Purification and immunochemical characterization of a 22 kilodalton surface antigen from Schistosoma mansoni

A 22 kDa antigen (Sm22) was purified from schistosomula membrane extracts by immunoaffinity chromatography with monoclonal antibody M.2. Western blotting suggested that the epitope bound by M.2 required a specific conformational folding of the molecule, which was sensitive to reducing agents. Two-dimensional electrophoresis of purified Sm22 demonstrated that the single 22 kDa protein recognized by M.2 on one-dimensional gel analysis was composed of at least two isomorphs. Additional Western blotting showed that Sm2 was one of the major antigens recognized by mouse anti-irradiated cercariae serum, and that this same serum recognized at least one epitope which was not sensitive to reducing agents. The mice vaccinated with irradiated cercariae were shown to be 75% protected from cercarial challenge. Sera from a rabbit immunized with Sm22 contained antibodies which bound to the surface of schistosomula and detected a single protein at 22 kDa by immunoprecipitation or Western blot. The rabbit anti-Sm22 sera also immunoprecipitated a 22 kDa in vitro translation product, indicating that at least one epitope on Sm22 is not dependent on glycosylation.     

Cloning and temperature-dependent expression in Escherichia coli of a Legionella pneumophila gene coding for a genus-common 60-kilodalton antigen.

All Legionella species express a 60-kilodalton (kDa) protein which contains a genus-specific epitope recognized by murine monoclonal antibody GW2X4B8B2H6. A genomic cosmid library of Legionella pneumophila chromosomal DNA was constructed in pHC79 and screened for 60-kDa antigen-expressing clones with the monoclonal antibody. A 3.2-kilobase EcoRI fragment from cosmid 14B11 expressing a 60-kDa protein was subcloned into pUC19 (pSH16), and deletion of a 1.2-kilobase HindIII fragment (pSH16A) generated a 33-kDa truncated polypeptide no longer reactive with the monoclonal antibody. Southern blot analysis of chromosomal DNA from selected Legionella species restricted with EcoRI and probed with the 1.2-kilobase fragment coding for the carboxyl region of the protein revealed DNA homology which was not observed with DNA from Escherichia coli. Maxicell analysis of pSH16 identified a second polypeptide of approximately 15 kDa expressed from a gene (htpA) upstream of the gene coding the 60-kDa protein (htpB). Both proteins were preferentially synthesized by L. pneumophila following heat shock (temperature shift from 25 to 42 degrees C), and under steady-state growth conditions the relative level of 60-kDa protein was unaffected by temperature. In E. coli, expression of a 60-kDa protein from pSH16 also increased following heat shock (25 to 42 degrees C), but under steady-state conditions expression was temperature dependent. Temperature-dependent expression from pSH16 was not observed in an rpoH (htpR) mutant strain of E. coli. The Legionella 60-kDa protein appears to be a heat shock protein which shares cross-reactive epitopes with the GroEL homolog of E. coli. In addition, a region of htpB encoding the 27-kDa carboxyl portion of the protein containing the monoclonal antibody-reactive epitope also contains DNA sequences unique to and conserved within the genus.     

Interprotein disulfide bonding between F and G glycoproteins of human respiratory syncytial virus

The envelope glycoprotein G, of human respiratory virus was purified by immunoaffinity chromatography using a monoclonal antibody reacting with G glycoprotein. The purified material was analyzed for its protein patterns and by western blot for its reactivity with specific monoclonal antibodies. In addition to the G specific proteins at 90 and 55 kilodalton (kDa) range, high molecular weight species were coeluted with G protein. Three high molecular weight species were noticed: one (140 kDa) reacting with fusion protein (F) monoclonal antibody and two other species (230 and 195 kDa) reacting with both fusion protein and G protein monoclonal antibodies. The protein reacting only with F monoclonal antibody consists of fusion protein dimer. Western blot and two dimensional gel electrophoretic analysis revealed that each of the other two complexes is composed of two moles of F protein and one mole of G protein. These two complexes differ in their molecular sizes depending on whether G is in the form of 90 or 55 kDa. Upon heat denaturation, fusion protein monomer (70 kDa) is released from the complex, leaving the two complexes, consisting of one mole of F protein and one mole of G protein (160 and 125 kDa species respectively). Disulfide-reducing agents are required to break the monomers of F and G complexes. These results provide a direct evidence for the presence of envelope glycoprotein complexes linked by interprotein disulfide bonding. This may have implications on the structural and functional properties of envelope glycoproteins.     

