The preparation of antibodies reactive against citrulline-containing charge isomers of myelin basic protein but not against the arginine-containing charge isomers.

Human myelin basic protein (MBP) is composed of several charge isomers, the result of various post-translational modifications. One of the charge isomers C-8, has been shown in our laboratory to contain six citrullinyl residues which replace arginyl residues at selected sites in the MBP. In order to determine the disposition of the citrulline-containing charge isomers in the myelin stack, we prepared specific antisera against the citrullinyl group. Since 9-fluorenylmethoxycarbonyl (Fmoc)-citrulline, required for the preparation of the synthetic peptides to be used for antibody production, was not commercially available, synthesis of the Fmoc-citrulline was a necessary prerequisite. The synthesis and purification of the N-9-fluorenylmethyloxycarbonyl derivative of citrulline is described. It was characterized by thin layer chromatography, 1H and 13C NMR spectroscopy, fast-atom bombardment mass spectroscopy, and thermal analyses. It was used in the automated peptide synthesis of a peptide Ala-Cit-His-Gly-Phe-Leu-Pro-Cit-His-Arg corresponding to residues 24-33 and Gly-Cit-Asp-Ser-Arg-Ser-Gly-Ser-Pro-Met-Ala-Cit-Arg, corresponding to residues 158-170 of the C-8 sequence, a naturally occurring charge isomer of human myelin basic protein, and a tetracitrulline peptide, Cit-Cit-Cit-Cit-Gly. The tetracitrulline peptide was used for the production of an antibody shown to react only with synthetic peptides and proteins containing citrulline. This antibody was used to distinguish between a citrulline-containing protein, C-8, a naturally occurring charge isomer of MBP, and a non-citrulline-containing charge isomer of MBP, C-1.     

Rapid release and unusual stability of immunodominant peptide 45-89 from citrullinated myelin basic protein.

Myelin basic protein (MBP) exists in a population of isoforms and isomers. The 18.5 kDa MBP-C1, the main human adult isoform, has 170 residues and is relatively unmodified, whereas the same isoform can be citrullinated on six arginine residues to create the MBP-C8 (MBP Cit6) isomer. MBP Cit6 dominates in MS brain, accounting for 45% rather than 25% of the population of MBP isomers. In the fulminant form of MS, known as Marburg's Disease, 18 of the 19 arginines in MBP are citrullinated (MBP Cit18). Citrullination of MBP could lead to instability of myelin or limited remyelination. In this investigation, the susceptibilities to degradation by cathepsin D of MBP Cit6 and MBP-C1, both from normal and MS brain tissue, and Marburg MBP Cit18 were compared. The pattern of digestion was similar, and no differences of corresponding isomers in normal and MS brain were noted. However, normal MBP Cit6 was degraded 10-fold more rapidly than MBP-C1, and MBP Cit18 was degraded even more rapidly. MBP peptide 45-89 was preserved regardless of isomer type or source. Its generation was directly related to the citrulline content of the MBP substrate being 4 times faster in normal MBP Cit6 and 35 times faster in Marburg MBP Cit18 than in normal MBP-C1. Peptide 45-89 from a citrullinated MBP exhibited more deamidation, and, regardless of source, showed an alpha-helix structure in a lipid mimetic environment. We postulate that the generation of MBP peptides, including those that are dominant and encephalitogenic, is directly related to deimination of arginine to citrulline in MBP.     

