Production and characterization of peptide antibodies

Proteins are effective immunogens for generation of antibodies. However, occasionally the native protein is known but not available for antibody production. In such cases synthetic peptides derived from the native protein are good alternatives for antibody production. These peptide antibodies are powerful tools in experimental biology and are easily produced to any peptide of choice. A widely used approach for production of peptide antibodies is to immunize animals with a synthetic peptide coupled to a carrier protein. Very important is the selection of the synthetic peptide, where factors such as structure, accessibility and amino acid composition are crucial. Since small peptides tend not to be immunogenic, it may be necessary to conjugate them to carrier proteins in order to enhance immune presentation. Several strategies for conjugation of peptide-carriers applied for immunization exist, including solid-phase peptide-carrier conjugation and peptide-carrier conjugation in solution. Upon immunization, adjuvants such as Al(OH)(3) are added together with the immunogenic peptide-carrier conjugate, which usually leads to high-titred antisera. Following immunization and peptide antibody purification, the antibodies are characterized based on their affinity or specificity. An efficient approach for characterization of peptide antibodies is epitope mapping using peptide based assays. This review describes standard solid-phase approaches for generation of peptide antibodies with special emphasis on peptide selection, generation of peptide conjugates for immunization and characterization of the resulting peptide antibodies.     

Site-directed antibodies as topographical probes of the gastric H,K-ATPase alpha-subunit.

Gastric acid is secreted by an ATP-driven H+ and K+ exchanger (H,K-ATPase), an integral apical membrane protein of parietal cells. Although the primary structure of the enzyme is known, its higher order structure is uncertain. In order to acquire topographical probes of native, microsomal H,K-ATPase, synthetic peptides corresponding to the 17 amino-terminal (N-peptide) and 16 carboxyl-terminal (C-peptide) residues of pig gastric H,K-ATPase alpha-subunit were coupled to keyhole limpet hemocyanin (KLH). Rabbits were immunized with peptide-KLH conjugates and their sera were tested for specificity by enzyme-linked immunosorbent assay (ELISA), immunoblotting, and immunocytochemistry. All sera showed high ELISA reactivities with synthetic peptides, peptide-BSA conjugates, and microsomal H,K-ATPase adsorbed to microtiter wells (some titers greater than 1:10(4)). Immunoblots of H,K-ATPase resolved by SDS-PAGE showed both N-peptide and C-peptide antibodies reacting with a single 94 kDa band. All sera selectively stained parietal cells in pig gastric mucosal sections. Preimmune sera gave negative or weak signals in all assays. In competition ELISAs, N-peptide antibodies, but not C-peptide antibodies, were displaced from the corresponding bound synthetic peptides by added microsomal H,K-ATPase. One of the N-peptide antibodies inhibited H,K-ATPase activity by more than 50%; binding of this antibody was decreased when ATP or K+ were bound to the enzyme. These results indicate a cytoplasmically-oriented alpha-subunit N-terminus which may participate conformationally in the H,K-ATPase catalytic cycle, and suggest that antibodies against synthetic H,K-ATPase peptides are potentially useful probes of native microsomal H,K-ATPase topography.     

Comparison of immunoadsorbents prepared by coupling sperm-whale myoglobin to a variety of insoluble polymers

Immunoadsorbents that were potentially suitable for the fractionation of antibodies to myoglobin were prepared by coupling myoglobin to aminoethylcellulose, bromoacetylcellulose, CM-cellulose, an ethylene-maleic anhydride co-polymer and Sepharose 4B. A comparison of the properties of the preparations indicated that Sepharose-myoglobin was superior, as an immunoadsorbent, to the other conjugates. Elution of adsorbed antibodies with m-propionic acid, pH2.5, gave yields of precipitable antibody higher than those achieved with other dissociating solvents. Propionic acid eluted 60-100% of the antibody adsorbed by Sepharose-myoglobin, and 60-90% of the eluted protein was precipitable antibody.     

Synthesis and characterization of hapten-protein conjugates for antibody production against small molecules

For the generation of antibodies against small hapten molecules, the hapten is cross-linked with some carrier protein to make it immunogenic. However, the formation of such conjugates is not always reproducible. This may lead to inconsistent hapten-protein stoichiometries, resulting in large variations in the generation of the desired antibodies. In the study described here the hapten (mercaptopropionic acid derivative of atrazine) was coupled to carrier protein at five different molar ratios. The hapten-protein conjugates prepared were characterized thoroughly by spectrophotometric absorption, fluorescence, matrix-assisted laser desorption ionization (MALDI), and gel electrophoresis methods, before being used for the immunization and assay purposes. Electrophoresis and fluorescence methods were very useful in detecting hapten-protein cross-linking while MALDI-MS and spectrophotometric detection provided qualitatively comparable hapten density. The production of specific antibodies was sought following the generation of appropriate hapten-protein conjugates. A high antibody titer with moderate antibody specificity was obtained with hapten density around 15 molecules per carrier protein. The study proved useful for monitoring the course of hapten-protein conjugation for the production of specific antibodies against small molecules.     

