Strategy for affinity maturation of an antibody with high evolvability to (4-hydroxy-3-nitrophenyl) acetyl hapten.

In order to quantitate the contribution of amino acid replacements to an increase in affinity during affinity maturation, we measured thermodynamic parameters of the antigen-antibody interaction for a group of anti-(4-hydroxy-3-nitrophenyl) acetyl monoclonal antibodies whose differences in amino acid sequences had arisen only from somatic hypermutation. We prepared a common ancestor and hypothetical intermediate clones that might occur on the affinity maturation pathway, by employing site-directed mutagenesis. Isothermal calorimetric titration of the antigen-antibody reaction revealed that antibody evolution proceeds in two steps. The first step is driven by a decrease in enthalpy, in which two amino acid replacements in the VL region play an essential role. Further accumulation of amino acid replacements in VH and VL regions during the second step induce a progressive increase in affinity, which is driven by an increase in entropy, which has a cooperative mutational effect.

Modification of short peptides using epsilon-aminocaproic acid for improved coating efficiency in indirect enzyme-linked immunosorbent assays (ELISA).

The hydrophobicity of short synthetic peptides of 5-10 residues was enhanced for high coating efficiency as antigens in indirect ELISA. To obtain enhanced hydrophobicity, coupling of epsilon-aminocaproic acids to the synthetic peptides was carried out during solid phase peptide synthesis. As a short peptide model, three analogues of a streptavidin binding peptide consisting of 5 amino acid residues were prepared with four epsilon-aminocaproic acid residues. HPLC analysis showed a dramatic increase in hydrophobicity after modification and the modified peptides showed a better adsorption ability than the unmodified peptides in indirect ELISA. The whole process from antigen coating to color development was carried out within 2.5 to 3 h by dissolving the peptide in methyl alcohol and evaporating the solvent in each well of the microplate. As an application of this method, a peptide assumed to function as one of the epitopes of the human 60 kDa Ro/SSA antigen was selected from hydrophilicity, acrophilicity and hydropathy plots. The peptide was synthesized having an epsilon-aminocaproic acid modification at both N and C terminal ends and was tested with 30 sera from patients with systemic lupus erythematosus (SLE), 20 normal sera and a standard anti-Ro/SSA serum. The ELISA results revealed that the method gave a high signal-to-background ratio without altering the specificity of the assay. Moreover, our process was far simpler and more rapid than conventional methods used in indirect ELISA. Thus this method could be useful in the development of techniques for the diagnosis of SLE.

Immunological properties of allergen chemically modified with synthetic copolymer of N-vinylpyrrolidone and maleic anhydride.

Several conjugates of model allergen ovalbumin (OA) and the copolymer of N-vinyl pyrrolidone and maleic anhydride (VMA) modified with epsilon-aminocaproic acid (Acp) were prepared in different OA/Acp-VMA ratios. All conjugates were separated by ultrafiltration and analyzed by HPLC. Their compositions were determined by amino acid analysis and UV spectrometry. To detect immunogenicity, all conjugates were injected intraperitoneally into (CBAxC57BL/6)F1 mice three times in 3-week intervals in OA doses equivalent to 0.5, 10, and 100 micrograms/mouse. Only the conjugate containing 20%OA (OA(20%)-Acp-VMA) did not induce significant quantities of anti-OA IgE, but did induce anti-OA IgG antibodies in dose-dependent manner comparable to that of unmodified OA. Mixtures of OA and Acp-VMA or OA modified only with VMA without Acp activation with Acp induced dose-dependent anti-OA IgE and IgG antibody formation comparable to that of OA. Using passive cutaneous anaphylaxis, RAST inhibition and leukocyte histamine release, a significant reduction of allergenicity was noted using OA(20%)-Acp-VMA. This conjugate stimulated activation of the OA-specific T-cell hybrid 3DO-548 comparable to that of unconjugated OA. During experimental allergen-specific hyposensitization with OA(20%)-Acp-VMA, suppression of anti-OA IgE response and elevation of anti-OA IgG responses were noted when compared with unmodified OA. Selective blockade of B-cell epitopes of allergen may occur using the carrier Acp-VMA to reduce allergenicity while not affecting T-cell epitopes, thereby preserving immunogenicity. This approach of chemical modification of allergen suggests new opportunities in the creation of preparations for allergen-specific immunotherapy.