ADP-Ribosylation of Human Myelin Basic Protein

Abstract: When isolated myelin membranes were ADP-ribosylated by [³²P]NAD⁺ either in the absence of toxin (by the membrane ADP-ribosyltransferase) or in the presence of cholera toxin, the same proteins were ADP-ribosylated in both cases and myelin basic protein (MBP) was the major radioactive product. Therefore, cholera toxin was considered a good model for ADP-ribosylation of myelin proteins. Although purified human MBP migrates as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular mass of 20 kDa, the microheterogeneity that is masked under these conditions can be clearly demonstrated on alkaline-urea gels at pH 10.6. At this pH, MBP is resolved into several components that differ one from the other by a single charge (charge isomers). These charge isomers can be resolved on CM52 columns at pH 10.6, and several can be ADP-ribosylated. Component 1 (C-1), the most cationic charge isomer, incorporated 1.79 mol of ADP-ribose/mol of protein. C-2 and C-3 (which differ from C-1 by the loss of one and two positive charges, respectively) incorporated slightly less at 1.67 and 1.63 mol of ADP-ribose/mol of protein, respectively, whereas C-8, the least cationic, incorporated less than 0.11 mol/mol of protein. In the presence of neutral hydroxylamine, the ADP-ribosyl bond was shown to have a half-life of about 80 min, suggesting an N-glycosidic linkage between ADP-ribose and an arginyl residue of the protein. As MBP contains several components that are ADP-ribosylated to different specific activities, the use of MBP, ADP-ribosylated in the natural membrane, to identify the sites involved would yield a mixture of peptides difficult to resolve. Therefore, to identify the sites ADP-ribosylated, an endoproteinase Lys-C digest of C-1 ADP-ribosylated by cholera toxin was prepared. Two radioactive peptides were isolated by reversed-phase HPLC. Amino acid and sequence analyses identified the radioactive peptides as residues 5–13 and 54–58 of the human sequence (sp. act., 0.89 and 0.62 nmol of ADP-ribose/nmol of peptide, respectively). The ADP-ribosylated residues were identified as Arg⁹ and Arg⁵⁴ by automated and manual Edman sequencing. Taken together with our previous observation that MBP binds GTP at a single site, these data suggest that MBP functions as part of a signal transduction system in myelin.     

Monoclonal Antibodies to Human Myelin Basic Protein

SJL/J and (SJL X PL) F1 hybrid mice were immunized with intact human myelin basic protein (MBP) or the three major peptic fragments of MBP, residues 1-38, 39-89, and 90-170. Immune spleen cells were fused with mouse myeloma P3 X 63Ag8 (NS1) cells in the presence of polyethylene glycol. Hybridoma supernatant culture fluids were screened for antibody to MBP by a solid-phase radioimmunoassay (RIA). The specificity of the monoclonal antibody (mAb) was characterized by RIA using the three major MBP peptic fragments and subfragments as well as MBP and MBP fragments of different species with known amino acid sequence differences. Six MBP mAbs were generated, one of them IgM isotype and the remainder IgG isotypes. One mAb each reacted against regions of residues 22-38, 39-69, 70-89, 90-116, and two reacted against residues 118-157. Immunoblots also showed that the five IgG mAbs were reactive against MBP and the peptic fragment of MBP containing the epitope. Immunohistochemical studies showed the IgG mAbs specifically stained myelinated fiber tracts in human brain tissue.     

Characterization of Myelin Basic Protein Charge Microheterogeneity in Developing Mouse Brain and in the Transgenic Shiverer Mutant

Abstract: Myelin basic protein (MBP) is a highly heterogeneous family of membrane proteins consisting of several isoforms resulting from alternative splicing and charge isomers arising from posttranslational modifications. Although well characterized in the bovine and human species, those in the mouse are not. With the availability of a number of transgenic and knockout mice, the need to understand the chemical nature of the MBPs has become very important. To isolate and characterize the MBP species in murine brain, two methods were adapted for use with the small amounts of MBP available from mice. The first was a scaled-down version of the preparative CM-52 chromatographic system commonly used to isolate MBP charge isomers; the second was an alkaline-urea slab gel technique that required five times less material than the conventional tube gel system and, from these gels, western blots were readily obtained. Murine MBP was resolved into two populations of charge isomers: the 18.5- and 14-kDa isoforms. Isolation and characterization of these charge isomers or components permitted us to assign possible posttranslational modifications to some of them. Component 1 (C-1), the most cationic isomer, had a molecular weight of 14,140.38 ± 0.79. C-2 consisted of two 14-kDa species, 14,136.37 ± 0.74 and 14,204.45 ± 0.70. Two variants, 14,215.57 ± 0.94 and 18,413.57 ± 0.76, constituted C-3. C-4, C-5, and C-8 (the least cationic isomer) each consisted of both 14- and 18.5-kDa isoforms. During myelinogenesis, the 18.5-kDa isoform appeared first (day 4); the 14-kDa isoform appeared at day 16 and subsequently became the dominant isoform. The transgenic shiverer mutant synthesized mainly the 18.5-kDa isoform, but none of the 14-kDa isoform, similar to the 4-day-old mouse. We concluded that the trangenic shiverer was able to initiate myelinogenesis with the 18.5-kDa isoform, but was unable to complete myelinogenesis because of the absence of the 14-kDa isoform.     