Immunodominant B-cell epitope in the Mce1A mammalian cell entry protein of Mycobacterium tuberculosis cross-reacting with glutathione S-transferase

The TB1-5 76C monoclonal antibody raised against a synthetic 60-mer peptide in the N-terminal part of the Mce1A mammalian cell entry protein of Mycobacterium tuberculosis has previously been shown to react with a linear epitope in the KRRITPKD region, residues 131-138 in Mce1A, and to cross-react with Mce1F. Six additional monoclonal antibodies raised against the same peptide were also shown to cross-react with Mce1F. Four of them reacted with a linear epitope in the same area, indicating that this area is immunodominant but showed distinct differrences in fine specificity. Two monoclonal antibodies did not react with synthetic peptides from this region on the solid phase in enzyme-linked immunosorbent assay, indicating greater influence of conformation on reactivity. None of the monoclonal antibodies reacted with 14-mer synthetic peptides from the corresponding area in Mce2A, Mce3A, Mce4A, M. avium, M. smegmatis or M. leprae. The reaction pattern of the monoclonal antibodies was analysed in relation to our model of the Mce1A molecule (AK Das et al. Biochem Biophys Res Commun 2003;302:442-7).The epitope is located on the surface of Mce1A, at the distal beta-strand-loop region in the beta-domain supporting its potential role in promoting uptake of M. tuberculosis in host cells. Monoclonal antibody TB1-5 19C cross-reacted with glutathione S-transferase of Schistosoma japonicum containing a PKE triplet. Monoclonal antibody TB1-5 76C gave a major band at about 44 kDa in Western blotting of M. tuberculosis sonicate, whereas polyclonal rabbit anti-Mce1A peptide antibodies reacting with the extended TTPKNPTKRRITPKDVI area of Mce1A showed a distinct band above the 160 kDa molecular mass standard.     

Anti-idiotypic antisera raised against monoclonal antibody specific for a p24 gag region epitope detects a common interspecies idiotype associated with anti-HIV responses.

One potential strategy for the control of human immunodeficiency virus (HIV) infection is immune network manipulation using anti-idiotypic antibodies: this study was undertaken to demonstrate experimentally the potential of such an approach which, in a more highly evolved form, could be used for the treatment of the acquired immune deficiency virus (AIDS) and related disorders. Anti-idiotypic antibodies were generated in rabbits against a murine monoclonal antibody identifying an epitope on the p24 gag core protein of HIV. After extensive absorption on affinity columns to remove isotype- and allotype-specific antibodies, the purified anti-idiotypic antibody preparation was shown to have specific complementarity with the immunizing mouse monoclonal antibody. This anti-idiotypic antibody was also shown to recognize a common idiotype associated with HIV-specific antibodies from both humans and chimpanzees infected with the AIDS virus. In addition a group of rats immunized with the anti-Id responded with significant antibody titers to recombinant derived p24 gag. These data indicate that at least a subpopulation of these polyclonal anti-Id antibodies structurally mimics an HIV gag region epitope and suggest that immunoregulation by anti-idiotypic antibodies may have therapeutic utility for the AIDS epidemic.     

Characterization of Trichinella spiralis antigens sharing an immunodominant, carbohydrate-associated determinant distinct from phosphorylcholine.

