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E D Day

Publications and source records attributed to E D Day.

At least 37 records · Page 2Linked to original sources
PubMed1984

Format determinants of synthetic myelin basic protein peptide S82 mimicked by a mixture of synthetic peptides S8 and S79.

Antibodies to synthetic myelin basic protein peptide S82 (TTHYG-SLPQKAQGHRPQDEG) did not react with synthetic peptide S8 (GSLPQKAQGHRPQDENG) and only partially so with synthetic peptide S79 (AQGHRPQDEG); however, the antibodies did react to a considerable extent with an equimolar mixture of S8 and S79. Since the anti-S82 antibodies had previously been shown to be directed to a non-sequential format determinant dependent on the conformation of secondary structure, it seems probable that the mixture of S8 and S79 assumed a format that neither one individually possessed to any great degree.

Amino Acid Sequence↗
PubMed1984

Affinity purification of two populations of antibodies against format determinants of synthetic myelin basic protein peptide S82 from S82-AH- and S82-CH-Sepharose 4B columns.

Two different kinds of immunosorbents were prepared that contained the synthetic myelin basic protein didecapeptide S82 (TTHYGSLPQKAQGHRDQDEG)--one coupled with AH-Sepharose 4B through hexanoate spacers to the C-terminal glycyl residue; the other, with CH-Sepharose 4B through hexanoate spacers to the N-terminal threonine residue. An antiserum rich in antibodies to a format determinant of S82 was passed through each column, and, by means of affinity purification, two homogeneous populations of anti-format antibodies were obtained, each with a binding affinity of 1 X 10(8)M-1 for S82. The population recovered from S82-AH-Sepharose 4B cross-reacted to a considerable extent with synthetic peptide S8 (GSLPQKAQGHRPQDENG) but only to a limited extent with S79 (AQGHRPQDEG). The population recovered from S82-CH-Sepharose 4B cross-reacted poorly, if at all, with S8. An equimoler mixture of S8 + S79, however, reacted well with either population of anti-format antibodies, thus showing that the mixture could mimic the format of S82. It was concluded that secondary structural conformation of S82 could be preserved during the coupling procedure and that the resulting immunosorbents could be used for the affinity purification of anti-S82 antibodies to the format determinants.

Animals↗
PubMed1984

Immunochemical cross-reactivity between intact purified myelin basic protein (MBP) and the synthetic encephalitogenic peptide S49.

Three antisera to myelin basic protein--a rabbit antiserum pool against rat myelin, a rabbit antiserum pool against rat myelin basic protein (MBP), and a monkey antiserum against bovine MBP--were found to contain detectable levels of antibodies that would bind radiolabeled S49 (GSLPQKAQRPQDENG). Strongly encephalitogenic in Lewis rat, S49 is a synthetic peptide representing residues 69-84 of bovine MBP with a deletion of glycine-76 and histidine-77 to make it analogous to rat and guinea pig MBPs. The rabbit antimyelin antiserum and the monkey anti-MBP antiserum contained antibodies directed against a non-sequential determinant that required asparagine 84, the glycine-histidine deletion, and residues 69-71 for maximal activity. S49-reactive antibodies from the rabbit anti-MBP antiserum were directed solely against a sequential determinant comprising residues 69-71, S49-reactive antibodies from all three antisera reacted in liquid phase with purified intact rat, guinea pig, and bovine MBP showing that the determinant is exposed for B cell recognition even in bovine MBP and can serve both as immunogen and reactant.

Amino Acid Sequence↗
PubMed1983

Immune lysis of reconstituted myelin basic protein--lipid vesicles and myelin vesicles.

Complement-mediated lysis of reconstituted lipid-myelin basic protein (BP) vesicles and myelin vesicles due to antibody raised against BP and isolated myelin is measured by determination of the amount of a water-soluble spin label, tempocholine chloride, released from the vesicles. The response is shown to be antigen-specific, antibody-dependent, and complement mediated. The relative response to different anti-BP antibody samples is similar to that determined by radioimmunoassay procedures. In contrast to immunoassays with BP in aqueous solution, this method measures immune recognition of the protein in either a synthetic or a natural membranous environment. This is important because this protein has been shown to have a different conformation when bound to lipid bilayers than in aqueous solution and its conformation depends on lipid composition. It is also a more rapid method because no separation of spin label still trapped in the vesicles and that released due to immune lysis is required. In synthetic membranes consisting of sphingomyelin, cholesterol, and an acidic lipid, either phosphatidylglycerol, phosphatidic acid, or phosphatidylserine, the response was greatest when the acidic lipid was phosphatidic acid. The response did not depend significantly on the antigen concentration expressed as molar ratio of BP to sphingomyelin, over the range 0.15:600 to 2:600, although it decreased at molar ratios less than 0.15:600. The antigen density required for immune lysis of vesicles containing this protein antigen is similar to that reported elsewhere for lipid antigens, although the time required for maximal lysis was greater. Both anti-BP and anti-myelin antibodies caused a greater specific complement-mediated response with synthetic vesicles than with myelin vesicles, which may be due to the different lipid and/or protein composition of myelin. Response was also obtained with the myelin vesicles, however, indicating that some determinants of BP can be recognized on the surface of the bilayer in isolated myelin by anti-BP.