Application of a non-species dependent ELISA for the detection of antibodies in sera of Burkholderia pseudomallei-immunized goats

A modified, non-species dependent ELISA was performed to detect antibodies in sera of Burkholderia pseudomallei-immunized goats using protein G- or protein A-peroxidase conjugates. The rise of antibody titers during the immunization period exhibited corresponding results by modified ELISA comparison to conventional ELISA and the IHA. Regarding the increase of antibody levels from the pre-immunized baseline to the post-immunized status, the antibody titer detected by modified ELISA was higher than IHA but lower than conventional ELISA. Further efforts are needed to improve the modified ELISA for the diagnosis of melioidosis in animals.     

Factors involved in the production of specific antibodies to estriol and estradiol

To determine what effect the site of attachment of the hapten would have on specificity, 6-oxoestrogens were synthesized and conjugated to several carrier proteins, using the mixed anhydride formed between isobutyl chloroformate and the steroid 6-oxime. Estriol was also treated with phosgene to produce a mixture of chloroformates followed by conjugation to bovine serum albumin. Rabbits were immunized, and the specificity of the anti-sera produced was established by cross-reaction measurements, using both direct binding and binding inhibitor assays. Comparison of the immune response showed that antibodies to the randomly linked estriol chloroformate had a high degree of cross-reactivity with estrone and estradiol (30–100%), while those produced to 6-oxo linked estriol showed only a 1–12% cross-reaction. Similar results were obtained for estradiol antibodies. Comparison of antibody formation and specificity of 6-oxoestriol linked to a polylysine copolymer, bovine serum albumin or rabbit serum albumin was made. Also, the antigen was given with complete Freund's adjuvant, adsorbed onto charcoal, entrapped within polyaery lamide gel or in vivo titrated with estrogen implants. Only estrogen specifically linked to bovine serum albumin in Freund's adjuvant gave satisfactory results, while the others yielded antibodies of decreasing titers or low specificity.It is concluded that specificity and titers seem to depend not only on the site of conjugation but also on the carrier, immunization procedure and certain other factors.     

Monoclonal antibodies to human interferon-gamma: production, affinity purification and radioimmunoassay

Human interferon-gamma (IFN-gamma) purified to electrophoretic homogeneity by a cation exchange h.p.l.c., was used for the development of monoclonal antibodies. Following immunization, spleen lymphocytes of two mice showing the highest binding and neutralizing titers were isolated, fused with NSO mouse myeloma cells and cloned. The screening of hybridomas was based on precipitation of the immune complexes with a second antibody and recovery of the biological activity of IFN-gamma from the precipitate. Twenty nine independent hybridomas secreting antibodies specific to IFN-gamma were obtained. Twelve out of these 29 hybridomas produced antibodies that neutralized the antiviral activity of pure as well as crude IFN-gamma. Moreover, IFN-gamma obtained by various induction procedures was neutralized as well, indicating that these various IFN-gamma subtypes are immunologically cross-reactive. Immune precipitation of partially purified 125I-labelled IFN-gamma by several monoclonal antibodies revealed two protein bands of 26,000 and 21,000 daltons. Immunoaffinity chromatography of IFN-gamma gave a 50-fold purification to a specific activity > or = 4 x 10(7) units/mg. Two of the monoclonal antibodies were found suitable for a sensitive and rapid double antibody solid-phase radioimmunoassay, allowing the detection of IFN-gamma at concentrations of at least 4 ng/ml (150 units/ml) within 8 h.     

Mouse immune response to meningococcal antigens

The mouse immune response against Neisseria meningitidis was studied by using an extract from group Y (Slaterus) known to contain protein antigens common to other meningococci. By using a solid-phase radioimmunoassay, high titers of specific IgM and IgG class antibodies were measured which lasted over 2 months after immunization. These antibodies cross-reacted with similar extracts from other meningococci groups. Bactericidal antibodies directed against protein antigens were also elicited after immunization and they belonged to IgM, IgG2a, and IgG2b isotypes. Cellular immunity, expressed as delayed type hypersensitivity under the conditions tested, could be detected neither in homologous nor heterologous reactions.     