Antibody to folic acid: increased specificity and sensitivity in ELISA by using epsilon-aminocaproic acid modified BSA as the carrier protein.

For raising high titre and specific antibody to haptens or drugs, epsilon-aminocaproic acid modified bovine serum albumin (epsilon-ACA-BSA) was prepared for use as a carrier protein. Folic acid (FA) was coupled to epsilon-ACA-BSA, Imj.BSA and BSA for raising antibodies in rabbits. Enhancement of FA immunogenicity with FA-ACA-BSA was observed. Apart from determination of titre by indirect ELISA, dose-response behaviour and specificity of these antisera were also compared. FA-ACA-BSA antibody showed high sensitivity and specificity. Using this antibody, an ELISA method for the determination of FA was developed. The study provides a simple approach to raise highly specific and high titre antibody against small molecules.

A monoclonal antibody to the epsilon-aminocaproic acid binding site on the kringle 4 region of human plasminogen that accelerates the activation of Glu1-plasminogen by urokinase.

A monoclonal antibody, 10-F-1, previously shown [V. A. Ploplis, H. S. Cummings, and F. J. Castellino (1982) Biochemistry 21, 5891-5897] to interact with a particular epsilon-aminocaproic acid (EACA)3 binding site on the kringle 4 (K4) region of human Glu1-plasminogen (Glu1-Pg), has been employed to assess the contribution of this particular EACA site toward the enhancement, by EACA and its analogs, of the urokinase (UK)-catalyzed activation of Glu1-Pg. As is the case with EACA-like compounds, the presence of antibody 10-F-1 accelerates the activation of Glu1-Pg by UK, but does not enhance the similar activation of Lys77-plasminogen. In the presence of concentrations of antibody 10-F-1 which saturate its binding site on Glu1-Pg, the Km of Glu1-Pg activation by UK is raised from 1.4 +/- 0.2 microM, a value obtained in the absence of antibody, to 17.0 +/- 2.0 microM. On the other hand, the kcat for this activation, 0.038 +/- 0.005 s-1, is elevated to 2.45 +/- 0.2 s-1 at saturating concentrations of antibody 10-F-1. The kcat/Km for activation under these conditions is 0.027 s-1 microM-1 in the absence of antibody, and 0.144 s-1 microM-1 in the presence of saturating levels of antibody 10-F-1. This demonstrates that the interaction of this antibody with its epitope results in a fivefold stimulation of the activation rate of Glu1-Pg by UK. The availability of antibody 10-F-1 allows for a specific means of probing the function of one of the four to five thermodynamically equivalent weak EACA sites on human plasminogen. From this particular study, it is concluded that the weak binding site for EACA on the K4 domain of Glu1-Pg is either in-part or in-whole responsible for the enhancing effect of EACA on human Glu1-Pg activation by UK.

Primary in vitro immunogenicity of liposomal model membranes in mouse spleen cell cultures.

Neither 2,4-dinitrophenyl-6-N-aminocaproylphosphatidylethanolamine (DNP-Cap-PE) nor fluoresceinthiocarbamylphosphatidylethanolamine (F1-PE) induces hapten-specific plaque-forming cells (PFC) when incubated with suspensions of spleen cells from unimmunized C57BL/6J mice. However, PFC are produced after incorporation of these synthetic lipid antigens into liposomal model membranes. The in vitro response is characterized by the following: a) it is time and dose dependent; b) the frequency of IgM PFC exceeds IgG PFC; c) both nonadherent and adherent cells are required (2-mercaptoethanol can replace the requirement for adherent cells in some experiments); d) depletion of thymus-derived cells by treatment with anti-theta antiserum plus complement does not diminish the response; e) spleen cells from nude BALB/c mice also produce PFC. Thus, the essential features of the in vivo immunogenicity of DNP-Cap-PE and F1-PE sensitized liposomes, which have been previously described, can be replicated in an in vitro cell culture system.