Animals↗
PubMed1983

A rabbit B cell determinant for a conserved portion of myelin basic protein, rabbit encephalitogenic sequence 65-74.

Synthetic peptide S24 (TTHYGSLPQKG) represents residues 65-74 of myelin basic protein (MBP) and contains the major determinant involved in the development of experimental allergic encephalomyelitis (EAE) in rabbits. This peptide is completely conserved in all nonprimate mammals for which sequence information is available. Although it is clear that peptides containing the S24 region are capable of inducing EAE, previous serologic studies have resulted in the conclusion that the determinant is "buried" or sequestered in intact MBP. Employing a liquid phase radioimmunoassay, we studied Ab responses to the S24 determinant in six rabbits injected with rat myelin. Two of the six animals developed small but measurable responses to the S24 determinant. In one of these rabbits, the response was boosted with a covalent conjugate of S82 and methylated BSA (MBSA). We also measured antibodies to the S24 determinant in rabbit antisera to human, monkey, dog, bovine, and the large and small forms of rat MBP. By nonequilibrium inhibition analysis, we determined that the antibody responses to these antigens were all directed to a determinant composed of residues 66-71 of MBP, and that intact MBP inhibits the binding of these antibodies to radiolabeled S24. The results demonstrate that the rabbit encephalitogenic region of myelin basic protein is exposed in the intact molecule both as an immunogen and as a reactant in liquid-phase assays; furthermore, they demonstrate that MBP antigenicity leading to B cell responses does not necessarily depend on sequence differences between the injected protein and its counterpart in the host species. The latter finding reinforces the contention of Atassi that autoantibody responses are not exclusive to "evolutionary hypervariable locations."

Amino Acid Sequence↗
PubMed1982

Oligoclonal IgG in rabbits with experimental allergic encephalomyelitis: non-reactivity of the bands with sensitizing neural antigens.

Oligoclonal IgG bands have recently been reported to occur in cerebrospinal fluid (CSF) and serum of rabbits with experimental allergic encephalomyelitis (EAE). To examine the specificity of these bands, a) individual bands eluted from rabbit CSF and sera were tested by radioimmunoassay (RIA) for anti-MBP activity and b) rabbit sera were absorbed with the neuroantigens used for sensitization. RIA of eluates from sequential agar gel slices of the entire IgG region of serum or CSF from MBP sensitized rabbits showed that anti-MBP activity occurred throughout the IgG region and did not localize to specific band-containing fractions. Furthermore, there was no change in banding patterns following absorption of EAE rabbit sera with washed brain homogenates, soluble MBP or MBP conjugated to Sepharose beads. Therefore, our results indicate that the oligoclonal IgG response in EAE is not preferentially directed against the sensitizing neuroantigen, and we raise the possibility of nonspecific B cell activation.

Animals↗
PubMed1982

Comparative levels of endogenous myelin basic protein-serum factors (MBP-SFs) in adult and suckling mice (B6CBAF1 and B6C3HF1, strains) and in neurologically mutant mice of the same genetic background.

Normal adult B6C3HF1 and B6CBAF1 mice contained at least 10 times higher levels (1.17 microM) of endogenous myelin basic protein-serum factors (MBP-SFs) than previously found in adult Lewis rats. In rat MBP-SF levels in the adult (0.03 microM) were much less than in the suckling animals (0.74 microM). In mice, by contrast, the adult (1.17 microM) and suckling (0.75 microM) levels were similar. Suckling mice from 9 different neurologically mutant strains and their clinically normal littermates had MBP-SF levels (0.5 microM) slightly below that of normal suckling mice of the same genetic background (0.75 microM).

Animals↗
PubMed1981

Equilibrium and nonequilibrium competitive inhibitions of antipeptide antibody binding by parent myelin basic protein and 18 related peptide sequences.

Equilibrium and nonequilibrium competitive inhibition analyses of a number of antisera to peptide S81 and S82 sequences were carried out through the use of inhibition radioimmunoassays with [125I]S81, [125I]S82, and [125I]S79 and a panel containing 18 related peptides and five myelin basic protein preparations. Two principal determinants were identified, one of them sequential, the other nonsequential. The sequential determinant involved a peptide at or near the C-terminal end of S82 that could be blocked by an interchange of asparagine for glycine at the C terminus. The nonsequential determinant was dominant for a number of rabbit and rat antisera, both anti-S82 and anti-S81, and was shared not only by S81 and S82 but also by S8 and S80, i.e., the family of residues of bovine MBP sequence 69-83. Neither determinant was expressed in any of the myelin basic protein preparations, and the nonsequential determinant was not expressed in peptide sequences smaller than S8.