The development of a radioimmunoassay for 12-L-hydroxyeicosatetraenoic acid

Antibodies directed toward 12-L-hydroxyeicosatetraenoic acid (12-L-HETE) were generated in rabbits by immunization with conjugates of 12-L-HETE and human serum albumin. The concentration of antibodies was determined by incubating immune plasma with 12-L-HETE that had been covalently linked to a solid support, washing the 12-L-HETE support, and measuring the quantity of bound antibodies by reaction with [¹²⁵I] Protein A. The addition of 0.5 ng-10 ng of fluid-phase 12-L-HETE to the standard mixture of solid-phase 12-L-HETE and anti-12-L-HETE plasma inhibited by 21–80% the binding of antibodies and consequently of [¹²⁵I]Protein A to the solid support. The 12-OH function positioned between two double bonds was the immunodominant determinant of this antigen-antibody reaction, but the carboxyl function also was recognized. This radioimmunoassay was used to detect and quantitate 12-L-HETE resolved by high pressure liquid chromatography.     

Immunological properties of uricase conjugated to neutral soluble polymers.

For a comparative study of immunological properties of protein-polymer conjugates, uricase was modified with (a) poly(N-vinylpyrrolidone) 6000 Da, (b) poly(N-acriloylmorpholine) 6000 Da, (c) branched monomethoxypoly(ethylene glycol) 10000 Da, and (d) linear monomethoxypoly(ethylene glycol) 5000 Da. Spectroscopic studies performed by UV, fluorescence, and circular dichroism did not show any relevant difference in protein conformation among the native and the conjugates. Immunological studies showed that both uricase antigenicity and immunogenicity were altered by polymer conjugation to an extent that depended upon the polymer composition; in particular, monomethoxypoly(ethylene glycol) 10000 Da remarkably reduced the protein antigenicity, while unexpectedly, the poly(N-vinylpyrrolidone) derivative presented higher antigenicity than the native protein. In Balb/c mice, the native protein elicited a rapid and intense immunoresponse whereas all the conjugates induced a lower production of anti-native uricase antibodies. The rank order of immunogenicity was native uricase > uricase-poly(N-vinylpyrrolidone) > or = uricase-poly(N-acriloylmorpholine) > uricase-monomethoxypoly(ethylene glycol) 5000 Da > uricase-monomethoxypoly(ethylene glycol) 10000 Da. The four conjugates also induced anti polymer immunoresponse. Anti poly(N-vinylpyrrolidone) and anti poly(N-acriloylmorpholine) antibodies were generated from the first immunization while low levels of anti polymer antibodies were found with both poly(ethylene glycol) conjugates only after the second immunization.     

Antibodies against neuroactive amino acids and neuropeptides. I. A new two-step procedure for their conjugation to carrier proteins and the production of an anti-Met-enkephalin antibody reactive with glutaraldehyde-fixed tissues.

We developed a new two-step procedure to couple haptens to bovine serum albumin (BSA) via glutaraldehyde (GA). After activation of BSA with excess GA and removal of unreacted GA, the hapten was bound to the activated protein in a second step. This two-step procedure is easy to use, the desired molecular ratio of coupled hapten to protein is conveniently adjusted, and no visible precipitation of the conjugate is detected. Using a low peptide concentration, nearly 50% of the inserted haptens are bound to the protein, and unbound expensive peptide can be recovered after Sephadex chromatography. Antisera to neuroactive amino acids (GABA, glycine, and glutamate) and neuropeptides (Met-enkephalin) were prepared by immunization of rabbits with these conjugates. Immunological analysis of immune sera by dot-blot and ELISA techniques and subsequent removal of crossreactivities by solid-phase adsorption yielded monospecific antibodies, which were further purified by affinity chromatography. The immunocytochemical specificities of these purified antibodies were verified in adjacent sections of GA-fixed rat spinal cord. Pre-embedding staining with anti-Met-enkephalin in combination with post-embedding staining for amino acids such as GABA allowed double staining of the two antigens in a single semi-thin section.     

Characteristics of the immune response to staphylococcal anatoxin in healthy persons

The immunization of young healthy males with adsorbed staphylococcal toxoid revealed the existence of two main types of postimmunization humoral response. One of them was characterized by an early (on days 7-14 after immunization) rise in the titer of antibodies with the subsequent gradual decrease of their content, while the number of circulating T-suppressors remained unchanged. The characteristic feature of the other type was a slow rise in the level of antitoxins by day 21 after immunization with the subsequent drop of their titers, preceded by a considerable increase in the number of circulating T-suppressors. The maximum antibody titer was definitely higher in the first type of response than in the second type (14.8 +/- 1.41 and 9.0 +/- 1.53 I. U./ml respectively). A single plasmapheresis procedure on day 21 after immunization produced no essential effect on the dynamics of the characteristics of cell-mediated and humoral immunity.     