The biochemical and immunochemical characteristics of T. spiralis molecules (group II antigens) sharing an immunodominant epitope were examined. Six major proteins, ranging from 43-68 kDa, and from pI 5.0-6.3, express the determinant. Together, they account for at least 3% by weight of the total protein in L1 larval homogenate. The antigens are glycosylated. Following periodate oxidation, they reacted with biotin aminocaproyl hydrazide, and treatment with trifluoromethanesulfonic acid decreased their Mr. Deglycosylated group II antigens lost immunoreactivity with a monoclonal antibody specific for the determinant, and oligosaccharides released by treatment with mild base blocked binding of the monoclonal antibody to native antigens. The determinant on one of the group II antigens (43 kDa) was removed by N-glycanase. Neither phosphorylcholine nor antibody to phosphorylcholine interfered with monoclonal antibody binding to native group II antigens. Together, these results suggest that the immunodominant group II antigen epitope is associated with N- and O-linked oligosaccharides, and that it is not phosphorylcholine.     

Triton X-114 phase fractionation of an integral membrane surface protein mediating monoclonal antibody killing of Mycoplasma hyorhinis

A previously defined immunoglobulin M(kappa) monoclonal antibody reacting with a surface epitope of Mycoplasma hyorhinis is shown in this report to mediate specific, complement-dependent mycoplasmacidal activity. Immunoblot analysis of mycoplasma components and their tryptic cleavage products showed that the epitope recognized was present on a protein with an apparent molecular weight of 23,000 (p23) and on a limit tryptic fragment of this protein with an apparent molecular weight of 18,000 (p18). Both p23 and p18 are shown by Triton X-114 phase fractionation to partition efficiently into the hydrophobic detergent phase. Other antigens bearing epitopes not expressed at the cell surface were present among the numerous hydrophilic proteins found in the aqueous phase. The external orientation and membrane association of the p23 antigen were further established by demonstrating that trypsin treatment of intact mycoplasmas generated the antigenic p18 fragment, which remained tightly associated with the organism. These results localize an epitope responsible for antibody-mediated mycoplasma killing onto a specific, surface-exposed region of an integral membrane protein of this organism. Since the monoclonal antibody used in this study does not bind to the surface of all strains of M. hyorhinis, the epitope identified also defines a structural marker of antigenic surface variation within this species, a feature previously observed during serological classification of the organism. Analysis of the antigenic and structural features of the p23 surface antigen may therefore be useful in establishing mechanisms of surface antigen variation among integral membrane proteins of mycoplasmas that could dictate important antigenic characteristics recognized during chronic disease caused by these agents.     

Identification of the immunoreactive peptide sequence for AgSK1, an adenocarcinoma-restricted antigen

SK1, a human immunoglobulin M (IgM) monoclonal antibody was derived from regional nodal lymphocytes of a Dukes B colon carcinoma patient. The antigen recognized by the human monoclonal antibody (HuMab) SK1, termed AgSK1, was shown to be a two-chain glycoprotein with an apparent molecular weight range of 42-46 kDa and preferentially expressed by human adenocarcinomas, particularly human gastrointestinal malignancies. To identify the gene encoding the AgSK1 antigenic epitope, a cDNA expression library constructed in lambda gt22A using mRNA from the colon carcinoma cell line HT29 was screened and one of the isolated clones encoding a 1.5-kb cDNA, which showed strong immunoreactivity with HuMab SK1, was selected for further analysis. This clone consisted of an amino terminal open reading frame of 54 amino acids and the carboxyl terminal 20 amino acids of this protein coding region contained the antigenic epitope recognized by HuMab SK1.     

The application of epitope mapping in the development of a new serological test for systemic candidosis.

A new serological test for systemic candidosis was developed by raising a rabbit antiserum probe against a specific epitope on Candida albicans, hsp 90. A major fragment at the carboxy terminal end of this immunodominant candidal antigen was epitope mapped by Geysen's method. An epitope, recognised by all infected patients with antibody to the 47 kDa antigen, was synthesized and conjugated to keyhole limpet haemocyanin. A rabbit was successfully immunized against this synthesized peptide epitope and this antiserum was compared, in a dot-immunobinding assay, with unfractionated hyperimmune rabbit antiserum to C. albicans and an affinity-purified rabbit antiserum to the 47 kDa antigen. The epitope-specific antibody probe was more sensitive than the hyperimmune candidal antiserum but less sensitive than the affinity-purified antibody against the 47 kDa antigen, which recognised multiple epitopes. This probe is technically easy to prepare in large amounts and gives no false positives.     

