scholarly journals Sequence Variation in the porA Gene of a Clone of Neisseria meningitidis during Epidemic Spread

2000 ◽  
Vol 7 (3) ◽  
pp. 390-395 ◽  
Author(s):  
J. Jelfs ◽  
R. Munro ◽  
E. Wedege ◽  
D. A. Caugant
Keyword(s):  
Monoclonal Antibodies ◽  
Neisseria Meningitidis ◽  
Vaccine Development ◽  
Stop Codon ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Epidemic Spread ◽  
Insertion Element ◽  
Variable Regions ◽  
Geographic Origins

ABSTRACT The ET-15 clone within the electrophoretic type (ET)-37 complex ofNeisseria meningitidis was first detected in Canada in 1986 and has since been associated with outbreaks of meningococcal disease in many parts of the world. While the majority of the strains of the ET-37 complex are serosubtype P1.5,2, serosubtype determination of ET-15 strains may often be incomplete, with either only one or none of the two variable regions (VRs) of the serosubtype PorA outer membrane protein reacting with monoclonal antibodies. DNA sequence analysis of the porA gene from ET-15 strains with one or both unidentified serosubtype determinants was undertaken to identify the genetic basis of the lack of reaction with the monoclonal antibodies. Fourteen different porA alleles were identified among 38 ET-15 strains from various geographic origins. The sequences corresponding to subtypes P1.5a,10d, P1.5,2, P1.5,10d, P1.5a,10k, and P1.5a,10a were identified in 18, 11, 2, 2, and 1 isolate, respectively. Of the remaining four strains, which all were nonserosubtypeable, two had a stop codon within the VR1 and the VR2, respectively, while in the other two the porA gene was interrupted by the insertion element, IS1301. Of the strains with P1.5,2 sequence, one had a stop codon between the VR1 and VR2, one had a four-amino-acid deletion outside the VR2, and another showed no expression of PorA on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Our results reveal that numerous genetic events have occurred in theporA gene of the ET-15 clone in the short time of its epidemic spread. The magnitude of microevolutionary mechanisms available in meningococci and the remarkable genetic flexibility of these bacteria need to be considered in relation to PorA vaccine development.

scholarly journals Production and characterisation of monoclonal antibodies against immunoglobulins of Cirrhinus mrigala (Hamilton 1822)

2020 ◽  
Vol 67 (2) ◽  
Author(s):  
Snatashree Mohanty ◽  
M. Makesh ◽  
K. V. Rajendran ◽  
P. P. Suresh Babu ◽  
Deepika Anand ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cross Reactivity ◽  
Catla Catla ◽  
Cirrhinus Mrigala ◽  
Indian Major Carps ◽  
Sds Page ◽  
Igg1 Isotype ◽  
Major Carps

Serum immunoglobulins (Ig) of mrigal Cirrhinus mrigala (Hamilton 1822) immunised with bovine serum albumin (BSA), were purified by affinity chromatography using BSA-CL agarose column. The purified mrigal Ig (m-Ig) was characterised under reducing condition by Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) which revealed two bands of 85 and 26 kDa corresponding to heavy and light chain, respectively. Following fusion of splenocytes from Balb/c mice immunised with purified m-Ig with myeloma cells, three hybridomas showing reactivity with m-Ig were cloned by limiting dilution. The monoclonal antibodies (MAbs) generated by these clones were designated as 3B2-E12, 3B2-F9 and 4C3-B2 and characterised by western blotting and isotyping. Western blot analysis of the supernatant from the three clones with purified m-Ig indicated that, all the three MAbs were specific to heavy chain. Isotyping revealed that 3B2-E12 MAb was of IgG1 isotype whereas the other two MAbs were of IgG2a isotype. Cross reactivity of anti-mrigal Ig MAb (3B2-E12) was observed with serum Ig of Catla catla and Labeo rohita indicating semi-conserved nature of Ig in Indian major carps.

scholarly journals First description of increased resistance in carbapenem-susceptible Klebsiella pneumoniae with imipenem treatment driven by outer membrane remodelling in China

2019 ◽  
Author(s):  
Xuebin Tian ◽  
Qiongdan Wang ◽  
Xiangkuo Zheng ◽  
Yajie Zhao ◽  
Renchi Fang ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Gel Electrophoresis ◽  
Stop Codon ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Premature Stop Codon ◽  
Underlying Mechanism ◽  
Systematic Research ◽  
Carbapenem Resistant ◽  
Before And After

