 recognises the C-terminal sequence. Combinations of iodinated antibodies with antibodies covalently linked to Sephacryl S300 were tested for their compatibility and potential for a sensitive assay. Two antibody combinations (1D1 plus 3H9 or 1A12) gave no dose-response curve indicating severe steric inhibition, whereas other combinations yielded assays with widely different detection limits (2-2400 ng ACTH/l). The combination of labelled 1D1 and solid-phase 2A3 gave the most sensitive assay and when optimised for antibody concentrations and incubation times the working range was 10-5 X 10(4) ng/l (CV less than 20%). The optimised sequential 2-step IRMA involves incubation of standard or test sample with labelled 1D1 for 18 h at 4 degrees C followed by incubation with solid-phase 2A3 for 2 h at room temperature, after which the labelled complex is separated by the sucrose layering technique. The detection limit of this IRMA was several 100-fold lower than by RIA using the same antibodies. The IRMA detected large molecular weight precursors containing the full ACTH sequence (22 000, 31 000 and 34 000) but not ACTH fragments (1-18, 1-24, 18-39). It is concluded that selected monoclonal antibodies provide a sensitive and rapid 2-site IRMA for intact ACTH and its precursors.     

Production of monoclonal antibodies to calcitonin and development of a two-site enzyme immunoassay

A procedure is described for the production of calcitonin-specific hybridomas which involves primary and secondary immunization of Balb/c mice in the hind footpads with free, synthetic human calcitonin and cell fusion of lymphocytes from popliteal lymphonodes with a P3 x 63 myeloma line. This protocol offers the following advantages: (a) it is short and easy to perform, (b) it requires small amounts of unconjugated antigen, and (c) it gives a high yield of antigen-specific IgG-secreting hybridomas. Routine screening was carried out by ELISA on solid phase calcitonin and binding of the monoclonals to free antigen was studied with calcitonin linked to biotin through a 13 carbon atom spacer. Monoclonal couples capable of simultaneously binding calcitonin in solution were found by pairing in all possible combinations 25 purified antibodies, in their unlabelled and biotinylated form, in a checkerboard matrix experimental system. Of the over 30 positive pairs identified, four were used in a one-step enzyme immunoassay for calcitonin determination on microtiter plates in a concn range between 0.1 and 5 ng/ml. With the detecting monoclonal directly conjugated to peroxidase with a heterobifunctional crosslinker, the range of the assay with one monoclonal pair was between 25 and 1000 pg/ml with an 18 hr incubation at 4 degrees C.     

Immunological detection of penicillin-binding protein 2' of methicillin-resistant staphylococci by using monoclonal antibodies prepared from synthetic peptides.

Two synthetic peptides 31 and 32 amino acids in length were prepared as deduced from a known amino acid sequence of penicillin-binding protein 2' (PBP2') of methicillin-resistant Staphylococcus aureus. Two monoclonal antibodies were generated from fused cells of myeloma cells and splenic cells of mice immunized with the synthetic peptides. Western blot (immunoblot) analysis demonstrated specific binding of the antibodies to PBP2' of a methicillin-resistant S. aureus strain. An immunoradiometric assay was developed by using these antibodies for simple detection of PBP2'.     

A two-site immunoradiometric assay for human pregnancy-associated plasma protein A (PAPP-A) using monoclonal antibodies

A rapid, sensitive immunoradiometric assay has been developed for human pregnancy-associated plasma protein A (PAPP-A) using a purified mouse monoclonal antibody as the tracer and a rabbit polyclonal antibody to this protein in the solid-phase antibody preparation. The assay showed no measurable cross-reaction (less than 0.1%) against a range of purified human placental proteins, and a good correlation with a previously described radioimmunoassay procedure when tested on samples taken throughout normal human pregnancies. No PAPP-A-like immunological activity could be detected in sera from non-pregnant women, confirming the absence of this protein from the circulation outside pregnancy.     

New monoclonal antibodies as probes for human cardiac troponin I: epitopic analysis with synthetic peptides.