Purified protein derivative (PPD) as an immunogen carrier elicits high antigen specificity to haptens.

The effectiveness of the carrier protein in eliciting antigen-specific antibodies was investigated. The effect of the carrier protein was independent of the conjugation chemistry involved. Keyhole limpet hemocyanin (KLH), purified protein derivative (PPD), and ovalbumin (OVA) were used as carrier proteins in the immunization of mice. Three antigens were studied: LY170881 (a small drug molecule), 4-[1'-cyanobenz(f)isoindolyl]butyric acid (CBI-butyric acid), and a seven residue peptide GPGRGPG (KLE1). The serum antibody response to the antigen or antigen:BSA conjugate was superior in the case where the PPD:antigen conjugates were used as the immunogen when compared to KLH and OVA. The specificity of the antibodies to the respective antigens vs cross-reactivity with the carrier protein was investigated. PPD-coupled antigen immunized mice generated a higher percentage of antigen-specific hybridomas compared to the other carrier proteins. These findings confirmed PPD as the best carrier molecule for the production of both polyclonal and monoclonal antibodies.     

Immunogenic properties of neomycin conjugates with macromolecular carriers]

To obtain diagnostic antibodies to neomycin, immunogenic properties of the neomycin conjugates with macromolecular carriers were studied. Bovine serum albumin and copolymers of N-vinylpyrrolidone with crotonic acid and N-hydroxyphthalimide ether of crotonic acid were used as carriers. It was shown that immunization of mice by the conjugates in combination with Freund's adjuvant resulted in production of neomycin antibodies, the titer being 1/80 to 1/130. When the antibiotic conjugates with the copolymers of N-vinylpyrrolidone were used and not the neomycin conjugates with the carrier of the protein nature, the neomycin antibodies were produced in the absence of Freund's full adjuvant. With the use of the isolated antibodies to neomycin a method for indirect solid phase enzyme immunoassay of neomycin was developed at the minimum detectable level of 25 ng/ml.     

Determination of antibodies to mycobacterial antigens in tuberculosis by erythrocyte immunoadsorption with a rosette marker

A solid-phase enzyme immunoassay system for the determination of antibodies to mycobacterial antigens, based on the method of erythrocyte immunoadsorption in microchambers for immunological reactions, has been developed. To detect antibodies specifically bound with the solid-phase antigen, the affinity rosettes of Staphylococcus aureus strain Cowan I, carrying protein A, with erythrocytes conjugated with human gamma globulin have been used. The significant correlation of the titers of 34 sera, determined by means of erythrocyte immunoadsorption, with extinction values obtained in the solid-phase enzyme immunoassay of antibodies to Mycobacterium tuberculosis has been established. The coincidence of the results in 92% of cases has been noted.     

Bisphosphonate conjugation to proteins as a means to impart bone affinity

Growth factors are endogenous proteins capable of stimulating new bone formation, but their clinical benefit for systemic stimulation of bone mass has not been demonstrated. The critical challenge is to deliver a significant dose of the proteins to bone after intravenous injection. This challenge may be overcome by derivatizing proteins with ligands that exhibit a high bone affinity (e.g., bisphosphonates). To demonstrate the feasibility of this approach, 1-amino-1,1-diphosphonate methane (aminoBP) was conjugated to a model protein, albumin. The conjugation was performed by (1) converting the amino group of aminoBP to a thiol group using 2-iminothiolane, (2) derivatizing the albumin amino groups with a thiol-reactive sulfosuccinimidyl-4-(N-maleimidomethyl)-1-cyclohexane carboxylate, and (3) reacting the derivatized albumin with thiolated aminoBP. Typically, 1-4 aminoBP molecules per albumin were obtained. The conjugated albumin exhibited a high affinity to hydroxyapatite that was proportional to the extent of conjugation. The conjugates were shown to exhibit a high affinity to bone matrix in vitro in a serum-containing medium. Once bound to bone matrix, the conjugates were found to desorb more slowly than the unmodified albumin, especially from bone whose organic matrix was removed by ashing. In conclusion, conjugation of bisphosphonates to albumin was shown to impart a high bone affinity to the protein, and such conjugates can be potentially targeted to bone.     

