Characterization of Chlamydia trachomatis antigens with monoclonal and polyclonal antibodies

1988 ◽  
Vol 34 (2) ◽  
pp. 141-147 ◽  
Author(s):  
Ian W. Maclean ◽  
Rosanna W. Peeling ◽  
Robert C. Brunham
Keyword(s):  
Chlamydia Trachomatis ◽  
Polyclonal Antibodies ◽  
Antigenic Specificity ◽  
Immunoblot Assay ◽  
Neutralizing Activity ◽  
Molecular Masses ◽  
Monospecific Antisera ◽  
Biologic Function

We used monoclonal antibodies (MAbs) to examine the antigenic specificity and biologic function of several Chlamydia trachomatis antigens. Thirteen distinct MAbs to eight C. trachomatis antigens were produced. Six MAbs reacted with unique epitopes on the major outer membrane protein (MOMP) and two of these had neutralizing activity. MAbs were produced to each of the chlamydial antigens with molecular masses of 10, 29, 32, 57, 60, 70, and 75 kilodaltons (kDa). These MAbs showed species and genus specificity in an immunoblot assay. None of the MAbs had neutralizing activity. The epitopes recognized on MOMP, 29-, and 10-kDa (presumably lipopolysaccharide) antigens were surface exposed. MAbs to the 75-kDa, 57-kDa, and MOMP antigens were used for immunoaffinity purification of these antigens to produce monospecific antisera in mice. With polyclonal sera, we found that the 75-kDa antigen was also immunoaccessible and that antibody to MOMP and 75-kDa antigens neutralized C. trachomatis infectivity. We conclude that, in addition to MOMP and lipopolysaccharide, antigens with molecular masses of 75 and 29 kDa are surface exposed. Antibodies to MOMP and 75-kDa antigens can neutralize the organism in vitro.

Identification and characterization of a novel Neospora caninum immune mapped protein 1

Parasitology ◽  
2012 ◽  
Vol 139 (8) ◽  
pp. 998-1004 ◽  
Author(s):  
X. CUI ◽  
T. LEI ◽  
D. Y. YANG ◽  
P. HAO ◽  
Q. LIU
Keyword(s):  
Neospora Caninum ◽  
Polyclonal Antibodies ◽  
Functional Protein ◽  
Reading Frame ◽  
Protein Motifs ◽  
Apicomplexan Parasites ◽  
Potential Vaccine ◽  
Palmitoylation Site

SUMMARYImmune mapped protein 1 (IMP1) is a newly discovered protein in Eimeria maxima. It is recognized as a potential vaccine candidate against E. maxima and a highly conserved protein in apicomplexan parasites. Although the Neospora caninum IMP1 (NcIMP1) orthologue of E. maxima IMP1 was predicted in the N. caninum genome, it was still not identified and characterized. In this study, cDNA sequence encoding NcIMP1 was cloned by RT-PCR from RNA isolated from Nc1 tachyzoites. NcIMP1 was encoded by an open reading frame of 1182 bp, which encoded a protein of 393 amino acids with a predicted molecular weight of 42·9 kDa. Sequence analysis showed that there was neither a signal peptide nor a transmembrane region present in the NcIMP1 amino acid sequence. However, several kinds of functional protein motifs, including an N-myristoylation site and a palmitoylation site were predicted. Recombinant NcIMP1 (rNcIMP1) was expressed in Escherichia coli and then purified rNcIMP1 was used to prepare specific antisera in mice. Mouse polyclonal antibodies raised against the rNcIMP1 recognized an approximate 43 kDa native IMP1 protein. Immunofluorescence analysis showed that NcIMP1 was localized on the membrane of N. caninum tachyzoites. The N-myristoylation site and the palmitoylation site were found to contribute to the localization of NcIMP1. Furthermore, the rNcIMP1-specific antibodies could inhibit cell invasion by N. caninum tachyzoites in vitro. All the results indicate that NcIMP1 is likely to be a membrane protein of N. caninum and may be involved in parasite invasion.

scholarly journals Synthesis and Characterization of Mercapturic Acid (N-acetyl-L-cysteine)-Aflatoxin B1 Adduct and Its Quantitation in Rat Urine by an Enzyme Immunoassay

