Structure of an Fab fragment against a C-terminal peptide of hCG at 2.0 A resolution.

3A2 is an antibody raised against human chorionic gonadotropin and recognizes a linear epitope on the C-terminal peptide of the human chorionic gonadotropin beta-subunit. Its three-dimensional structure has been determined to 2-A resolution using molecular replacement and refined to a conventional R-factor of 18.2%. The protein exhibits the typical immunoglobulin fold, and the model contains 944 ordered water molecules and one sulfate ion. A comparison of the complementarity-determining regions of the Fab3A2 with those from the Protein Data Bank following the canonical structure method reveals a canonical main chain conformation. This antibody belongs to the canonical structure class (combination of canonical conformations of the complementarity determining loops) that shows a preference for haptens and not for peptides. However, the shape of the surface of the antigen binding loops resembles that of an anti-peptide antibody.

Epitope mapping and serodiagnosis using Ata- and (Lys)7 peptides.

The general applicability of the new peptide immobilization strategy in which the peptide of interest is N-terminally extended with an acetyl-thio-acetyl group or (poly)-Lys extension during synthesis, has been demonstrated in epitope-mapping experiments and serodiagnosis. Ala-scanning experiments and minimal epitope determination showed that the antigenicity of Ata-extended peptides derived from the human chorionic gonadotropin (hCG) and hepatitis B virus (HBV) amino acid sequence, was superior to the free parent peptides. Further, it could be shown that the choice of the epitope-mapping procedure (peptide in solution or immobilized on a solid support) may lead to a different perception of which residues constitute the epitope. In addition, a time-consuming conjugation process could be circumvented since the ELISA reactivity of BSA-conjugates was comparable to that of Ata-extended peptides. In the serodiagnosis using sera from various HIV-positive individuals, the lysyl-peptide showed a signal/noise ratio 10 times higher than the parent peptide, indicating that sensitivity increased as a result of this N-terminal lysyl tail. In all experiments we observed that antibody detection could be performed at roughly 10 times lower amounts of peptide when N-terminally linked to an Ata-group or lysyl-extension compared to the free parent peptide or the BSA-conjugated equivalent.

The influence of binding capacity and affinity on the improved performance of N-terminally extended hCG peptides, determined by ELISA-based procedures.

The improvement of peptide-ELISA responses by the use of small synthetic peptides elongated at the N-terminus with an Ata-group or a (Lys)7 extension has been analyzed. For this purpose, binding capacity and affinity were evaluated by specific ELISA procedures. The ELISA experiments on binding capacity, performed with saturating antibody concentrations, revealed a difference of more than three orders of magnitude in binding capacity between the parent peptides and the N-terminally linked peptides, in favor of the latter peptides. Antibody affinity values were determined by a liquid-phase equilibrium method as well as by a solid-phase equilibrium method. N-terminal extension of the peptides had almost no effect on the affinity when equilibrium between the peptide and the antibody was reached in solution. In contrast, solid-phase affinity was greatly enhanced when the N-terminally linked peptides were adsorbed to the polystyrene surface. This enhancement was determined by the N-terminal extension and the peptide amino acid sequence (40 to 600 times higher). Thus, the use of N-terminally extended peptides can greatly increase the performance of a peptide-ELISA through improved surface effects, resulting in higher binding capacity and functional affinity.

Adsorption studies of tritium-labeled peptides on polystyrene surfaces.

In this study, three presentation formats of an epitope peptide (hepta-peptide), derived from the human chorionic gonadotropin amino acid sequence, were compared for adsorption to the polystyrene wells of a microELISA plate. The peptides had either a free N-terminus, an Ata-group or a linear (Lys)7-extension at the N-terminal. In order to measure the adsorption properties, all peptides were tritiated by synthesizing an additional 3H-labeled glycyl residue to the N-terminus of their peptide sequence. Over a broad range of peptide concentrations used as coat solution, extension of the peptide by an Ata-group consistently increased adsorption by a factor of 1.5 to 3 compared to the free parent peptide. Of the three peptides studied, the Ata-peptide showed the highest surface coverage of 0.6 mg/m2 when 1.0 mmol/l was offered as the concentration of peptide in the coating solution. The highest surface coverage observed for the parent peptide was 0.4 mg/m2 (at 1.5 mmol/l). The lysyl (K7) peptide showed a maximum plateau value of 0.2 mg/m2, and therefore the lysyl (K7) extension reduced the peptide surface coverage at relatively high coat concentrations (above 0.1 mmol/l) compared to the parent peptide. At lower input concentrations (below 0.1 micromol/l), however, the packing density of the lysyl (K7) peptide was up to 25 times higher when compared to the other two peptide analogs. We conclude that better adsorption as well as improved antibody binding activity and (functional) affinity could explain the higher reactivity observed in ELISA procedures when peptides are N-terminally extended by an Ata-group or lysyl (K7) extension.

Direct coating of poly(lys) or acetyl-thio-acetyl peptides to polystyrene: the effects in an enzyme-linked immunosorbent assay.