Definition of the epitope recognized by the Plasmodium falciparum-reactive human monoclonal antibody 33G2.

The human monoclonal antibody 33G2 has earlier been shown to inhibit merozoite reinvasion of red blood cells in Plasmodium falciparum cultures in vitro and to inhibit cytoadherence of infected red blood cells to melanoma cells in vitro. 33G2 cross-reacts with a family of P. falciparum antigens, Ag332, Pf11.1 and Pf155/RESA, sharing a common feature of repeated sequences consisting of regularly spaced pairs of glutamic acid. Peptides corresponding to residues 2-19 of the known amino acid sequence of Ag332 have been shown earlier to have the highest inhibitory capacity of antibody binding to infected red blood cells. Using the PEPSCAN method, overlapping hepta-, hexa-, penta- and tetrapeptides corresponding to residues 1-19 of the known sequence of Ag332 were synthesized. Antibody fine specificity was examined by synthesizing an octapeptide (residues 1-8) and all possible single amino acid substitutions. The monoclonal antibody was shown to react with a linear 5-amino acid-long sequence corresponding to Ag332 residues 3-7: VTEEI. These amino acids were irreplaceable or only partially replaceable in the replacement set analysis. Furthermore, epitope analogs corresponding to sequences contained within the Pf11.1 repeats and overlapping heptapeptides corresponding to Pf155/RESA repeats were synthesized. Reactivity to epitope analogs and Pf155/RESA peptides provided information which may explain antibody cross-reactivity. The defined epitope of this monoclonal antibody is of interest as a potential B cell epitope for the development of a malaria subunit vaccine.     

Epitope analysis of peanut allergen Ara h1 with human monoclonal IgM antibody clone #86

A human-mouse hybridoma clone #86 secreting IgM-class human monoclonal antibody to peanut allergen protein Ara h1 was newly established. To detect an antibody-binding sequence (epitope) on Ara h1, the monoclonal antibody #86 was reacted with multi-pin apparatus with a series of peptides synthesized from the amino acid sequence of Ara h1. The antibody #86 was found to bind to a peptide with amino acid sequence of 481EEEEDEDEEEEGSNREVRRY500. Further analysis with shorter pin-peptides with ten amino acid-long showed that the peptides reacted with the antibody #86 contained a sequence of 485DEDEEEE491. This might be an essential linear sequence of this epitope. When the 485DED487 part of the peptide was replaced by alanine, decreased binding of antibody #86 was observed.     

The plasma cell associated antigen detectable by antibody VS38 is the p63 rough endoplasmic reticulum protein.

AIMS: To characterise the 64 kDa intracellular antigen present on normal and neoplastic plasma cells detected by monoclonal antibody VS38 and by another antibody, MC186, of similar reactivity. METHODS: The VS38 monoclonal antibody was used to screen a bacterially expressed peripheral blood cDNA library, and the immunocytochemical staining of the two antibodies was compared with those raised specifically to the protein identified as the VS38 antigen. RESULTS: A partial cDNA encoding the VS38 antigen was cloned and shown to be identical to the human p63 gene. p63 is a non-glycated, reversibly palmitoylated type II transmembrane protein which is found in rough endoplasmic reticulum. Antibody MC186 also recognised this protein and both VS38 and MC186 together with two antibodies raised to p63 gave identical immunostaining patterns. CONCLUSIONS: The VS38 antigen was identified as the rough endoplasmic reticulum protein p63. While it is not exclusively expressed on plasma cells, the presence of p63 distinguishes plasma cells from other lymphoid cells because of their high secretory activity.     

Characterization of gp195 processed products purified from Plasmodium falciparum culture supernates

Schizonts of the malaria parasite Plasmodium falciparum synthesize a 195 kDa surface glycoprotein (gp195) that is processed into several smaller products including one of 83 kDa, which, in the case of the Camp strain, is sequentially processed into 73 and 67 kDa products. gp195 and its processing intermediates larger than 83 kDa were not precipitated from culture supernates, but the 83 and 73 kDa products were precipitated by three monoclonal antibodies (McAbs). The 83 and 73 kDa products were affinity purified from culture supernates by adsorbing to McAb 7B2 coupled to Affigel 10 and eluting either with 0.2 N acetic acid, pH 2.8, or with 3 M potassium isothiocyanate (KSCN). The epitope recognized by McAb 7B2 was denatured by acid elution but could be regenerated by treating with 8 M urea followed by dialysis. The implications of renaturing antigens to regenerate epitopes should be considered in studies on the purification, function and immunogenicity of malaria antigens.     