Characterization of Basic Proteins from Goldfish Myelin

Abstract: Myelin basic protein (MBP) from common goldfish ( Carassius auratus ) myelin was extracted with dilute mineral acid. Immunological cross-reactivity of the goldfish MBP, with polyclonal antisera raised against bovine MBP, suggested that the goldfish protein has epitopes for these antibodies. It also reacted with a monoclonal antibody specific for a seven amino acid epitope (130–137) conserved in the MBP of most mammalian species. To characterize the charge heterogeneity of this protein, we iodinated the protein with ¹²⁵I and chromatographed it on a carboxymethyl cellulose-52 column together with a nonlabeled acid soluble fraction prepared from human white matter as a carrier protein. All of the goldfish protein was recovered in the unbound fraction, demonstrating that it was less cationic than the carrier protein (human MBP). We have also examined the urea alkaline gel profile of the goldfish MBP together with the human C-1, C-2, C-3, C-4, and C-8 components. The results from these experiments indicated that this MBP extracted from goldfish brain myelin lacked the microhet-erogeneity that is associated with MBPs from higher vertebrates. The MBPs from goldfish myelin were separated into their isoforms by reversed-phase HPLC. Amino acid compositions were determined for both the 17- and 14-kDa goldfish proteins. Amino acid analysis revealed similarities with the compositions of other MBPs; however, the serine content in both the 17- and 14-kDa proteins was higher than that of the human C-1, the mouse C-1 protein, and the shark proteins. The HPLC-purified 14-kDa goldfish protein was chemically cleaved with CNBr for partial sequence analysis. Even from the limited sequence obtained, the sequence ATAST was found in goldfish, which is also present in human, rabbit, and guinea pig MBPs.     

Primary structure of equine myelin basic protein by mass spectrometry

Equine myelin basic protein (MBP) has been isolated from spinal cord and shown to consist of a number of components (charge isomers) by alkaline-urea gel electrophoresis. Mass analyses of several of these components showed that each was posttranslationally modified and some have been identified. Component 1, the most cationic charge isomer, was sequenced by a combination of liquid chromatography and mass spectrometry of peptides obtained by proteolytic digestion. At 172 residues it is slightly larger than the bovine (169) and the human (170). A major difference between bovine and equine sequences was the replacement of AQGH (bovine residues 76-79) by SRDG (equine). A number of other replacements involving single amino acids were also found. Methylated arginine (residue 108 equine) was found as both the mono- and the dimethylated derivative and represents the first MS/MS evidence for this modification in any MBP.     

Conformational Correlates of the Epitopes of Human Myelin Basic Protein Peptide 80–89

Different epitopes residing within the decapeptide of residues 80-89 of human myelin basic protein (MBP) exist in the MBP-like material detected in human CSF and urine. In the present study, the structure of human MBP peptide 80-89 was examined by a combination of physical measurements and correlated with its varying immunochemical reaction with three polyclonal antisera. At least two epitopes are present in the decapeptide. Progressive shortening and reduction in net negative charge of MBP peptide 80-89 to form peptides 81-89, 82-89, 83-89, and 84-89 revealed an epitope not present in intact MBP. Circular dichroism and Fourier-transform infrared of these MBP peptides in water demonstrated random structure that was partially changed to beta-structure in the shorter peptides. In methanol, used as a model for a lipid environment, the random structure was diminished and was replaced by alpha-helix and beta-structure, especially in the shorter peptides. The findings indicate that the range of epitopes present in this decapeptide is influenced by conformation, which, unexpectedly, becomes progressively less random as the peptide becomes smaller, especially in a hydrophobic environment. This behavior has implications for the immunochemical detection of small antigens or antibodies to them in tissue extracts or body fluids.     