Direct adsorption of small peptides to polystyrene surfaces is often not satisfactory. Therefore, a simple and general coating procedure to improve the coating efficiency of small synthetic peptide antigens to polystyrene is described. In this study, the binding capacities of four small synthetic peptides N-terminally linked to various moieties during synthesis were compared to their parent counterparts in terms of the amount of peptide coat concentration required to achieve 50% of the maximum enzyme-linked immunosorbent assay signal. Elongation of a short epitope sequence by an N-terminal acetyl-thio-acetyl (Ata) group or a lysyl moiety resulted in an enormous reduction in peptide coat concentration for all tested peptides of net two to four orders of magnitude when corrected for chain elongation. The optimal length of the lysyl moiety depended on the length of the model peptide. Replacement of both extensions by analogues (i.e., Ata analogues and other basic amino acid residues in the case of the lysyl moiety) was possible without reducing their enhancing properties to a great extent. Additional experiments showed that a lysyl moiety consisting of a linear stretch of seven lysyl moiety consisting of a linear stretch of seven lysyl residues was more effective in comparison to a branched lysyl construct and could easily compete with the multiple antigen peptide approach.

Purification of rubella virus E1-E2 protein complexes by immunoaffinity chromatography.

A murine monoclonal antibody directed against the E1 membrane glycoprotein of rubella virus was immobilized on an N-hydroxysuccinimide-activated chromatographic support. The antibody was used to purify rubella virus E1-E2 protein complexes from Tween-80/diethyl ether extracts of cell culture supernatants containing virus particles. The adsorption behaviour of immunosorbents with ligand densities of 2.9, 5.4 and 11.1 mg monoclonal antibody per millilitre of gel was investigated using batchwise conditions. Then the immunoaffinity purification process was optimized with regard to adsorption efficiency by adjusting the flow rate, the bed height and the amount of sample loaded onto the column. The optimized immunoaffinity purification process which is reproducible and relatively simple (one-step) had a yield of 73%, a concentration factor of 5-8 and a purification factor of about 2600. No mouse IgG due to ligand leakage could be detected in the immunopurified product using an enzyme immunoassay. High-performance size exclusion chromatography, sodium dodecyl sulphate polyacrylamide gel electrophoresis, immunoblotting and electron microscopy showed that the immunopurified product contained rosette-like structures formed by complexes of E1 and E2 proteins. The product retained its hemagglutinating activity and proved to be suitable for application in a fluorescent enzyme immunoassay for determination of anti-rubella IgG in human serum.

Assessment of the functional affinity constant of monoclonal antibodies using an improved enzyme-linked immunosorbent assay.

The use of an enzyme-linked immunosorbent assay (ELISA) for the determination of affinity constants implies heterogeneous measurements. Therefore, despite their simplicity, direct solid-phase binding assays are not common. Many investigators have serious, and mostly justified, reservations about the application of solid-phase affinity methods. They refer to problems such as diffusion effects and difficulties in reaching equilibrium due to heterogeneous binding and co-operativity. Accordingly, functional affinity determinations are often described as meaningless. These objections apply to the measurement of the affinity of a monoclonal antibody using the enzyme-linked immunosorbent assay of Beatty et al. (J. Immunol. Methods (1987) 100, 173), which is based on the effect of antibody affinity on the sigmoidal dose response curve. The affinity constant is calculated by mathematical equations, based on the Law of Mass Action and the authors made a number of important assumptions--avoiding the above mentioned problems--in order to justify the use of the Law of Mass Action. By carefully examining these assumptions we have developed an improved ELISA procedure for functional affinity determinations on the basis of a primary coating with the antigen only. the coating conditions were validated by employing gold labelled colloidal particles and physical counting of the bound particles under the scanning electron microscope. Since monovalent binding between human chorionic gonadotropin and its monoclonal antibody could be achieved under equilibrium conditions, the application of the Law of Mass Action and hence of the Beatty formula became possible. We conclude that under these conditions functional affinity determinations are appropriate.

Preparation and characterization of monoclonal antibodies against ornithine decarboxylase.

In order to develop a method for the immunocytochemical detection of ornithine decarboxylase (ODC), EC 4.1.1.17, we have prepared and characterized monoclonal antibodies (MAbs) against ODC. The primary structure of rat ODC (Rattus Norvegicus) was used for the selection of an epitope by computer calculations. The epitope (P16), a hexadecapeptide representing ODC-(345-360), was synthesized by means of solid phase peptide synthesis and coupled to a carrier protein. A bovine serum albumin conjugate of the P16 peptide was used as the immunogen for the production of MAbs in mice. Hybridoma clones were screened and the specificity of the monoclonal antibodies was tested in an ELISA utilizing a thyroglobulin conjugate of the hexadecapeptide. Two hybridoma cell lines were developed, i.e., MP16-2 and MP16-3. The epitope specificity of the MAbs produced by these cell lines was characterized in an ELISA using a set of small peptides representing parts of the P16 hexadecapeptide chain. MP16-2 recognized the ODC-(355-360) portion whereas MP16-3 reacted with the ODC-(345-350) part of the hexadecapeptide. Further studies showed that both MAbs also recognized native ODC but not the inhibited (i.e., ODC labelled with 3H-DFMO) enzyme indicating that the selected epitope was associated with the active site of ODC or a locus in its direct vicinity.