Amino Acid Sequence↗
PubMed1981

Endogenous myelin basic protein-serum factors (MBP-SFs) and anti-MBP antibodies in humans. Occurrence in sera of clinically well subjects and patients with multiple sclerosis.

Sera of normal subjects and patients wtih multiple sclerosis (MS) have been frequently found to contain picomolar quantities of endogenous myelin basic protein-serum factors (MBP-SFs). These serum factors, collectively representing a heterogeneous spectrum, were detected and measured by means of a competitive inhibition radioimmunoassay (RIA) designed to distinguish their respective binding affinities with anti-MBP reagent antiserum. Anti-MBP antibodies in these same normal and patient sera were also detected and their differing binding affinities determined. In general, when sera of normal subjects were found to contain free MBP-SFs, the reagent anti-MBP antibodies in the reagent antiserum used to detect them were of relatively high binding affinity (8 X 10(8) M-1). When normal sera were found to contain free anti-MBP antibodies, the affinities of such antibodies were invariably lower (0.06-0.7 X 10(8) M-1). In contrast, sera of patients with active MS and exhibiting clinical fluctuations in their disease, infrequently contained high or medium high affinity MBP-SFs, whereas higher affinity anti-MBP antibodies were commonly detected. These patterns of MBP-SFs and anti-MBP antibodies in normal and MS human sera resemble those previously observed in studies of normal Lewis rats and rats developing experimental allergic encephalomyelitis (EAE). The findings here reported provide additional support for the view that circulating endogenous MBP-SFs may function as neuroautotolerogens that restrict expansion of MBP-reactive lymphoid cell clones having potentially injurious effector activity for central nervous system (CNS) tissue.

Adult↗
PubMed1981

Endogenous myelin basic protein-serum factors (MBP-SFs) in Lewis rats. Evidence for their heterogeneity and reactivity with anti-MBP antibodies of different affinities.

MBP-SF, previously described as an endogenous myelin basic protein-serum factor in Lewis rats with a suggested function as a neuroautotolerogen, appears not to be a single factor but a heterogeneous collection of serum factors (MBP-SFs), most probably small fragments of MBP, each cross-reactive with a different region of the multideterminant parent molecule. The heterogeneity of the MBP-SFs in any serum sample is defined and limited by the spectrum of binding affinities of the antibody populations represented in a given reagent anti-MBP antiserum. Some samples of normal Lewis rat serum have been found to contain high affinity MBP-SFs which coexist with low affinity anti-MBP antibodies whereas other sera have shown the reversed pattern, viz. low affinity MBP-SFs and high affinity antibodies. Additional sera have been found to contain MBP-SFs of several different affinities. In time-course studies of rats sensitized to neuroantigen-adjuvant a variety of MBP-SFs and anti-MBP antibodies of different affinities may be observed in sequentially collected sera from a given animal. In no animal has any serum sample been found to contain the full spectrum of MBP-SFs. Although some MBP-SFs have been found to increase temporarily during the 2nd week after neuroantigen/CFA sensitization, all MBP-SFs tend to disappear in the 2nd week and to be replaced by anti-MBP antibodies of differing affinities 3-4 weeks following sensitization.

Animals↗
PubMed1981

Studies of rat antibodies specific for myelin basic protein (MBP). Antibody-dependent cell-mediated lysis of MBP-sensitized targets in vitro.

Rats immunized with rat myelin basic protein (MBP) in an encephalitogenic regimen produce antibodies which cooperate with spleen cells from unimmunized rats in the specific lysis of MBP-sensitized chicken erythrocytes (MBP-CRBC) in vitro. Antibodies against MBP capable of participating in antibody-dependent cell-mediated cytotoxicity (ADCC) were detected in serum 9 days post immunization, and prior to the onset of experimental allergic encephalomyelitis (EAE). Measurements based on a sensitive radioimmunoassay technique had previously established that anti-MBP activity emerges by day 9, early enough to participate in events leading up to EAE. The ADCC assay was designed to further characterize functions of immunoglobulins associated with the humoral response to MBP, with emphasis on the cytophilic properties of early antibodies. The assay described was found to be nearly as sensitive as the RIA and could detect specific syngeneic rat anti-MBP at concentrations of about 0.03 pmoles/ml serum. Whether ADCC-type reactions take place in the central nervous system during acute EAE is not known; however, the presence of early cytophilic antibody in serum suggests that humoral immunity may play an ancillary role to that demonstrated for T-cell immunity in EAE.