Immune response to a major epitope of p24 during infection with human immunodeficiency virus type 1 and implications for diagnosis and prognosis.

A sequential inhibition enzyme-linked immunoassay (SIEIA) using a peroxidase-conjugated monoclonal antibody reacting to the sequence AAEWDRVHP of p24HIV-1 (amino acids 209 to 217 of p55) was developed in order to detect and determine the titer of antibody to this epitope in various populations of human immunodeficiency virus type 1 (HIV-1)-positive patients. There was a good correlation between SIEIA and a commercially available competition assay that uses recombinant p24 protein and polyclonal antibody to HIV-1 antigen, demonstrating the importance of the described epitope. Analysis of sera from French patients showed a decline of antibody to the AAEWDRVHP sequence associated with the progression of AIDS. No decrease was observed with serum samples from African patients. An immune response to the epitope was detected by SIEIA early in the course of seroconversion. Although our SIEIA uses a single p24 epitope, these data are in accordance with previously published studies in which antibodies to the whole p24 were analyzed. Sera reacting to p24 only (indeterminate profiles by Western blot [immunoblot]) did not bind to AAEWDRVHP. This epitope, which is conserved between HIV-1 and HIV-2/simian immunodeficiency virus, appears to be a major antigenic domain of p24. The area containing the sequence AAEWDRVHP and the corresponding monoclonal antibody may serve as a convenient alternative to whole purified p24 and polyclonal antibody in diagnostic and prognostic assays.     

A recombinant surface protein of Babesia bovis elicits bovine antibodies that react with live merozoites

Ten monoclonal antibodies (MoAbs) were generated against five surface-exposed proteins (16 kDa, 42 kDa, 44 kDa, 60 kDa, 225 kDa) on merozoites of Babesia bovis. A genomic library constructed in the lambda gt11 expression vector was screened with MoAbs in a plaque immunoassay for identification of clones expressing recombinant surface proteins. Two recombinant clones were identified (lambda Bo44-15 and lambda Bo44-16) that encoded a protein recognized by a MoAb specific for an epitope on the native 44-kDa surface protein. Southern blot analysis using radiolabeled Bo44-15 DNA (1.25 kb) against merozoite DNA and bovine leukocyte DNA confirmed the parasite-specificity of the cloned insert and revealed multiple bands of hybridization with merozoite DNA. Western blot analyses of lambda Bo44-15 lysogen preparations demonstrated that recombinant protein production in this clone was IPTG-induced and that the recombinant molecule was a beta-galactosidase fusion protein. Additionally, recombinant 44-kDa protein, purified by immunoaffinity chromatography, reacted with specific MoAb in Western blot assay indicating that the integrity of the epitope was retained during purification. Immune sera from calves immunized with purified recombinant Bo44-15 protein immunoprecipitated metabolically radiolabeled merozoite protein of 44 kDa indicating that antibody induced by recombinant Bo44-15 protein recognized native 44-kDa protein. Also, these sera reacted with the surface of live merozoites as evidenced by indirect immunofluorescence assay. Serum antibody titers determined by this assay had a wide range.     

Phenotypic diversity in the alpha C protein of group B streptococci.