Forty monoclonal antibodies (MAbs) specific for human cardiac troponin I (TnI) were selected to develop a new alternative for specific biological diagnosis of acute myocardial infarction. Using an immunoenzymatic sandwich assay, these MAbs were employed in the mapping of human cardiac TnI and showed six different epitopes. Parts of the TnI peptide sequences were synthesised; the sequences were chosen from the published sequences of mammalian TnI. Immunological assays showed that 8 out of 40 MAbs recognised a RAYATEPHAK (P2) N-terminus cardiac-specific sequence of human TnI. The information obtained from epitopic mapping of TnI and the properties of the peptides allowed pairs of MAbs to be selected for the development of a future specific TnI assay.     

Detection of human antibodies against Plasmodium falciparum sporozoites using synthetic peptides.

A large peptide consisting of about 40 (Asn-Ala-Asn-Pro) repeats of Plasmodium falciparum circumsporozoite protein, (NANP)40, was synthesized. It was recognized specifically by monoclonal antibodies produced against P. falciparum sporozoites. Moreover, this peptide strongly inhibited the binding of such monoclonal antibodies to antigens present in a sporozoite extract. The (NANP)40 peptide was employed without any carrier to develop an enzyme-linked immunosorbent assay to detect sporozoite-specific serum antibodies arising after natural malaria infections. Antibodies were detected in a high percentage (43.1%) of European patients suffering from acute P. falciparum malaria and in Africans living in an area of Gabon endemic for malaria. In the latter group, the frequency of antisporozoite antibodies increased with age, reaching 65.9% in individuals more than 40 years old. There was a significant correlation between the results obtained with an immunofluorescence assay with glutaraldehyde-fixed sporozoites and those obtained by enzyme-linked immunosorbent assay with (NANP)40. Therefore, such synthetic peptides representing the repetitive epitope of P. falciparum circumsporozoite protein can be used for the detection of antisporozoite antibodies and for the epidemiological studies required to obtain base-line data concerning the immune status of individuals before their participation in a sporozoite vaccine trial.     

A sensitive two-site sandwich enzyme immunoassay for human angiotensin converting enzyme utilizing monoclonal antibodies.

A sensitive enzyme immunoassay was developed for human angiotensin converting enzyme. Monoclonal antibodies specific for two unique converting enzyme epitopes were utilized to develop a two-site sandwich enzyme immunoassay. Alkaline phosphatase conjugated to the detecting antibody hydrolyzes nicotinamide adenine dinucleotide phosphate (NADP) to NAD. Subsequently, NAD is cycled between its reduced and oxidized forms by an alcohol dehydrogenase/diaphorase catalyzed redox cycle. Each cycle converts iodonitrotetrazolium violet to a highly colored formazan which is quantitated. With this assay, as little as 94 pg/ml of native converting enzyme is detectable without interference from either therapeutic or endogenous converting enzyme inhibitors.     

Production and characterization of monoclonal antibodies to anti-human chorionic somatomammotropin by immunization with two free synthetic peptides

The peptides corresponding to the fragments 135-140 and 166-174 of human chorionic somatomammotropin (hCS) were synthesized, and used to raise monoclonal antibodies to the native hCS molecule. The synthetic peptides were injected into BALB/c mice in the free form, i.e. not conjugated to a carrier, and the spleens were fused with Sp2/01Ag8 myeloma line to produce monoclonal antibodies. The antibodies produced belonged to the IgM and IgG classes and, once purified by affinity chromatography on hCS-Sepharose, they were covalently coupled to macroporous polystyrene beads and characterized by competitive radioimmunoassay. Their affinity constants were determined by elaborating the radioimmunoassay data by nonlinear regression analysis and they were found to range from 10(5) to 10(6) M-1. The evaluation of the affinity constant of the antibodies produced is always important as a measure of the immunogenicity of an antigen, particularly when synthetic peptides are used as immunogens.     

Neutralization of diverse HIV-1 strains by monoclonal antibodies raised against a gp41 synthetic peptide

Three IgM monoclonal antibodies raised against synthetic peptide analogs of a hydrophilic region of the gp41 transmembrane env protein of HIV-1 neutralize different HIV-1 isolates but not HIV-2 isolates, as determined by HIV titration and by syncytial inhibition assays. VSV (HIV-1) pseudotypes, however, were not neutralized, indicating that gp41 was not accessible to these antibodies on the pseudotype particles. The antibodies affect early steps in adsorption and penetration of HIV-1.     

Localization of sequence-determined neoepitopes and neutrophil digestion fragments of C-reactive protein utilizing monoclonal antibodies and synthetic peptides.