A Hydroxyproline-Containing Protein from Shark Brain That Is Related to Myelin Basic Protein

Myelin basic protein (MBP) from shark (Chondricthyes) consists of a simpler mixture of charge isomers than human MBP. About two-thirds of the total amount applied to a CM-52 cellulose cation-exchange column was recovered in the unbound fraction of the column; the remaining one-third bound to column and was eluted as a single OD280 peak. This bound material did not sow the usual pattern of charge microheterogeneity found with human or bovine MBP. The unbound fraction was composed of a high molecular weight protein (55-60 kDa), which constituted most of this protein fraction and a low molecular weight protein (approximately 18 kDa). The amino acid composition of our unbound fraction was similar to that reported earlier. The Glx (glutamic acid + glutamine) was increased about threefold whereas the Arg content was only about 25% of that of the 18.5 kDa variant of bovine or human origin. The presence of hydroxyproline (1.2 residues/100) in this protein was noteworthy, identification of which was achieved by amino acid analysis in two different systems and by mass spectrometry. In the precolumn derivatization method, hydroxyproline eluted at 2.7 min; in the postcolumn derivatization method it eluted at 12.2 min. Identification of hydroxyproline was completed by fast atom bombardment-mass spectral analysis. The effect of hydroxyproline on the secondary structure of this protein is being studied. Verification that this high molecular weight protein contained MBP sequences within its primary structure was confirmed by immunological methods.(ABSTRACT TRUNCATED AT 250 WORDS)     

Monoclonal Antibodies Against Myelin Proteolipid Protein: Identification and Characterization of Two Major Determinants

This report describes the preparation and characterization of a panel of monoclonal antibodies (mAbs) against the myelin proteolipid protein (PLP). A Lewis rat was immunized with bovine proteolipid apoprotein and 27 mAbs were selected based on their reactivity against bovine PLP on enzyme-linked immunosorbent assays. Eleven mAbs recognized the PLP carboxyl-terminal sequence when tested against a panel of synthetic peptides in a solid-phase assay. A carboxyl-terminal pentapeptide (residues 272-276) was sufficient for antibody binding and the terminal phenylalanine residue was found particularly important. Deletion, modification, or replacement of this residue markedly reduced or obliterated antigen-antibody interaction. Nine mAbs reacted with a second antigenic determinant, residues 209-217, but these could be identified only by competitive immunoassays. This peptide was a more effective inhibitor than the longer peptides 202-217 and 205-221, suggesting that flanking residues may interfere with peptide-antibody interaction. Seven antibodies did not react with any of the synthetic peptides tested and their determinants remain unidentified. Immunoblot analysis showed that the mAbs reacted with both the PLP and the DM-20 isoforms. Twenty-three of the mAbs were of the immunoglobulin G2a or b isotype; the remaining antibodies were immunoglobulin M and all of these were specific for residues 209-217. Cultured murine oligodendrocytes were stained by most of the mAbs tested, but the most intense reactivity was observed with the carboxyl-terminus-specific mAbs. The immunocytochemical analyses demonstrate that the mAbs react with the native PLP in situ and show their potential usefulness for studies of the cell biology of myelin and oligodendrocytes.     

Post-translational Modifications of Chicken Myelin Basic Protein Charge Components

Purified myelin basic protein (MBP) from various species contains several post-translationally modified forms termed charge components or charge isomers. Chicken MBP contains four charge components denoted as C1, C2, C3 and C8. (The C8 isomer is a complex mixture and was not investigated in this study.) These findings are in contrast to those found for human, bovine and other mammalian MBP’s. Mammalian MBP’s, each of which contain seven or eight charge components depending on the analysis of the CM-52 chromatographic curves and the PAGE gels obtained under basic pH conditions. Chicken MBP components C1, C2 and C3 were treated with trypsin and endoproteinase Glu-C. The resulting digests were analyzed by capillary liquid chromatography combined with either an ion trap tandem mass spectrometer or with a Fourier transform ion cyclotron resonance mass spectrometer. This instrumentation permitted establishing the amino acid composition and the determination of the post-translational modifications for each of the three charge components C1-C3. With the exception of N-terminal acetylation, the post-translational modifications were partial. The C1 component lacks any phosphorylated sites, a finding in agreement with the analysis of other MBP species. It also had a single methylation at R105 as did the components C2 and C3. The C2 component contains ten phosphorylated sites (S7, S18, S33, S64, S73, T96, S113, S141, S164, and S168), and modified arginine to citrulline residues at R24, and R165. Component C3 contains eight phosphorylated sites (S7, S33, S64, T96, S113, S141, S164, and S168), and citrulline residues at Arginine 41, R24 and R165. Partial deamidation of glutamine residues Q71, Q101 and Q146 were present in addition to asparagine N90 that was found in all three charge components. The glutamine at residue 3 is partially deamidated in isomers C1 and C2, whereas glutamine 74 and asparagine 83 were found not to be deamidated. Comparison of the PTM’s of MBP’s isolated from several vertebrate species reveals marked differences in their phosphate content. Chicken MBP does not share any phosphorylated sites with dogfish MBP; However, it does contain phosphorylated serine and threonine residues in common with mammalian MBP.     

