Subcloning DT40 by limiting dilution

Author(s):  
Jean-Marie Buerstedde
Keyword(s):  
1993 ◽  
Vol 27 (3-4) ◽  
pp. 65-68 ◽  
Author(s):  
B. H. Kwa ◽  
M. Moyad ◽  
M. A. Pentella ◽  
J. B. Rose

Cryptosporidium parvum is an important patliogen of diarrlieal disease which has been implicated in several outbreaks associated with contamination of surface waters. In monitoring for C. parvum in drinking water sources, it is important to asce tain the viability, and more importantly, the infectivity of low numbers of recovered oocysts. Groups of 10 Balb/C nude (nu/nu) mice, 4-8 weeks old at time of inoculation, were infected with C. parvum oocysts from naturally infected calves and purified using Sheather's sucrose gradients. Oocysts were counted using the Merifluor IFA kit (Meridian). Each group of 10 mice were infected with 1,10,100 and 1000 oocysts respectively. Numbers of oocysts per inoculation were determined by limiting dilution, and parallel inocula were counted microscopically to ascertain the accuracy of the dilutions. Two uninfected nude mice were kept in each cage to serve as controls. Mouse stools were collected every 4 days, concentrated using the Fekal Kontrate Concentration Kit (Meridian) and oocysts were counted with a UV microscope using the Merifluor IFA Kit (Meridian). Oocyst counts were expressed in terms of number of oocyst/g feces. Mice inoculated with 1000 oocysts began to shed oocysts on day 32, mice inoculated with 100 oocysts began to shed on days 44-48, mice inoculated with 10 oocysts began to shed on days 56-60, and mice inoculated with 1 oocyst shed on days 68-88. All infected mice continued to shed oocysts intermittently and with variable oocyst counts until day 180 when the experiment was terminated. This study established that it is possible to infect nude mice with very low numbers, down to a single oocyst. We are currently in the process of correlating the nude mouse assay with other viability assays.


2013 ◽  
Vol 23 (4) ◽  
pp. 921-936 ◽  
Author(s):  
Hongmei Yang ◽  
David J. Topham ◽  
Jeanne Holden-Wiltse ◽  
Hulin Wu

1989 ◽  
Vol 5 (2) ◽  
pp. 161-161
Author(s):  
A. Sette ◽  
L. Adorini ◽  
C. Mancini ◽  
G. Doria

1984 ◽  
Vol 160 (1) ◽  
pp. 1-11 ◽  
Author(s):  
M Slaoui ◽  
O Leo ◽  
J Marvel ◽  
M Moser ◽  
J Hiernaux ◽  
...  

We have shown that, by suitable idiotypic manipulation, BALB/c mice can express the major cross-reactive idiotype (CRI) of A/J mice in response to azophenylarsonate (Ars). In order to know if the CRIA idiotype is present in the potential repertoire of BALB/c before any intentional selection, we used polyclonal activation in vitro and limiting dilution analysis. The readout was done with two monoclonal anti-CRIA antibodies that recognize distinct idiotopes on a CRIA+ A/J germline-encoded monoclonal antibody. We studied the frequency of CRIA+ lipopolysaccharide (LPS)-reactive cells in the spleens of nonimmune and immune A/J mice and in the spleens of naive and manipulated (i.e., producing CRIA+ antibodies) BALB/c mice. A/J and BALB/c naive individuals presented very high frequencies of Ars-specific B cells while the frequency of CRIA+ B cells was only a minor subset (0.5%) of the total Ars-specific subset in the two strains. When A/J mice were immunized with Ars-keyhole limpet hemocyanin, a clear preferential expansion of the CRIA+ minor subset of A/J mice was observed (100x). No such enhancement was observed in BALB/c mice similarly treated. Manipulated BALB/c mice presented a higher frequency of CRIA+ anti-Ars B cells than naive or antigen-immunized BALB/c individuals.


2003 ◽  
Vol 51 (2) ◽  
pp. 237-244 ◽  
Author(s):  
K. Yeşilbağ ◽  
I. Burgu

In this study, 15 bovine viral diarrhoea viruses (BVDV) isolated from the field in Turkey were characterised for their biotype, cloned and eventually analysed for their epitopic composition in terms of glycoprotein E2. Immunoplaque assay, plaque assay, limiting dilution and streptavidin-biotin-peroxidase techniques were used for biotype characterisation, cloning of cytopathic (cp) and noncytopathic (ncp) biotypes and epitope analysis, respectively. While 14 out of 15 BVDV isolates were distinguished as ncp biotype, 1 isolate was found to be containing both biotypes (cp + ncp). According to the reactivity patterns of isolates with 15 monoclonal antibodies, 4 different antigenic groups could be formed. There were no antigenic differences between the isolates derived from the same animal with various time intervals. On the other hand, biotype clones isolated from the same animal exhibited difference in one epitope. This is the first study describing antigenic characterisation of BVDV field isolates in Turkey.


1995 ◽  
Vol 181 (1) ◽  
pp. 79-91 ◽  
Author(s):  
P J Lehner ◽  
E C Wang ◽  
P A Moss ◽  
S Williams ◽  
K Platt ◽  
...  

The major histocompatibility complex class I-restricted cytotoxic T lymphocyte (CTL) response is important in the clearance of viral infections in humans. After influenza A infection, a peptide from the matrix protein, M58-66, is presented in the context of the MHC allele HLA-A0201 and the resulting CTL response is detectable in most HLA-A0201 subjects. An initial study suggested that M58-66-specific CTL clones show conserved T cell receptor (TCR) alpha and beta gene segments. We have addressed the significance of this observation by determining the expression of V beta 17 during the development of M58-66-specific CTL lines in 21 unrelated HLA-A0201 subjects, and analyzing TCR usage by M58-66-specific CTL clones. TCR V beta 17 was the dominant V beta segment used and CD8 V beta 17 expansion correlated with M58-66-specific lysis. Limiting dilution analysis from five subjects showed the M58-66 CTL precursor frequency to vary between 1/54,000 and less than 1/250,000, and that up to 85% of the matrix peptide (M58-66)-specific CTL used the V beta 17 gene segment. The M58-66 specific CTL response was dependent on previous viral exposure and specific V beta 17 expansion, as it was not found in cord blood, despite a readily expandable V beta 17+ CD8+ T cell subpopulation. Sequence analysis of 38 M58-66-specific V beta 17 transcripts from 13 subjects revealed extensive conservation in the CDR3 region including conservation of an arginine-serine motif. To test the dependence of this CTL response on the V beta 17 gene segment, peripheral blood lymphocytes were depleted of CD8+ TCR V beta 17+ cells, before the generation of M58-66-specific CTL. In most cases such depletion blocked or severely reduced the generation of the M58-66-specific response, and under limiting dilution conditions could abolish M58-66-specific CTL precursors. These studies reveal the dependence of this natural human immune response on a particular TCR gene segment.


2005 ◽  
Vol 174 (6) ◽  
pp. 3421-3431 ◽  
Author(s):  
Thierry Bonnefoix ◽  
Philippe Bonnefoix ◽  
Pascal Perron ◽  
Jian-Qing Mi ◽  
Wan Fai Ng ◽  
...  

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