Group B streptococci (GBS) is the leading cause of neonatal sepsis and meningitis. C proteins are an immunologically important group of surface-associated antigens in GBS that remain incompletely characterized. Two C proteins have been designated alpha and beta on the basis of protease susceptibility. We recently used a monoclonal antibody to describe a protective epitope of the GBS alpha (or trypsin-resistant) C protein in the prototype Ia/c GBS strain. In the present study, we examined 51 GBS isolates for expression of C-protein alpha and beta antigens. The alpha antigen, as detected with monoclonal antibody in sodium dodecyl sulfate (SDS) extracts, appears as a heterogeneous series of proteins spaced 8 kDa apart on SDS-polyacrylamide gel electrophoresis, but has a maximum molecular mass that varies among strains from 62.5 to 167 kDa. By immunoblotting with human immunoglobulin A, polyclonal antiserum, or monoclonal antibody, the beta antigen, in contrast, appears as a single protein of molecular mass between 124 and 134 kDa. The amount of alpha antigen expressed by each strain was quantified by enzyme immunoassay inhibition and was found to vary markedly from strain to strain. The susceptibility of strains of GBS to opsonization and killing by human polymorphonuclear leukocytes in the presence of either complement alone or complement with alpha-specific monoclonal antibody was examined. Strains expressing the alpha antigen were less readily killed in the absence of specific antibody than were alpha-negative strains. Killing in the presence of alpha-specific monoclonal antibody was found to correlate directly with the maximum molecular mass of the alpha antigen and with the quantity of antigen on the bacterial cell surface. Isolates of GBS that express the alpha C protein vary widely in the quantity and molecular mass of the alpha antigen produced, and this heterogeneity appears to have biologic importance.     

Characterization of a monoclonal antibody panel shows that the myotonic dystrophy protein kinase, DMPK, is expressed almost exclusively in muscle and heart

Myotonic dystrophy (DM) is a multisystemic disorder caused by an inherited CTG repeat expansion which affects three genes encoding the DM protein kinase (DMPK), a homeobox protein Six5 and a protein containing WD repeats. Using a panel of 16 monoclonal antibodies against several different DMPK epitopes we detected DMPK, as a single protein of approximately 80 kDa, only in skeletal muscle, cardiac muscle and, to a lesser extent, smooth muscle. Many earlier reports of DMPK with different sizes and tissue distributions appear to be due to antibody cross-reactions with more abundant proteins. One such antibody, MANDM1, was used to isolate two related protein kinases, MRCK alpha and beta, from a human brain cDNA library and the shared epitope was located at the catalytic site of DMPK using a phage-displayed random peptide library. The peptide library also identified an epitope shared between DMPK and a 55 kDa muscle-specific protein. The results suggest that effects of the repeat expansion on the DMPK gene may be responsible for muscle and heart features of DM, whereas clinical changes in other tissues may be due to effects on the other two genes.     

Distribution in tissue sections of the human groEL stress-protein homologue.

A monoclonal antibody (ML30) raised against the 65 kDa heat-shock protein of mycobacteria showed widespread staining of sections from standard paraffin-embedded human tissues. The staining had a granular pattern and was particularly marked in cells with abundant mitochondria. Increased staining was observed in the synovial lining, histocytes and in the endothelium of reactive and rheumatoid synovium; it was also increased in the reactive lung alveolar lining. It is suggested that the antibody identifies an epitope in mitochondria of a protein homologous with the groEL heat-shock protein of bacteria.     

A rhoptry antigen of Plasmodium falciparum contains conserved and variable epitopes recognized by inhibitory monoclonal antibodies

Four monoclonal antibodies produced against Plasmodium falciparum recognize an antigen in merozoites that is localized in rhoptries, as judged by a punctate, double dot fluorescence pattern. All four antibodies bound to the same affinity purified antigen in a two site immunoradiometric assay. Immunoprecipitation of antigen by monoclonal antibody followed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis yielded protein bands of 80, 66 and 42 kDa. Western blotting gave bands of 80 and 66 kDa only with three of the antibodies: the fourth did not blot. Based on protease inhibitor data the 66 kDa band is considered to be a cleavage product of the 80 kDa band, but the 42 kDa band does not appear to derive from the latter and may be a coprecipitation product. This group of antigens labels with both [35S]methionine and [3H]histidine. Two of the monoclonal antibodies inhibited merozoite invasion of erythrocytes. One of these inhibitors recognizes a variable epitope, whereas the second recognizes a highly conserved epitope present in all 106 primary isolates of P. falciparum tested from Brazil, Thailand and Papua New Guinea.