A simple, rapid and inexpensive technique to bind small peptides to polystyrene surfaces for immunoenzymatic assays

Synthetic peptides are widely used in indirect ELISA to detect and characterize specific antibodies in biological samples. Small peptides are not efficiently immobilized on plastic surfaces by simple adsorption, and the conjugation to carrier proteins with different binding techniques is the method of choice. Common techniques to conjugate peptide antigens to carrier proteins and to subsequently purify such complexes are time consuming, expensive, and occasionally abrogate immunogenicity of peptides. In this report we describe a simple, fast and inexpensive alternative protocol to immobilize synthetic peptides to plastic surfaces for standard ELISA. The technique is based on use of maleimide-activated bovine serum albumin or keyhole limpet hemocyanin as a protein anchor adsorbed on the polystyrene surface of the microtiter plate. Following adsorption of the carrier protein, sulfhydryl-containing peptides are cross-linked with an in-well reaction, allowing their correct orientation and availability to antibody binding, avoiding the time consuming steps needed to purify the hapten-carrier complexes. The immunoreactivity of peptides was tested by using both monoclonal and polyclonal antibodies in standard ELISA assays, and compared with established coating methods.     

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.     

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.     

Immunologic methods for quantitative estimation of small peptides and their application to bradykinin.

A simple strategy was developed for the immunologic quantitative determination of small, biologically active peptides utilizing bradykinin (BK) as the model peptide prototype. Methods were developed for the preparation of a peptide-carrier complex suitable for immunization and for immobilization of peptides onto the plastic surface of enzyme-linked immunosorbent assay (ELISA) plates. An avidin-bound biotinylated peptide complex was used for raising peptide antibodies with high titers (1:4000) in the rabbit. The peptide BK was coupled to synthetic polymeric carriers poly-D-lysine (PL) and poly-D-lysine-succinylated (PLS) via the BK carboxy and amino terminus, respectively, with the aid of a water soluble carbodiimide. These carriers with antigen peptide side chains as well as avidin-biotinyl-peptide complexes were efficient surface immobilizing reagents for microwell plastic plates used in the detection of kinins by ELISA. Monoclonal antibodies reacted competitively with kinins in plates coated with either PL-BK or PLS-BK. In contrast, rabbit (polyclonal) antibodies reacted specifically in the plates coated with PLS-BK but only a non-specific reaction could be obtained with the PL-BK coated plates (i.e., could not be displaced with BK). Based on results using synthetic BK analogues, the carboxy terminal half of the BK molecule appears to be the stronger antigenic determinant in both mouse and rabbit systems. The polyclonal antibodies demonstrated a greater affinity to bradykinin compared to the monoclonal antibodies. Their use improved the sensitivity of the ELISA for kinin determination by one order of magnitude. Kinin levels determined in plasma tryptic digests by ELISA with the polyclonal antibodies and PLS-BK system were in agreement with published values.     

Polystyrene, poly-L-lysine and nylon as adsorptive surfaces for the binding of whole cells of Mycobacterium tuberculosis H37 RV to ELISA plates

Several methods of coating whole cells of Mycobacterium tuberculosis H37 RV to ELISA microtitre plates were compared with the aim of developing an ELISA screening assay for murine monoclonal antibodies in culture supernatants and human antibodies in patient sera. Undercoats of nylon or poly-L-lysine were compared to polystyrene as adsorptive surfaces for the bacteria, the effect of increased ionic strength and iclusion of SDS in the coating buffer measured, and methanol (70%) and glutaraldehyde (5%) investigated for their efficiency as fixatives of the bacterial monolayers. The results suggest PBS as a satisfactory coating buffer for the bacterial cells on polystyrene, and 70% methanol the preferred fixative for the dried antigen-coated plates.     

Development of a screening system for detection of somatic mutations. II. The use of peptides and insoluble protein fragments in a non-competitive solid-phase enzyme immunoassay

A system proposed for measurement of mutational risk consists in detection of hemoglobin mutations expressed in erythrocytes. For this detection the production of antibodies specific for Hb variants is essential. Recently we reported a sensitive solid-phase EIA for the production and selection of polyclonal and monoclonal antibodies specific for hemoglobin determinants. An important characteristic of this EIA was the coating of water-insoluble proteins to polystyrene microtiter plates. Here we report that with this system, insoluble protein fragments and small peptides may also be covalently coated to a polystyrene surface. Coating is independent of the length of the peptides. This allows direct, non-competitive titration of the antibody response to small peptides and avoids the drawbacks of competitive assay.     