2009 ◽  
Vol 92 (2) ◽  
pp. 487-495 ◽  
Author(s):  
Sujatha Nayak ◽  
Penmatsa Tanuja ◽  
Rao Beedu Sashidhar
Keyword(s):  
Enzyme Immunoassay ◽  
Aflatoxin B1 ◽  
Polyclonal Antibodies ◽  
Mercapturic Acid ◽  
Rat Urine ◽  
In Vitro Synthesis ◽  
Layer Chromatography ◽  
Serum Albumen

Abstract A simple and sensitive indirect noncompetitive enzyme immunoassay to quantitate mercapturic acid-aflatoxin B1 (AFB1) adduct in rat urine is reported. A novel procedure was developed for in vitro synthesis of an immunogen, bovine serum albumen-glutathione-aflatoxin B1 (BSA-GSH-AFB1) using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride. Sulphydryl group's analysis confirmed the conjugation of SH groups to AFB1. Thin-layer chromatography and spectral analysis (absorption, fluorescence, and Fourier transform infrared) of the conjugates further confirmed the formation of the adducts. Polyclonal antibodies specific to mercapturic acid-AFB1 adduct were produced against BSA-GSH-AFB1. The assay was found to be linear in the range of 100 pg100 ng of the analyte (y ab x). A 50 displacement of BSA-GSH-AFB1 antibodies was achieved at an inhibitory concentration (IC50)of 11.9 ng GSH-AFB1 (r2 0.98) and 1.22 ng N-acetyl-L-cysteine (NAC)-AFB1 (r2 0.98). Spiking 5 g/mL of reference standard to the control rat urine showed a recovery of 98 2. The immunoassay was validated in a rodent model exposed to a single oral dose of 1 mg/kg body mass of pure AFB1. The excretion of NAC-AFB1 adduct was quantitated at the end of 24 h. The concentration of the NAC-AFB1 adduct excreted in urine as determined by the immunoassay was found to be in the range of 3.225.97 g/mg creatinine. The present method may find wide application as a biochemical tool in molecular epidemiological and intervention studies with respect to human exposure to dietary aflatoxins.

scholarly journals Detection and characterization of proteins encoded by the second ORF of the M2 gene of pneumoviruses

1999 ◽  
Vol 80 (8) ◽  
pp. 2011-2016 ◽  
Author(s):  
G. Ahmadian ◽  
P. Chambers ◽  
A. J. Easton
Keyword(s):  
Inclusion Bodies ◽  
Virus Protein ◽  
Sequence Identity ◽  
Avian Pneumovirus ◽  
Orf2 Protein ◽  
Monospecific Antisera ◽  
Gst Fusion Proteins

The nucleotide sequence of the M2 gene of pneumonia virus of mice (PVM) was determined. The sequence showed that the gene encoded a protein of 176 amino acids with a predicted molecular mass of 20165 Da from a major ORF, which is smaller than the equivalent proteins encoded by human, bovine and ovine respiratory syncytial (RS) viruses. The PVM M2 protein is conserved, having 41% similarity to the equivalent human RS virus protein. In common with the M2 genes of the RS viruses and avian pneumovirus (APV), the PVM mRNA also contained a second ORF (ORF2) that partially overlaps the first ORF and which is capable of encoding a 98 residue polypeptide. No significant sequence identity could be detected between the putative M2 ORF2 proteins of PVM, APV and the RS viruses. The expression of the M2 ORF2 proteins of the pneumoviruses was investigated by using monospecific antisera raised against GST fusion proteins. Western blot analysis demonstrated the presence of polypeptides encoded by M2 ORF2 of PVM and RS virus corresponding with those predicted by in vitro translation studies, but this was not the case for APV. The PVM polypeptide was present as three distinct products in vivo. The PVM and RS virus polypeptides were also detected in cells by immunofluorescence, which showed that both were present in the cytoplasm with a degree of localization in inclusion bodies. No APV M2 ORF2 protein could be detected in vivo. The RS virus M2 ORF2 polypeptide was shown to accumulate during infection and the potential implications of this are discussed.

scholarly journals Characterization of INS-15, A Metalloprotease Potentially Involved in the Invasion of Cryptosporidium parvum

2019 ◽  
Vol 7 (10) ◽  
pp. 452 ◽  
Author(s):  
Xu ◽  
Guo ◽  
Li ◽  
Zhang ◽  
Wu ◽  
...  
Keyword(s):  
Cryptosporidium Parvum ◽  
Polyclonal Antibodies ◽  
Protozoan Parasite ◽  
Partial Reduction ◽  
E Coli ◽  
Severe Diarrhea ◽  
Domain I