Immuno-crossreactivity and toxicity assessment of conjugation products of the cyanobacterial toxin, microcystin-LR

Immunoassays are increasingly used to investigate the production, properties and fates of the cyanobacterial hepatotoxic microcystins in vitro and in vivo. Responses of an ELISA immunoassay to microcystins have been determined using the authentic toxin antigen, microcystin-LR, and conjugation products between the toxin and glutathione, cysteine-glycine and cysteine. The antibodies against microcystin-LR crossreacted with the toxin conjugation products with similar affinities (96-112%) to that of microcystin-LR, when assayed at a concentration of 1 microg l(-1). Toxicity assessment of the conjugates, in comparison to microcystin-LR, indicated a reduction according to mouse bioassay. In vitro protein phosphatase inhibition assay indicated that the conjugates possessed approximately 3-9-fold lower toxicity than microcystin-LR.     

Conjugation, immunoreactivity, and immunogenicity of calix[4]arenes; model study to potential calix[4]arene-based Ac3+ chelators.

For the development of calix[4]arene-based radiotherapeutic agents, the conjugation to biomolecules and immunogenicity in mice of potential 225Ac3+-chelating calix[4]arenes were studied. A calix[4]arene triethyl ester isothiocyanate and a bis(calix[4]arene) hexacarboxylic acid, containing a masked thiol functionality, were used in conjugation experiments to a mouse monoclonal antibody and serum albumins. All characterization techniques indicate that only the calix[4]arene carboxylic acid is successfully conjugated to the biomolecules. The immunoreactivity of the conjugates is not impaired when up to 6 equiv of calixarene are bound to the monoclonal antibody. Animal tests indicated that the immunogenicity toward the calix[4]arene is strongly influenced by the nature of the carrier, the dosage, and the injection method. No immune response occurred when a homologous carrier was used or when a heterologous carrier was applied at a dosage of 10 microg per immunization via intravenous injection. Under all other conditions, the presence of antibodies directed against the calix[4]arene was demonstrated. Thus, for the application in radioimmunotherapy, the conjugation of a calix[4]arene to a humanized antibody will probably not lead to an immune response, and the immunoreactivity will not be disturbed.     

Efficient strategies for the conjugation of oligonucleotides to antibodies enabling highly sensitive protein detection

Three methods for the conjugation of oligonucleotides to antibodies and the subsequent application of these conjugates to protein detection at attomole levels in immunoassays are described. The methods are based on chemical modification of both antibody and oligonucleotide. Aldehydes were introduced onto antibodies by modification of primary amines or oxidation of carbohydrate residues. Aldehyde- or hydrazine-modified oligonucleotides were prepared either during phosphoramidite synthesis or by post-synthesis derivatization. Conjugation between the modified oligonucleotide and antibody resulted in the formation of a hydrazone bond that proved to be stable over long periods of time under physiological conditions. The binding activity of each antibody-oligonucleotide conjugate was determined to be comparable to the corresponding unmodified antibody using a standard sandwich ELISA. Each oligonucleotide contained a unique DNA sequence flanked by universal primers at both ends and was assigned to a specific antibody. Highly sensitive immunoassays were performed by immobilizing analyte for each conjugate onto a solid support with cognate capture antibodies. Binding of the antibody-oligonucleotide conjugate to the immobilized analyte allowed for amplification of the attached DNA. Products of amplification were visualized using gel electrophoresis, thus denoting the presence of bound analyte. The preferred conjugation method was used to generate a set of antibody-oligonucleotide conjugates suitable for high-sensitivity protein detection.     

Synthesis and structural characterization of bioactive peptide conjugates using thioether linkage approaches.

Applications of cysteine-insertion and thioether linkage approaches to the preparation of a number of bioactive peptide conjugates are reported. Peptides containing epitopes from (i) herpes simplex virus type 1 glycoprotein D, (ii) a specific N-terminal beta-amyloid epitope recognized by therapeutically active antibodies, and (iii) a GnRH-III peptide from sea lamprey with antitumour activity, were elongated with Cys residues and attached to a chloroacetylated tetratuftsin derivative carrier via a thioether linkage either directly, or by insertion of a spacer. The structures and molecular homogeneity of all the peptide conjugates were ascertained by HPLC, MALDI and electrospray mass spectrometry. The use of a spacer such as an oligoglycine or GFLG-tetrapeptide gave an increased yield in the conjugation reaction and enhanced reaction rates. In the formation of cysteinyl-thioether linkages, it was found that the position of flanking Cys residues markedly influenced the conjugation reaction and the formation of intermolecular epitope disulfide-dimers. C-terminal Cys residues gave thioether conjugates with significantly diminished epitope-dimerization, while Cys at the N-terminal caused rapid disulfide-dimerization, thereby preventing efficient conjugation.