Rapid and efficient identification of epitopes/mimotopes from random peptide libraries

Phage-displayed random peptide libraries are important tools in identifying novel epitopes/mimotopes that may lead to the determination of antigen specificity. In this approach, high-affinity phage peptides are enriched by affinity selection (panning) on a monoclonal antibody. To facilitate identification of all potential phage peptides specific for recombinant monoclonal antibodies (rAbs) previously generated from clonally expanded plasma cells from the cerebrospinal fluid of patients with multiple sclerosis (MS), we developed a high-throughput method to determine phage specificity. In contrast to the 8-9 days needed in the standard large-scale method of amplifying phage clones for ELISA, the high-throughput method takes only 1 day. ELISA using phage clones amplified directly in 96-well plates avoids large-scale phage purification and enables rapid identification of specific epitopes/mimotopes. This technique will expedite identification of MS-specific peptides that can be used to discover the corresponding protein antigens.     

Peptide ELISA for measuring antibodies to N-protein of porcine reproductive and respiratory syndrome virus

Indirect and competition ELISAs with synthetic peptides were used to characterize the epitopes of the N-protein of porcine reproductive and respiratory syndrome virus (PRRSV) that are recognized by a battery of monoclonal antibodies (mAbs) and by antibodies from infected pigs. Four linear epitopes recognized by mAbs have been identified in the most hydrophilic segment of the N-protein (AA25-57). Similarly, at least four linear epitopes in this segment are immunogenic in PRRSV-infected pigs, but only one corresponds to an epitope recognized by one of the mAbs (AA36-45). Antibody formation to these epitopes varied greatly between individual pigs. Most infected pigs generated antibodies that bound to both peptides and HerdChek plates, which are commonly used in the sero-diagnosis of PRRSV infections, but the time course of formation of peptide binding antibodies and antibodies that react with HerdChek plates differed greatly between pigs. This suggests that, although the peptide and HerdChek ELISAs may detect antibodies to some of the same epitopes, they also seem to detect antibodies to epitopes that are uniquely expressed by one and not the other. Some mAbs fail to bind to HerdChek ELISA plates and this is also the case for certain pig antibodies. Peptide ELISA results identified four herds in which most or all pigs possessed N-protein peptide binding antibodies, even though they were HerdChek ELISA sero-negative and exhibited no other signs of PRRSV infection. Thus PRRSV infections may be more widespread than presently realized involving strains that cause asymptomatic infections. The peptide ELISA is useful as an adjunct to the HerdChek ELISA or it could replace it since only two serum samples among 450 tested were HerdChek ELISA positive but peptide ELISA negative. The peptide ELISA is also considerably cheaper than the HerdChek ELISA, more flexible and can provide information on the epitope specificity of the reacting antibodies.     

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.     

Use of two monoclonal antibodies in an ELISA test for the detection of antibodies to bovine leukaemia virus envelope protein gp51

A competition ELISA technique involving two monoclonal anti-gp51 antibodies has been developed for the detection of bovine leukaemia virus (BLV) antibodies. Precoated gp51 antigen-microtitre plates were obtained by incubation of plastic adsorbed monoclonal antibody with a non-purified BLV preparation. Samples to be tested were incubated in the wells of the gp51-coated plates; the presence of anti-gp51 antibodies was indicated by competition for antigen binding with an enzyme linked monoclonal antibody directed to an important epitope on gp51. This test is as sensitive as a routinely used indirect ELISA test; it is highly specific, reliable and easy to perform.     

Enzyme-linked immunosorbent determination of autoantibodies to zona pellucida as a possible cause of infertility in women

An enzyme-linked immunosorbent assay (ELISA) for determination of antibodies against the zona pellucida was developed and compared with the already available indirect immunofluorescence (IIF) technique. Sera from 100 women with explained and unexplained infertility were screened for the presence of autoantibodies to the zona pellucida by ELISA and IIF techniques. Porcine/goat zonae immobilized on activated microtitre plates or solubilized zona pellucida antigens adsorbed on poly-L-lysine-coated microtitre plates were used as a solid phase in an ELISA. Assay of anti-zona pellucida antibodies in xenogeneic and allogeneic sera was performed by incubation of test samples with the solid phase against human serum supplied by WHO as a reference positive control, followed by incubation with staphylococcal protein A conjugated to horseradish peroxidase. The ELISA was effectively used to screen the production of monoclonal antibodies from mouse myeloma X mouse splenocyte hybridomas. The sensitivity of the ELISA was more than 2500-fold greater than that of the IIF technique. Significantly high titres of autoantibodies to zona pellucida were found in patients with unexplained infertility as compared with patients with a known cause of infertility, and their normal counterparts.     