Cryptosporidium parvum is a protozoan parasite that can cause moderate-to-severe diarrhea. Insulinase-like proteases (INS) are one of the largest protein families within the small proteome of the pathogen. However, their roles in C. parvum biology remain un-elucidated. In this study, a member of the protein family, INS-15 of C. parvum encoded by cgd3_4260, was cloned, expressed and characterized to understand its function. INS-15 and its domain I were expressed in Escherichia coli and polyclonal antibodies against the domain I and one specific polypeptide were prepared in rabbits. The role of INS-15 protein in the C. parvum invasion was preliminarily studied. Recombinant INS-15 protein and its domain I were successfully expressed in E. coli, together with various degraded products. The cgd3_4260 gene had a peak expression at 2 h of in vitro C. parvum culture, while the INS-15 protein was expressed in the mid-anterior region of sporozoites and the area of merozoites opposite to the nucleus. Anti-INS-15 domain I antibodies reduced the invasion of C. parvum sporozoites by over 40%. The anterior location of INS-15 in invasion stages and partial reduction of in vitro growth indicate that INS-15 plays some roles in the invasion or early development of C. parvum.

scholarly journals Characterization of prenylated protein methyltransferase in Leishmania

1999 ◽  
Vol 342 (3) ◽  
pp. 513-518 ◽  
Author(s):  
Marie-Pierre HASNE ◽  
Françoise LAWRENCE
Keyword(s):  
Protein Kinase C ◽  
Leishmania Donovani ◽  
Post Translational Modification ◽  
Methyltransferase Activity ◽  
Kinase C ◽  
Protein Methyltransferase ◽  
Molecular Masses ◽  
Endogenous Substrates

Prenylated protein methyltransferase, an enzyme involved in the post-translational modification of many signalling proteins, has been characterized in a parasitic flagellated protozoan, Leishmania donovani. The activity of this enzyme was monitored by the methylation of an artificial substrate, an S-prenylated cysteine analogue, with S-adenosyl-L-[methyl-3H]methionine as methyl donor. More than 85% of the methyltransferase activity was associated with membranes. The enzyme methylates N-acetyl-S-trans,trans-farnesyl-L-cysteine and N-acetyl-S-all-trans-geranylgeranyl-L-cysteine, but N-acetyl-S-trans,trans-geranyl-L-cysteine only very weakly. In contrast with the enzyme from mammals, the leishmanial enzyme had a greater affinity for the farnesylated substrate than for the geranylgeranylated one. Activity in vitro was not modulated by cAMP, protein kinase C activator or guanosine 5′-[γ-thio]triphosphate. An analysis of the endogenous substrates showed that the carboxymethylated proteins were also isoprenylated. The main carboxymethylated proteins have molecular masses of 95, 68, 55, 46, 34-23, 18 and less than 14 kDa. Treatment of cells with N-acetyl-S-trans,trans-farnesyl-L-cysteine decreased the carboxymethylation level, whereas treatment with guanosine 5′-[γ-thio]triphosphate increased the carboxymethylation of various proteins, particularly those of molecular masses 30-20 kDa.

Role of phosphoprotein phosphatases in the corpus luteum: I Identification and characterisation of serine/threonine phosphoprotein phosphatases in isolated rat luteal cells

1996 ◽  
Vol 150 (2) ◽  
pp. 205-211 ◽  
Author(s):  
S L Ford ◽  
D R E Abayasekara ◽  
S J Persaud ◽  
P M Jones
Keyword(s):  
Polyclonal Antibodies ◽  
Luteal Cell ◽  
Luteal Cells ◽  
Cell Functions ◽  
Phosphoprotein Phosphatases ◽  
Sds Page ◽  
Molecular Masses ◽  
Dose Dependent Decrease