Targeting of anti-citrullinated protein/peptide antibodies in rheumatoid arthritis using peptides mimicking endogenously citrullinated fibrinogen antigens

IntroductionWe have previously identified endogenously citrullinated peptides derived from fibrinogen in rheumatoid arthritis (RA) synovial tissues. In this study, we have investigated the auto-antigenicity of four of those citrullinated peptides, and explored their feasibility to target anti-citrullinated protein/peptide antibodies (ACPA).MethodsThe autoantigenic potential of the fibrinogen peptides was investigated by screening 927 serum samples from the Epidemiological Investigation of RA (EIRA) cohort on a peptide microarray based on the ImmunoCAP ISAC® system. In order to assay for ACPA blocking, two independent pools of purified ACPA were incubated with the respective targeting peptide prior to binding to cyclic citrullinated peptide (CCP)2 using the CCPlus® ELISA kit.ResultsTwo peptides derived from the fibrinogen α chain, Arg573Cit (563-583) and Arg591Cit (580-600), referred to as Cit573 and Cit591, and two peptides from the fibrinogen β chain, Arg72Cit (62-81) and Arg74Cit (62-81) (Cit72 and Cit74), displayed 65 %, 15 %, 35 %, and 53 % of immune reactivity among CCP2-positive RA sera, respectively. In CCP2-negative RA sera, a positive reactivity was detected in 5 % (Cit573), 6 % (Cit591), 8 % (Cit72), and 4 % (Cit74). In the competition assay, Cit573 and Cit591 peptides reduced ACPA binding to CCP2 by a maximum of 84 % and 63 % respectively. An additive effect was observed when these peptides were combined. In contrast, Cit74 and Cit72 were less effective. Cyclization of the peptide structure containing Cit573 significantly increased the blocking efficiency.ConclusionsHere we demonstrate extensive autoantibody reactivity against in vivo citrullinated fibrinogen epitopes, and further show the potential use of these peptides for antagonizing ACPA.     

Cell-mediated immunoreactivity of tumor-bearing mice with myelin basic protein and related peptides

Spleen cells (SC) from tumor-bearing mice and mice immunized with porcine myelin basic protein (MBP) reacted in vitro in E-rosette augmentation assays with MBP and certain of its constituent peptides. Peptides 1-115, 43-169, 64-83, 113-121, and 153-161 reacted significantly with both types of SC, while peptide 1-19 reacted only with SC from MBP-immunized mice. The phenomenon of specific inhibition of peptide reactivity by a moderate excess of a related protein was used to identify peptides as accessible epitopes of that protein. Peptide 113-121 was specifically inhibited by excess MBP when reacted with both types of SC, whereas peptide 64-83 was inhibited by excess MBP only when reacted with SC from MBP-immunized mice. These reactions suggest that the immunizing antigen in tumor-bearing mice is related to MBP but differs in epitopes associated with peptides 1-19 and 64-83.     

An idiotype shared by monoclonal antibodies to different peptides of human myelin basic protein.

