A panel of antigens of muscle larvae of Trichinella spiralis and T. pseudospiralis as revealed by two-dimensional Western blot and immunoelectron microscopy

Parasitology ◽  
1999 ◽  
Vol 118 (6) ◽  
pp. 615-622 ◽  
Author(s):  
Z. WU ◽  
I. NAGANO ◽  
Y. TAKAHASHI
Keyword(s):  
Western Blot ◽  
Trichinella Spiralis ◽  
Cross Reactivity ◽  
Microscopical Study ◽  
Detectable Level ◽  
Antigenic Peptides ◽  
Two Dimensional ◽  
Western Blots ◽  
Muscle Larvae ◽  
Isoelectric Points

This study characterized antigens of Trichinella spiralis and T. pseudospiralis muscle larvae recognized by mice infected with the worms. Two-dimensional (2-D) Western blot analysis revealed some profile of antigenic peptides including: (1) molecular weight (MW); (2) isoelectric points (pI), (3) reactivity to well-defined monoclonal antibodies (mAb) and (4) cross-reactivity between the 2 species. Antigenic peptides of T. spiralis consisted of about 100 spots. The MW ranged from 22 to 80 kDa, and pI ranged from 4 to 7. The mAb against TSL-1 stained most of the T. spiralis excretory–secretory (E–S) peptides migrating at 40, 45 and 50 kDa, and the mAb against TSL-4 stained non-E–S peptides. Antigenic peptides of T. pseudospiralis consisted of about 20 to 30 peptide spots. The MW ranged from 25 to 80 kDa, and pI ranged from 4 to 7. The mAb against TSL-1 stained most of the T. pseudospiralis E–S peptides migrating at 35 and 45 kDa, and the mAb against TSL-4 stained non-E–S peptides. Two-dimensional Western blots showed that the E–S products of T. spiralis and T. pseudospiralis were highly cross-reactive with each other. The non-E–S peptides were, however, not recognized by T. pseudospiralis-infected sera but were recognized by T. spiralis-infected sera. An immunoelectron microscopical study showed the similar result that stichocyte granules and cuticle surface (known to contain E–S antigen) had cross-reactive antigens between the two species. T. pseudospiralis-infected sera stained very weakly the cuticle inner layers and haemolymph (known to contain non-E–S antigen). This evidence implies that mice infected with T. pseudospiralis do not evoke antibodies against non-E–S antigen at the detectable level.

scholarly journals A comparison of antigenic peptides in muscle larvae of severalTrichinellaspecies by two-dimensional western-blot analysis with monoclonal antibodies

Parasite ◽  
2001 ◽  
Vol 8 ◽  
pp. S117-S119 ◽  
Author(s):  
M.A. Dea-Ayuela ◽  
F.M. Ubeira ◽  
A. Pitarch ◽  
C. Gil ◽  
A.R. Martínez-Fernández ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Antigenic Peptides ◽  
Two Dimensional ◽  
Muscle Larvae

scholarly journals Antithrombin Milano: a new variant with monomeric and dimeric inactive antithrombin III

Blood ◽  
1985 ◽  
Vol 65 (2) ◽  
pp. 496-500 ◽  
Author(s):  
M Wolf ◽  
C Boyer ◽  
A Tripodi ◽  
D Meyer ◽  
MJ Larrieu ◽  
...  
Keyword(s):  
Antithrombin Iii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cross Reactivity ◽  
Two Dimensional ◽  
Western Blots ◽  
Sds Page ◽  
New Variant ◽  
At Iii ◽  
Monospecific Antisera

Abstract A qualitative defect of antithrombin III (AT III) has been demonstrated over three generations in eight members of an Italian family by the discrepancy between a normal amount of antigen and decreased antithrombin and anti-Xa activity in the presence or in the absence of heparin. By two-dimensional immunoelectrophoresis in the absence of heparin, two peaks of AT III were present in all patients' plasma. AT III was purified from normal and propositus plasma by sulfate dextran precipitation followed by heparin affinity chromatography. The elution profile of the patient's AT III was abnormal and allowed the separation of two populations of AT III, normal and abnormal. The first fraction (normal AT III) contained AT III activity, migrated as a single peak by two-dimensional immunoelectrophoresis and by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE), demonstrated a single band with a molecular weight (mol wt) identical to that of normal AT III (60,000). Conversely, the last fraction, devoid of AT III activity, migrated as a single abnormal peak by two-dimensional immunoelectrophoresis in the absence of heparin. By SDS-PAGE, two bands were observed: one with a mol wt of 60,000 and a second one with a mol wt of 120,000. Western blots clearly demonstrated cross-reactivity of the 120,000 and 60,000 mol wt bands with monospecific antisera to human AT III. Reduction of the 120,000 mol wt band converted it to a single 60,000 mol wt band, suggesting the presence of an abnormal dimeric form of AT III. The name AT III Milano is proposed for this new variant.

scholarly journals Preparation and characterization of monoclonal antibodies against ES antigens of Trichinella spiralis

2014 ◽  
Vol 51 (4) ◽  
pp. 253-261 ◽  
Author(s):  
J. Cui ◽  
F. Jing ◽  
G. Fu ◽  
H. Ren ◽  
L. Liu ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Western Blot ◽  
Post Infection ◽  
Trichinella Spiralis ◽  
Internal Organs ◽  
Cross Reactions ◽  
Muscle Larvae ◽  
Somatic Antigens