A method of membrane preparation for immunoassay

Membranes prepared from a variety of solid tissues were used as solid-phase antigens for ELISA or RIA after fixation onto polylysine-primed 96-well plates. The preservation of antigens in these membrane preparations was tested by reactivity in ELISA using 2 monoclonal antibodies: W6/32, which recognizes an HLA framework antigen (a protein antigen) and anti-SSEA-1, directed to a carbohydrate antigen carried on glycoproteins. Levels of antigen deposition and usefulness as solid-phase antigens were assessed for ELISA as compared to RIA. Coated plates may be frozen for many months with preservation of antigenic activity. This method is relatively simple, rapid, and is useful for preparation of tissue antigens for immunoassay, especially for screening monoclonal antibodies.     

Measurement of mouse anti-phospholipid antibodies to solid-phase microspheres by both flow cytofluorometry and Alcian blue-pretreated microtitre plates in an ELISA

Conventional solid-phase immunoassays measuring interactions between anti-phospholipid antibodies and phospholipids are generally characterized by problems of reproducibility and high levels of non-specific binding. Here we describe two immunoassays based on the use of phospholipids in the form of solid-phase microspheres to measure the presence of anti-phospholipid antibodies in sera. Following the production of antibodies in mice against liposomes containing lipid A, we show that flow cytofluorometric analysis provides a reproducible and sensitive way to detect anti-phospholipid antibodies. We also present a sensitive, rapid and reproducible enzyme-linked immunosorbent assay (ELISA) using Alcian blue pretreated microtitre plates and solid-phase microspheres as coating antigen. This ELISA permitted the detection of antibodies to 1/1000 dilution, while untreated plates gave negative results. Such modified ELISA procedures may be applicable to other types of molecule exhibiting solid-phase binding problems e.g. synthetic peptides (J. Immunol. Methods 175 (1994) 131–135).     

Rapid characterization of protein epitopes recognized by monoclonal antibodies using direct probing on thin-layer and paper chromatograms

A simple method for the comparison and identification of protein epitopes recognized by monoclonal antibodies directly on thin-layer plates and 3MM paper chromatograms is described. Enzyme digests of myelin basic protein were separated on thin-layer plates and 3MM paper, fixed with glutaraldehyde and probed directly with affinity-purified mouse monoclonal antibodies. Detection of the immunoreactive peptides was enhanced using a second rabbit anti-mouse immunoglobulin and finally located using an alkaline phosphatase-conjugated anti-rabbit immunoglobulin. By probing the same enzyme digests of MBP with various monoclonal antibodies raised against MBP, a different binding 'pattern' of reactive peptides is rapidly obtained for monoclonal antibodies of differing specificities. This procedure was extended to the identification of the antigenic determinant using synthetic peptides. The major advantages of this procedure are its simplicity, non-radioactive nature and speed. Furthermore, there is the possibility of sequencing immunoreactive peptides eluted from the 3MM paper.     

A rapid ELISA for measurement of antibodies to nucleic acid antigens using UV-treated polystyrene microplates

Pretreatment of polystyrene microplate wells with certain doses of UV light enhances their capacity for binding to single-stranded DNA, double stranded DNA and various synthetic polynucleotides. The use of UV-irradiated plates to immobilize nucleic acid antigens provides a simple, rapid, and specific ELISA for measuring anti-nucleic acid antibodies. The assay is at least as sensitive as the more complex method of precoating plates with poly(L-lysine). It is useful for detection of anti-DNA antibodies in sera of systemic lupus erythematous patients, as well as in culture fluids of murine and human anti-DNA-secreting hybridomas.     

Improved in vitro methods to predict the in vivo toxicity in man of therapeutic monoclonal antibodies including TGN1412

TGN1412 is a “superagonistic” CD28 monoclonal antibody (IgG4) that caused serious adverse events at its first time in human clinical trial. In the present study, different in vitro methods for detecting and quantifying unwanted pro-inflammatory activity of therapeutic monoclonal antibodies (mAbs) such as TGN1412 are described. The antibody of interest is immobilised by wet-coating or air-drying onto polypropylene or polystyrene 96-well plates prior to the addition of human peripheral blood mononuclear cells (PBMCs). The cells are incubated for 16–24 h with the immobilised antibody which allows the accumulation of pro-inflammatory cytokines, quantified by enzyme-linked immunoabsorbent assay (ELISA), in response to the antibody. Cytokine responses stimulated by TGN1412 immobilised by air-drying onto polypropylene and polystyrene plates were much larger than responses to TGN1412 wet-coated onto polypropylene and polystyrene plates, respectively. In additional experiments with other mAbs associated with clinical reactions, air-dried mAbs stimulated larger tumour necrosis factor-α (TNF) responses than antibodies added in aqueous phase. Also, TGN1412 air-dried onto plastic plates stimulated large proliferative responses of 3-day cultures of lymphocytes. It was concluded that immobilising mAbs by air-drying offers a useful in vitro method for detecting and quantifying pro-inflammatory activities of therapeutic mAbs.     