A mAb of the IgG1/kappa isotype was raised against human myelin basic protein (MBP) peptide acetyl 1-9. This mAb, termed F23, reacted with human MBP and human MBP peptides acetyl 1-9, 1-14, and 1-44, but not with MBP peptides 10-19, 80-89, or 45-89. According to the guidelines of the molecular recognition theory, a complementary peptide to human MBP peptide 1-9 was synthesized and used to raise murine mAb with anti-Id activity. Two mAb anti-Id, F25F7 and F25C8, both of the IgM/kappa isotype, were selected for further study. These anti-Id reacted with F23, the mAb for which they were selected, and also reacted with another mAb, which was of the IgG1/kappa isotype and was raised to human MBP peptide 80-89. There was no reaction with another control mAb of the IgG1/kappa isotype or murine myeloma IgG1. By immunoblotting techniques, it was demonstrated that the Id on each of the mAbs to MBP peptides was located on the kappa L chain but also could be recognized in nonreduced IgG. The cross-reactive anti-Id suppressed antibody secretion of Id-producing hybridoma cells in an Id-specific manner, and kinetic studies suggest an intracellular mechanism for the suppression. These cross-reactive Id among antibodies to different MBP peptides imply that the same V region genes of kappa L chains are involved in the selection of antibodies to an autoantigen, like MBP, and may play a role in the modulation of immune responses against MBP in certain inflammatory demyelinating diseases.     

Comparison of recombinant human immunodeficiency virus gag precursor and gag/env fusion proteins and a synthetic env peptide as diagnostic reagents

Diagnostic reagents for detection of human immunodeficiency virus (HIV) exposure with improved reliability may be provided by viral encoded proteins produced by recombinant DNA techniques or by synthetic peptides corresponding to appropriate viral epitopes. We have expressed at high levels in E. coli a gag gene segment corresponding to approximately 97% of the p55 gag precursor protein, as well as a novel gag/env fusion protein that contains antigenic determinants in common with gag p24, env gp41, and env gp120. The gag and gag/env proteins were purified from insoluble inclusion bodies by sequential extraction with increasing concentrations of urea. These components were tested for reactivity with antisera to HIV proteins and peptides. We have also chemically synthesized a peptide corresponding to env residues 578-608, representing a portion of env gp41. The final preparation of gag and gag/env proteins in 8 M urea reacted with sheep anti-HTLV-III p24 gag antibodies and acquired immune deficiency syndrome (AIDS) patient sera. The gag/env fusion protein also reacted with rabbit anti-HIV env 500-511 peptide antibody. Both recombinant proteins and the env peptide were suitable as reagents for evaluation of serum samples by enzyme-linked immunosorbent assay (ELISA). Results of ELISA assays utilizing the recombinant viral proteins and synthetic peptide were in good agreement with results obtained using disrupted virus as antigen in ELISA assays and immunoblotting.     

The disulfide-rich region of platelet glycoprotein (GP) IIIa contains hydrophilic peptide sequences that bind anti-GPIIIa autoantibodies from patients with immune thrombocytopenic purpura (ITP)

Immune thrombocytopenic purpura (ITP) is an autoimmune blood disease caused by autoantibody-mediated destruction of blood platelets. Platelet glycoprotein (GP) IIb/IIIa is a common target for antiplatelet autoantibodies. The present studies were undertaken (1). to confirm whether the disulfide rich repeat region of GPIIIa contains target epitopes for antiplatelet antibodies in patients with ITP; (2). to determine whether these antigens were defined by peptide sequences in the absence of post-translational modification; and (3). to correlate observed immunologic reactivity with the recently solved X-ray crystallographic structure of an analogous integrin complex, the vitronectin receptor, alpha(V)beta(3). Recombinant fusion proteins of four GPIIIa extracellular sequences were prepared and purified. Immunoblotting results with purified recombinant peptides showed potent reactivity of 16 of 24 ITP patient serum anti-GPIIb/IIIa antibodies with the fusion protein containing the GPIIIa sequence of residues from 468 to 691. These results are consistent with a report by Kekomaki et al. that a 50 kDa chymotryptic digestion product of GPIIIa isolated from blood platelets contains target epitopes for serum antiplatelet antibodies in 16 of 33 ITP patients. Smaller peptides including residues 446-501 and residues 593-691 each reacted with only 5 of the 24 patient sera; furthermore all but 3 of these interactions were very weak. Visualization of the conformation of the extracellular portion of alpha(V)beta(3) reveals the location of the 222-residue antigenic GPIIIa (beta(3)) peptide 'B' at the immediately extracellular region of the protein that includes a beta-tail domain and several integrin-EGF domains. In summary, predictions of hydrophilicity, surface accessibility and antigenicity and the three dimensional structure of the beta(3) integrin correlate with autoantibody binding to a recombinant GPIIIa peptide 'B' containing residues 468-691.     