AbstractHybridomas secreting monoclonal antibodies (MAbs) against excretory-secretory (ES) antigen of Trichinella spiralis muscle larvae were produced. The 12 monoclones (designated 3A11, 7D9, 7F6, 5A7, 6H7, 39F3, 38H2, 37E7, 35B9, 29A11, 39C9 and 39A6) secreted IgM, IgG3 and IgG2b, respectively. Western blot using T. spiralis ES antigens showed that ten of 12 MAbs recognized the bands between 119.8 − 19.3kDa (mainly 68.4 − 28.7kDa) and two MAbs (3A11, 37E7) did not recognize any protein constituents of ES antigens. Nine of 12 MAbs also recognized the larval antigens of T. nativa, T. pseudospiralis and T. nelsoni at 40 days post-infection (dpi). No cross-reactions were found with the somatic antigens of adult worms of P. westermani, C. sinensis, and T. solium cysticercus. However, three (3A11, 6H7 and 39A6) were cross-reacted with the somatic antigens of adult worms of S. japonicum. Immunofluorescent location showed that the nine MAbs reacted with the cuticle and internal organs of T. spiralis larvae. Additionally, five and eight MAbs generated in this study can recognize the early antigens of pre-encapsulated T. spiralis larvae at 11 dpi and 13 dpi, respectively. The generation and characterization of the MAbs against T. spiralis ES antigens provide foundation for the development of specific early serodiagnostic techniques for trichinellosis.

scholarly journals Characteristic band pattern in western blots for specific detection of anti-Trichinella spiralis antibodies in different host species

2014 ◽  
Vol 64 (1) ◽  
pp. 33-43 ◽  
Author(s):  
Nataša Ilić ◽  
Alisa Gruden-Movsesijan ◽  
Milena Živojinović ◽  
Ljiljana Sofronić-Milosavljević
Keyword(s):  
Host Species ◽  
Trichinella Spiralis ◽  
Standard Test ◽  
Antibody Detection ◽  
Reference Laboratory ◽  
Serum Samples ◽  
Western Blots ◽  
Muscle Larvae ◽  
Molecular Masses ◽  
Inhibition Studies

Abstract Western blot (Wb) is considered to be the gold standard test for Trichinella infection serology, since this method allows specific Trichinella antigens to be distinguished from cross-reactive antigens. This is not the case with widely used antibody assay techniques - indirect immunofluorescence and ELISA - which are sensitive, but subject to crossreactions that make the interpretation of weakly positive results difficult. Application of Trichinella spiralis muscle larvae excretory-secretory (ES) antigens for the specific antibody detection in ELISA resulted in improved specificity compared to that of crude worm extract that was previously in use, but since production of ES has not yet been standardized, differences among laboratories occur. For this reason, the Wb profile of serum samples from different T. spiralis infected host species: human, horse, swine and dog, was investigated in the Serbian National Reference Laboratory for Trichinellosis (NRLT). The common feature of the obtained Wb profiles was the appearance of a triad of bands with molecular masses (Mw) of 45, 49, and 53 kDa. The very same triad was recognized by a monoclonal antibody (mAb) 7C2C5 specific for an immunodominant epitope unique to the muscle larvae stage of all species in the genus Trichinella. Inhibition studies confirmed that mAb and anti-Trichinella antibodies from sera competed for the same parasite epitope. Based on the obtained results, the NRLT introduced the recognition of the above mention triad as the basis for specific anti-Trichinella antibodies detection in the sera of infected hosts.

Molecular cloning and expression of the full-length tropomyosin gene from Trichinella spiralis

2003 ◽  
Vol 77 (1) ◽  
pp. 57-63 ◽  
Author(s):  
T. Nakada ◽  
I. Nagano ◽  
Z. Wu ◽  
Y. Takahashi
Keyword(s):  
Trichinella Spiralis ◽  
Expression System ◽  
Muscle Layer ◽  
Full Length ◽  
Cloning And Expression ◽  
Western Blots ◽  
Reading Frame ◽  
Muscle Larvae ◽  
Taxonomical Position ◽  
Good Agreement

AbstractA clone, designated as TsTM, was selected from the cDNA library of newborn larvae (NBL) of Trichinella spiralis through immunoscreening against infected sera. The clone contained a cDNA transcript of 855 bp in length with a single open reading frame, which encoded 285-amino acids (33 kDa in the estimated molecular weight). A sequence analysis revealed that the clone TsTM encoded the full-length of tropomyosin gene. The phylogenetic analysis of the tropomyosin gene was in good agreement with the classical taxonomical position of T. spiralis. The fusion proteins encoded by the clone TsTM were produced in an Escherichia coli expression system and affinity purified, and the antibody was raised against the protein for the following studies. The antibody against the fusion protein positively bound to the hypodermal muscle layer in immunolocalization analysis, and the 35 kDa band in crude extracts of muscle larvae but not in excretory and secretory (ES) products on Western blots. The antigenicity of the clone TsTM was recognized by host mice but exhibited little species specificity.

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