Secretion rate independent evaluation of IgM antibody avidity at the level of single immunocytes.

The hemolytic plaque inhibition assay has been performed on spleen cells from mice immunized with TNP-HRBC to evaluate avidity of anti-TNP IgM antibodies. At different times after immunization direct plaques were inhibited by soluble TNP-EACA, TNP61-BGG, or anti-mu antiserum. Analysis of the inhibition data provided independent estimates of antibody avidity and secretion rate. Avidity was found to increase with time, to reach a maximum when the antibody response attained the peak value, and then to decline as the response was waning. There was a decrease followed by increase of the secretion rate concomitant with the rise and fall of the antibody response and avidity.

Specific suppression of anti-hapten reaginic antibody titers with hapten-coated liposomes.

Reaginic antibodies to DNP and ovalbumin (OA) were induced in B6D2F1 mice by a single i.p. injection of 1 microgram of DNP3-OA suspended with 1 mg of A1(OH)3 in 0.5 ml of saline. The anti-DNP reaginic antibody titers were markedly depressed by treatment of mice with DNP-coated liposomes. This treatment, however, did not affect the level of antibody formation to OA.

Comparative immunogenic properties of N-substituted phosphatidylethanolamine derivatives and liposomal model membranes.

This study describes some of the parameters that quantitatively or qualitatively influence the immunogenicity in guinea pigs of synthetic lipid antigens: phosphatidylethanolamine (PE) derivatives in which the amino (N) group has been substituted with either dinitrophenyl (DNP), dinitrophenylaminocaproyl (DNP-Cap), fluoresceinthiocarbamyl (Fl), or mono (p-azobenzenearsonic acid) throsyl (ABA-Tyr) residues. Previous experiments have shown that the non-covalent insertion of DNP-Cap-PE and ABA-Tyr-PE into the same lipid bilayers of sphingomyelincholesterol-dicetylphosphate liposomes markedly enhanced anti-DNP-Cap antibody formation over that produced by liposomes sensitized with only DNP-Cap-PE. The humoral response to Fl-PE and CNP-PE-sensitized liposomes is also augmented by the simultaneous incorporation of ABA-Tyr-PE. Moreover, micelles containing both DNP-Cap-PE and ABA-Tyr-PE induce more antibodies to the DNP-Cap deteminant than do micelles of DNP-Cap-PE alone, or a mixture of DNP-Cap-PE and ABA-Tyr-PE micelles. Nevertheless, in regard to a humoral response, liposomes were more potent immunogens than were their micellar counterparts. Of all the N-substituted derivatives examined so far, ABA-Tyr-PE is unique in that it can elicit cell-mediated immunity in addition to antibodies. The cellular response to ABA-Tyr-PE is not, however, stimulated by incorporation into liposomal bilayers and requires administration of either micelles or liposomes in complete Freund's adjuvant. In contrast, the ability of ABA-Tyr-PE to enhance a humoral response to another N-substituted PE derivative present in the same immunogen is also observed when the latter are given with incomplete Freund's adjuvant. The relationship of these findings to the immunogenicity of naturally occurring lipid antigens, as well as conventional immunogens having at least one determinant covalently attached to a protein carrier is discussed.

A micro-titer red-cell-linked-antigen-antiglobulin-test for DNP specific immunoglobulins.

A micro-titer red-cell-linked-antigen--antiglobulin test is described for the measurement of DNP specific immunoglobulins in mouse plasma samples.