丙型肝炎病毒核壳蛋白抗原的化学合成及其在血清学诊断中的应用

选取丙型肝炎病毒(HCV)核壳蛋白区两个可能含有优势抗原表位的19和16氨基酸的肽殴(C-19肽和C-16肽),利用固相多肽合成技术进行了化学合成。经用反相高压液相层析(HPLC)及脉冲液相多肽测序技术检定合成肽产品的纯度及序列后,以C-19和C-16肽作为包被抗原组装成检测HCV血清抗体的ELISA诊断试剂。用所组装的合成肽试剂检测中国药品生物制品检定所提供的HCV标准参比血清,并比较利用该试剂和美国Abbott实验室生产的HCV第二代EIA诊断试剂平行检测109份献血员血清的结果,表明C-19肽能够特异、重复、灵敏地检出HCV血清抗体,可以作为我国第一代HCV诊断试剂的合成肽抗原。     

Targeting of specific domains of diphtheria toxin by site-directed antibodies.

Antibodies highly selective for two functionally distinct regions of diphtheria toxin (DTx) were prepared using synthetic peptide conjugates as immunogens. Three peptides were selected for synthesis: sequence DTx141-157 on fragment A, which contains the putative protein elongation factor (EF-2) ADP-ribosyltransferase site; DTx224-237 on fragment B, selected on the basis of forming a predicted surface loop; and DTx513-526 on fragment B, forming a part of the region containing the putative receptor binding domain. All of the anti-peptide antibodies recognized the corresponding peptide, and also reacted with the toxin, specifically with the fragment containing the sequence against which they were raised, confirming the utility of this approach in generating fragment-specific antibodies. The anti-peptide antibody with the highest binding titre both to the peptide and to the native toxin was the one prepared against the sequence with the highest surface and loop likelihood indices of the three peptides selected. The similarity of the reactivity profiles with peptide and native and denatured toxin is consistent with the prediction that the region selected occurs in a surface loop and that the structure of the peptide is similar to the conformation of this region in the native protein. The epitopes for two of the anti-peptide antibodies were mapped. The results indicated that even though the antisera were raised to peptides containing 14 amino acids (aa) they were directed predominantly against a narrow region within the peptide, consisting of only 5-6 aa residues.(ABSTRACT TRUNCATED AT 250 WORDS)     

Epitopes of peptide 43-88 of guinea pig myelin basic protein: localization with monoclonal antibodies

Two monoclonal antibodies, one raised by immunization with mouse myelin basic protein (MBP) and the second raised by immunization with peptide 68-88 of guinea pig MBP, were compared with respect to specificity. The former antibody (15.32) cross-reacted completely with rat, guinea pig, human, and bovine MBP. It also reacted with peptide 43-88 from each MBP. The latter antibody (22.17) was nonreactive with MBP, but cross-reacted with peptide 43-88 from rat, human, guinea pig, and bovine MBP. When tested with small peptides derived from peptide 43-88, antibody 22.17 reacted with an epitope in the C-terminal region. Antibody 15.32 reacted with an epitope in the N-terminal half of the peptide. The data show that 22.17 reacted with a unique epitope associated only with free peptide, whereas 15.32 recognized an epitope common to both peptide 43-88 and MBP.     

Rat and Mouse Monoclonal Antibodies to Human Myelin Basic Protein

BALB/c mice and Lewis rats were immunized with human myelin basic protein and its N- and C-terminal fragments. Mouse X mouse fusions produced seven monoclonal antibodies, all of the IgG class and directed against the N-terminal fragment. Five of the antibodies seemed to be against the same epitope, between amino acid residues 92 and 118. One antibody bound between residues 45 and 91, and the remaining antibody reacted with both peptides 1-44 and 45-91. Three monoclonal antibodies, all of the IgM class, were obtained by rat X rat hybridization. Two monoclonal antibodies, raised against whole myelin basic protein and the C-terminal fragment, respectively, each bound to peptide 118-178. The remaining antibody, raised against the N-terminal fragment, bound to peptide 45-91. These monoclonal antibodies are of interest for use in clinical radioimmunoassays and for immunohistochemical investigation of the structural relationships of the myelin sheath.