The sequence A alpha-(154-159) of fibrinogen is capable of accelerating the t-PA catalysed activation of plasminogen.

The rate of activation of plasminogen by tissue-type plasminogen activator (t-PA) is greatly increased by fibrin, but much less by fibrinogen. Fibrin(ogen) fragments such as the fibrin(ogen) cyanogen bromide fragment FCB-2 and FCB-5, and a synthetic peptide with the sequence of fibrinogen A alpha-(148-160), a constituent of FCB-2, also have rate-enhancing properties. In order to find a possibly smaller, still stimulating site within A alpha-(148-160) we synthesized successive linear amino-terminally acylated hexapeptides [i.e. A alpha-(148-153), A alpha-(149-154)'d, .... A alpha-(155-160)] from the sequence A alpha-(148-160). The only hexapeptide within the sequence A alpha-(148-160) capable of enhancing the rate of plasminogen-to-plasmin conversion by t-PA appears to be the amino-terminally acylated peptide comprising the sequence A alpha-(154-159). This peptide enhances the plasminogen activation rate six-fold; half-maximal activation rate is reached at a peptide concentration of 56 microM.

The sequence gamma-(312-324) is a fibrin-specific epitope.

Fibrin accelerates the activation of plasminogen catalyzed by tissue-type plasminogen activator much stronger than fibrinogen. Detailed studies showed that (part of) this rate-enhancing effect of fibrin is brought about by two sites in the fibrin molecule: one in A alpha-(148-160) and one in the gamma-chain stretch 311-379 (also known as FCB-5). During the fibrinogen-to-fibrin conversion, A alpha-(148-160) appears to become accessible, because a monoclonal antibody against synthetic A alpha-(148-160) reacts with fibrin, but not with fibrinogen. Because a similar situation may exist for (at least parts of) FCB-5, we have prepared a monoclonal antibody against a part (ie, gamma-[312-324]) of FCB-5, and found that this is fibrin-specific and does not bind fibrinogen. We conclude that gamma-(312-324) is hidden in fibrinogen and is exposed by the formation of fibrin.

Use of Mpc-amino acids in solid phase peptide synthesis leads to improved coupling efficiencies.

The Mpc-group has a somewhat better stability than the Fmoc-group, resists catalytic hydrogenolysis, is highly stable in acidic media and its elimination product does not polymerize spontaneously. In a direct comparison of coupling efficiencies obtained in solid phase peptide syntheses using Mpc- or Fmoc-amino acids it is shown that the use of Mpc-amino acids leads to better coupling efficiencies and, consequently, a more homogeneous peptide. An improved synthesis of Mpc-ONSu and of Mpc-amino acid derivatives is presented.

Structural requirements of fibrinogen A alpha-(148-160) for the enhancement of the rate of plasminogen activation by tPA.

Fibrin, not fibrinogen, enhances the rate of tPA catalysed plasminogen activation. In earlier studies we have shown that a site involved in this rate enhancement is located in a tridecapeptide, i.e. fibrinogen A alpha-(148-160). This sequence comprises a special charge distribution in which a stretch with alternating neutral and acidic amino acids is embraced by basic amino acids. In this study we found that the disruption of charge distribution as caused by replacing valine 152 by other (charged and/or polar) amino acids leads to loss of rate-enhancing capacity. Also lysine at position A alpha-157 was replaced by lysine derivatives and other amino acids. We found that the side chain of the amino acid at position A alpha-157 must contain no (as in glycine) or one carbon atom without substitution (alanine). When the side chain contains two or more carbon atoms, there should also be a polar group in the side chain. We also synthesized a series of hexapeptides covering the sequence of A alpha-(148-160), and found that only A alpha-(154-159) is stimulatory, notwithstanding the fact that the peptides A alpha-(152-157), A alpha-(153-158) and A alpha-(155-160) also contain lysine A alpha-157. We conclude that the shortest peptide with stimulation activity is A alpha-(154-159); that the charge distribution in A alpha-(148-160) is important; that it is not lysine A alpha-157 per se that is crucial, but rather the properties and orientation of the side chain of A alpha-157.

The sequence A alpha-(148-160) in fibrin, but not in fibrinogen, is accessible to monoclonal antibodies.

Fibrin, but not fibrinogen, accelerates the activation of plasminogen catalyzed by tissue-type plaminogen activator. Previous work showed that essential information for this accelerating capacity of fibrin resides in the sequence corresponding to residues 148-160 of the A alpha chain of fibrinogen [A alpha-(148-160)]. Our working hypothesis, based on those findings, is that A alpha-(148-160) is buried in fibrinogen and becomes accessible to proteins such as plasminogen and/or tissue-type plasminogen activator when fibrinogen is converted to fibrin. To test this hypothesis we have raised a monoclonal antibody against synthetic A alpha-(148-160) and found that this antibody reacts with fibrin and not with fibrinogen. This finding shows that A alpha-(148-160) becomes accessible when fibrinogen is converted to fibrin and that A alpha-(148-160) is a fibrin-specific neoantigenic determinant.