Animals↗
PubMed1981

Affinity purification of an acylated and radiolabelled synthetic derivative of residues 75-83 of bovine myelin basic protein, [125I]S79. A model for the purification of picomole quantities of specific peptide fragments of myelin basic protein and antibodies against them.

Among the antibodies contained in a rabbit antiserum to synthetic peptide sequence TTHYGSLPQKAQGHRPQDEG (S82) of bovine myelin basic protein (residues 65-83 plus glycine), was a population reactive with a C-terminal determinant of S82 and cross-reactive with S79 (AQGHRPQDEG) but not S6 (AQGHRPQDENG). This antibody population was purified 153-fold by affinity chromatography from a minicolumn containing S79 coupled to CH-Sepharose 4B(TM) and eluted with 3 M MgCl2. The purified antibodies were then coupled to CNBr-activated Sepharose 4B(TM) and used to purify 125I-labelled, acylated S79 ([125I]S79), 3 M MgCl2 once again having been used to elute the labelled ligand. Sips distribution studies revealed appreciable heterogeneity of binding affinities of unpurified antibodies in their reaction with affinity-purified [125I]S79 or of purified antibodies in their reaction with unpurified [125I]S79 (heterogeneity constant a = 0.34 and 0.36, respectively). In contrast Sips distribution data indicated considerable restriction of binding of the purified antibodies in their reaction with purified labelled ligand (a = 0.92) with an average affinity constant of K0 = 1.56 X 10(8) M-1. The results indicate that the heterogeneous spectrum of binding affinities originally displayed by the unpurified S79-reactive antibodies in their reaction with unpurified labelled S79 was due both to the presence of some antibodies characterized by high affinity binding (K0 greater than 10(9) M-1) and of some labelled ligand with low binding affinity. The affinity chromatographic method as here described should prove advantageous in purifying and eventually characterizing picomolar amounts of serum factors, previously postulated to be fragments of myelin basic protein, that are reactive with reagent antibodies up to an affinity level of 10(8) M-1.

Antibodies↗
PubMed1981

Immunogenicity of synthetic peptide sequences S81 and S82 (residues 68-83 and 65-83) of bovine myelin basic protein. Time-course of antibody responses in rats and rabbits.

The timing and intensity of the antibody responses to the marker determinants of synthetic peptide S81 and S82 sequences of bovine myelin basic protein (MBP) (residues 68-83 and 65-83, respectively) were studied in 20 Lewis rats and 6 rabbits. All rats immunized with either peptide in CFA responded with antibody development. All rabbits immunized with S82 and CFA developed both antibodies and experimental allergic encephalomyelitis. In contrast only one rabbit developed antibodies against S81 and none of the S81-challenged rabbits developed disease. On the basis of extrapolation of linear time-response curves to zero activity, the time of appearance of anti-peptide antibody activity in the Lewis rats was 15.1 +/- 1.7 days after a single immunization, a week longer than the normal latent period before appearance of anti-MBP antibodies. The time of appearance of anti-S82 antibody activity in rabbits exhibiting linear response curves was 18 days, 4 days after a booster immunization with S82 in incomplete Freund's adjuvant. The development of clinical signs of experimental allergic encephalomyelitis occurred within 4 weeks after initial challenge (a few days after boosting) and continued for 8--13 days in all S82-immunized rabbits.

Amino Acid Sequence↗
PubMed1981

Equilibrium competitive inhibition analysis of synthetic peptide antigens from myelin basic protein as affected by the dual-dilution phenomenon.

It was shown that 125I-labelled and unlabelled forms of synthetic encephalitogenic peptide S82 (residues 65-83 plus glycine) of bovine myelin basic protein (MBP-Bov) were equally competitive in dual-dilution radioimmunoassays with rat- and rabbit-anti-S82 antisera without causing much deviation even at the extremes of the dual-dilution binding curves (solved in terms of total S82). With other antisera the deviations caused by the addition of unlabelled S82 were much greater than would be expected among repetitive assays with labelled antigen alone, and the excessive deviations were usually more prominent in one region of the dual-dilution binding curve than in another. Thus, establishing equivalence between labelled and unlabelled antigen with respect to one antiserum even at several dilutions does not establish proportionate sharing with respect to all antisera at all antigen concentrations. A method of dual-dilution equilibrium competitive inhibition analysis was devised that took this precaution into account. By means of the method, synthetic MBP-Bov peptides representing different parts of the S82 sequence were compared with homologous S82 peptide for their inhibitory effects upon dually diluted [125I]S82-anti-S82 systems. By this process several different S82 determinants were pinpointed, some with high affinity antibodies, others with low affinity antibodies, yet others equally well at high or low affinity.

Animals↗