We recently described 17 anti-CRP mAb, seven to native- (or conformational) and 10 to neo- (or sequence-determined) epitopes, including several anti-neo-CRP mAb specific for CRP peptide 199-206. In the present study, four new anti-native- and four new anti-neo-CRP mAb were generated and characterized by ELISA reactivity with native and modified human and rabbit CRP, as well as binding to pronase fragments of human CRP in Western blots. Assays with 17 synthetic CRP peptides identified anti-neo-CRP mAb specific for peptides 1-16, 14-24 and 137-152, respectively. The anti-neo-CRP mAb were reacted with fragments obtained by digesting CRP with multiple additional enzymes, including Staphylococcal V8 protease, trypsin, elastase, plasmin, thrombin and alpha-chymotrypsin. Native CRP was remarkably resistant to enzymic digestion, particularly in the presence of calcium, but was readily cleavable upon denaturation. Twenty-three informative fragments served to further distinguish mAb reactivity with at least four additional neo-CRP epitopes, which presumptively included residues in the regions of amino acids 22-45, 41-61, 114-121 and 130-138, respectively. The eight epitopes identified corresponded well with predicted regions of CRP antigenicity. In addition, at least six distinct native or conformation-determined epitopes were delineated. Reactivity of the anti-neo-CRP mAb with fragments of CRP generated by PMN enzymes indicated that regions sensitive to cleavage by neutrophil enzymes are located at approximately 3, 10 and 16 kD from the amino terminus of the CRP subunit. We expect that the anti-CRP mAb described and mapped herein will be useful tools for the elucidation of CRP structure and function.     

A rapid, sensitive two-site immunometric assay for TSH using monoclonal antibodies: investigation of factors affecting optimisation

Twenty-three monoclonal antibodies specific for human thyrotropin (TSH) have been prepared and characterised. Four different epitopes have been identified, 3 of which are sufficiently distinct to allow simultaneous binding of the corresponding antibodies. Only 1 epitope is expressed on free TSH beta-subunit. The best antibodies have affinities for TSH approaching 10(11) M-1 and cross-reactions with lutropin and chorionic gonadotropin of 0.2% or less. A 2-site immunoradiometric assay for TSH was established using 2 monoclonal antibodies, one of which was absorbed to plastic tubes and the other labelled with 125I. Several parameters affecting assay performance were investigated, including conditions for adsorption of antibodies to various plastics, selection of antibody combinations, concentration of labelled antibody and incubation protocol. The optimised assay covered a working range of 1-60 mU/1 (0.17-10 ng/ml) TSH in a 4 h, single incubation protocol, with no significant interference from other glycoprotein hormones at their maximum physiological or pathological concentrations.     

A two-site sandwich immunoradiometric assay of squirrel monkey (Saimiri sciureus) IgM using monoclonal antibodies

Nine hybrid clones secreting antibodies to squirrel monkey (Saimiri sciureus) IgM were produced and two of these (1F1G5 and 5H11B3) were selected for further studies. These non-precipitating monoclonal antibodies reacted with two distinct repetitive antigenic determinants, probably of the conformational type, only present on the native or SDS-denatured IgM molecule. Reduction of the pentamer with 2-mercaptoethanol led to complete destruction of the corresponding epitopes. 1F1G5 antibodies from ascitic fluids were used in the purification of monkey IgM by affinity chromatography. The characteristics of 1F1G5 and 5H11B3 MAbs permitted the development of a solid-phase two-site immunoradiometric assay for the measurement of IgM levels in serum specimens taken from healthy animal donors of both sexes.     

Use of synthetic peptides to map sequential epitopes recognized by monoclonal antibodies on the bovine leukemia virus external glycoprotein.

Six sequential epitopes (A, B, B', D, D', E) were previously defined on the bovine leukemia virus (BLV) envelope glycoprotein gp51 by their reactivity with monoclonal antibodies. A panel of synthetic peptides covering almost the entire sequence of gp51 was tested in enzyme-linked immunosorbent assays in order to localize these epitopes. E was shown to be included in peptide 142-161 (MCF4), B and B' in peptide 195-205, D and D' in peptide 218-237 (MCF6), and A in peptide 249-268 (MCF7). These results extend and confirm previous observations suggesting that the sequential epitopes recognized by our battery of monoclonal antibodies are located in the carboxylic half of BLV gp51.