Recognition of pertussis toxin by antibodies to synthetic peptides

Eight synthetic peptides, selected from the amino acid sequence of pertussis toxin (PT) subunits S1, S2, S3 and S4, were assessed for their ability to induce protein-recognizing and neutralizing antibodies. Seven of these peptides, prepared as conjugates of either keyhole limpet haemocyanin or tetanus toxoid, induced significant levels of antibody, all of which reacted with SDS-denatured PT on Western blots. Six of the antibodies bound to PT-coated ELISA plates; this binding was inhibited by homologous peptide antigen. However, none of the antibodies, including those directed against the N-terminus of subunit S1, were able to attenuate in vivo or in vitro toxin-dependent activity. Further investigation revealed that only one antibody, specific for the C-terminus of S1 (peptide Slc, 237-255), could recognize the conformation of native PT in solution. The other five antipeptide antibodies which reacted with PT-coated ELISA plates did not recognize PT when captured onto ELISA plates via either a monoclonal antibody or fetuin, unless the conformation of the toxin had been relaxed by reduction with dithiothreitol. Conversely, the native PT-recognizing response of peptide Slc did not bind the conformationally relaxed PT molecule. From this study, it appears likely that a peptide capable of inducing PT-neutralizing antibody must closely resemble the conformation of the cognate sequence in the native protein.     

Influence of the peptide insolubilization method on detection of anti-peptide antibodies in ELISA. Evaluation of nonspecific interactions

Different methods of peptide insolubilization in solid phase were compared in ELISA, to verify the influence of the peptide antigen presentation in the interaction with related antibodies. Our studies were performed using as model the peptide fragment 163-171 of human Interleukin 1 beta, and polyclonal or monoclonal anti-peptide antibodies. It was found that the peptide, N-terminally linked to a protein carrier before the adsorption on microtiter wells, interacted with specific polyclonal and monoclonal antibodies with high sensitivity and specificity. In contrast the recognition of similar random conjugates, prepared using a bivalent cross-linking reagent or the peptide covalently linked to poly-L-Lysine-pretreated wells, was hampered generally by very high levels of nonspecific binding. On the other hand, the free peptide adsorbed directly to the solid phase interacted with antibodies with very low sensitivity and specificity. Nonspecific interactions were found in particular between peptides and hyperimmune sera or nonrelated monoclonal antibodies. On the contrary pre-immune sera and normal mouse immunoglobulins never showed significant interactions with any of peptides. This nonspecificity was also overcome when N-terminally linked peptide-protein conjugates were used for the assay.     

Enzyme immunoassay for human chorionic gonadotropin using monoclonal antibodies elicited with a synthetic peptide

We recently described the generation of mouse monoclonal antibodies directed against hCG using a synthetic peptide immunogen: the carboxy-terminal peptide (CTP) of beta-hCG. The CTP, residues 109-145, is a portion unique to hCG and is not present in other glycoprotein hormones. These monoclonal antibodies react with beta-hCG-CTP and whole hCG but do not react with hLH. In the present study, a two-site sandwich ELISA specific for hCG has been developed using these CTP-induced monoclonal antibodies. Two monoclonal antibodies were paired: beta-hCG-CTP-a6 was immobilized to give a hCG-specific solid phase, and a biotinylated monoclonal antibody against beta-hCG was used as the indicator antibody. This second antibody was introduced during the hCG-capture step because of its agonist effect on hCG-capture by beta-hCG-CTP-a6 solid phase. This ELISA system can detect 2.2 IU/l hCG in a serum-free medium and 10.4 IU/l in a medium containing 20% human serum. We did not observe any cross-reactivity with hLH up to concentrations of 5000 IU/l. The hCG-ELISA correlated well (r = 0.99) with a control RIA in an assay of 78 human sera from healthy males, pregnant or non-pregnant females and from patients with choriocarcinoma, hydatidiform mole, or teratocarcinoma.     

Use of monoclonal antibody in an ELISA test for the detection of antibodies to bovine leukaemia virus

A variant of the ELISA technique, involving a monoclonal anti-gp51 antibody yields a highly sensitive method for the detection of bovine leukaemia virus (BLV) antibodies. The gp51 antigen-coated microtitre plates are obtained by incubation of plastic-adsorbed monoclonal antibodies with a non-purified mixture of BLV antigens. Sera to be tested are incubated in the wells of the gp51-coated plates and bound antibodies are revealed by an enzyme-linked antibovine immunoglobulin reagent. This test is as sensitive as liquid phase radioimmunoassay using the same gp51 antigen and thus appears as a highly sensitive, practical, rapid and cheap method for the detection of BLV antibodies.