Abstract Although the role of protein kinases and phosphorylation in steroidogenesis has received much attention, very little is known about the activities of phosphoprotein phosphatases (PP) and dephosphorylation in steroidogenic tissues. The aims of the present study were therefore to identify which of those serine/threonine PPs more commonly involved in intracellular signalling are expressed in rat luteal cells; to quantify, in vitro, the effects of inhibitors on PP activity extracted from purified rat luteal cells; and to measure the effects of PP inhibitors on the phosphorylation of endogenous luteal cell proteins. Polyclonal antibodies raised against the catalytic subunits of PP types 1 and 2A, and a monoclonal antibody raised against the Ca2+-binding subunit of PP2B, were used to identify immunoreactive proteins that migrated on SDS-PAGE with approximate molecular masses of 37, 34 and 16 kDa, corresponding well with the reported molecular mass of PP1, PP2A and PP2B respectively. Five selective inhibitors of PP1/PP2A: okadaic acid, calyculin A, cantharidin, tautomycin and microcystin-RR, each caused a dose-dependent decrease in the activity of PPs in luteal cell homogenates, and also enhanced 32P incorporation into numerous luteal cell proteins; most notably, proteins with approximate molecular masses of 20 and 22 kDa. The results of this study suggest that PPs may play an important role in the regulation of rat luteal cell functions. Journal of Endocrinology (1996) 150, 205–211

scholarly journals Preliminary Characterization of Two Small Insulinase-Like Proteases in Cryptosporidium parvum

2021 ◽  
Vol 12 ◽  
Author(s):  
Rui Xu ◽  
Cong Lai ◽  
Fuxian Yang ◽  
Qiang Zhang ◽  
Na Li ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cryptosporidium Parvum ◽  
Polyclonal Antibodies ◽  
Apical Region ◽  
Transcription Level ◽  
Severe Diarrhea ◽  
Dense Granules ◽  
Genes Encoding

Cryptosporidium parvum is a major cause of moderate-to-severe diarrhea in humans and animals. Its compact genome contains 22 genes encoding divergent insulinase-like proteases (INS), which are poorly characterized. In this study, two small members of this family, INS-21 encoded by cgd7_2080 and INS-23 encoded by cgd5_3400, were cloned, expressed, and characterized to understand their functions. Recombinant INS-21 and INS-23 were expressed in Escherichia coli and polyclonal antibodies against these two proteins were prepared. The cgd7_2080 gene had a high transcription level during 0–2 h of in vitro C. parvum culture, while cgd5_3400 was highly transcribed at 0–6 h. INS-21 was mostly located in the apical region of sporozoites and merozoites whereas INS-23 was found as spots in sporozoites and merozoites. The immunoelectron microscopy confirmed the expression of INS-21 in the apical region of sporozoites while INS-23 appeared to be expressed in the dense granules of sporozoites. The neutralization efficiency was approximately 35%, when the cultures were treated with anti-INS23 antibodies. These results suggest that INS-21 and INS-23 are expressed in different organelles and might have different functions in the development of C. parvum.

Characterization of a recombinant porcine follistatin in a heat shock expression system

2002 ◽  
Vol 82 (3) ◽  
pp. 295-304
Author(s):  
C. R. Christensen ◽  
J. Kowalski ◽  
P. J. Chedrese ◽  
V. Misra ◽  
B. Laarveld ◽  
...  
Keyword(s):  
Heat Shock ◽  
Ovarian Function ◽  
Expression System ◽  
Porcine Granulosa Cells ◽  
Molecular Masses ◽  
Estradiol 17Β

The role of follistatin in ovarian function has yet to be fully elucidated; it is present in low concentration in vivo and it is difficult to obtain suitable quantities of follistatin for characterization. We have cloned porcine follistatin cDNA in an expression system that uses the heat shock protein promoter BoHSP70. Recombinant follistatin with apparent molecular masses of 39, 46, 48, 50 kDa was expressed and secreted into culture medium at concentrations of 400–500 μg × 20 mL-1 × 150 cm-2 flask (4 × 107 cells). In ligand blots, the recombinant follistatin was shown to be immunologically similar to native follistatin and to bind to recombinant activin A. Porcine granulosa cells dissected from 1–3 mm follicles from immature gilts were cultured with recombinant follistatin or with a follistatin monoclonal antibody to examine the activity of the recombinant follistatin. At a physiologically relevant concentration of 1 μg mL-1 the recombinant porcine follistatin suppressed the accumulation of estradiol-17β (P < 0.05). There was a trend for estradiol-17β accumulation in the presence of 10 μg mL-1 of monoclonal anti-follistatin antibody (P = 0.1). This expression system consistently produced large quantities of recombinant porcine follistatin, which is immunologically and biologically similar to follistatin, and is capable of independently inhibiting estratiol-17βproduction in vitro. Key words: Porcine, follistatin, heat shock promoter, glycoprotein, ovary, estradiol

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