Abstract The emergence of carbapenem-resistant Kelbsiella pneumoniae (CRKP) posed threats to human health. Although there are numerous studies regarding porin alteration in association with the production of ESBLs and/or AmpC β-lactamase, a systematic research about the treatment-emergence of porins alteration in antibiotic resistance does not exist yet. The aim of this study was to investigate the underlying mechanism and evolution of resistance of K. pneumoniae during carbapenem treatment. Here, we reported three strains (FK-2624, FK-2723 and FK-2820) isolated from one patient before and after imipenem treatment during hospitalization. Antibiotic susceptibility testing indicated that FK-2624 was susceptible to almost antimicrobials but fosfomycin; FK-2723 and FK-2820 were MDR. After imipenem therapy, FK-2820 was evolved to carbapenem-resistant. PCR and Whole-Genome sequencing ( WGS) indicated that resistance genes bla SHV , oqxA and fosA5 were detected in FK-2624, in addition, FK-2723 and FK-2820 harbored bla DHA , qnrB , aac (6’)-Ib . Virulence factors K57, ybtA, mrkD, entB and iroN were detected simultaneously in all of three strains. The results of pairwise comparisons , multi-locus sequencing typing (MLST) and pulsed-field gel electrophoresis (PFGE) revealed high homology among the isolates. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) results showed that isolate FK-2820 lacked OmpK 36 as there was a premature stop codon of the outer membrane porin encoding gene ompk36 confirmed by sequencing. Real-time RT-PCR revealed that the expression of ompK36 in FK-2820 was 0.093 times the control isolate ATCC 13883. Our study highlighted that the alteration of outer membrane porins due to the 14-day use of imipenem clinically play a potential role in leading to the carbapenems-resistance of FK-2820.

Isolation, Cultivation, and Characterization of Borrelia burgdorferi from Rodents and Ticks in the Charleston Area of South Carolina

2000 ◽  
Vol 38 (1) ◽  
pp. 120-124
Author(s):  
J. H. Oliver ◽  
K. L. Clark ◽  
F. W. Chandler ◽  
L. Tao ◽  
A. M. James ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Borrelia Burgdorferi ◽  
Reference Strain ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Western Blots ◽  
Cotton Rat ◽  
Cotton Mouse ◽  
B 31

ABSTRACT Twenty-eight Borrelia burgdorferi isolates from the Charleston, S.C., area are described. This represents the first report and characterization of the Lyme disease spirochete from that state. The isolates were obtained from December 1994 through December 1995 from the tick Ixodes scapularis , collected from vegetation, and from the rodents Peromyscus gossypinus (cotton mouse), Neotoma floridana (eastern wood rat), and Sigmodon hispidus (cotton rat). All isolates were screened immunologically by indirect immunofluorescence with monoclonal antibodies to B. burgdorferi -specific outer surface protein A (OspA) (antibodies H5332 and H3TS) and B. burgdorferi -specific OspB (antibodies H6831 and H614), a Borrelia (genus)-specific antiflagellin antibody (H9724), Borrelia hermsii -specific antibodies (H9826 and H4825), and two polyclonal antibodies (one to Borrelia species and another to B. burgdorferi ). Six of the isolates were analyzed by exposing Western blots to monoclonal antibodies H5332, H3TS, H6831, and H9724. All isolates were also analyzed by PCR with five pairs of primers known to amplify selected DNA target sequences specifically reported to be present in the reference strain, B. burgdorferi B-31. The protein profiles of six of the isolates (two from ticks, one from a cotton mouse, two from wood rats, and one from a cotton rat) also were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We conclude that the 28 Charleston isolates are B. burgdorferi sensu stricto based on their similarities to the B. burgdorferi B-31 reference strain.

scholarly journals Unique role of the complement receptor CR1 in the degradation of C3b associated with immune complexes.

1982 ◽  
Vol 156 (6) ◽  
pp. 1739-1754 ◽  
Author(s):  
M E Medof ◽  
K Iida ◽  
C Mold ◽  
V Nussenzweig
Keyword(s):  
Monoclonal Antibodies ◽  
Immune Complexes ◽  
Solid Phase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Alpha Chain ◽  
Sds Page ◽  
High Concentrations ◽  
Polypeptide Chains

The main finding of this paper is that CR1, the membrane receptor for C3b and C4b, together with C3b/C4b-inactivator (I), degrades C3b bound to immune complexes (C3b*). Two fragments are generated: C3c, which is released from the immune complexes, and C3d*. The C3c fragment released from the cell intermediate EAC1423b prepared with 125I-C3 was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and radioautography. It has a 135,000 mol wt and contains disulfide bonded labeled polypeptide chains of 75,000 and 31,000 mol wt, which presumably represent the beta and a fragment of the alpha-chain of C3b*. Silver staining of the SDS-PAGE gels revealed other C3-derived bands with 39-42,000 mol wt. Human erythrocytes + I also cleave C3b* into C3c and C3d*. The activity of the erythrocytes is CR1 mediated because it can be totally inhibited by monoclonal antibodies to CR1. In contrast with these results, I together with the serum protein beta 1H (H) transform EAC1423b into hemolytically inactive EAC1423bi and cleave the alpha' chain of C3b* into fragments of 70,000 and 40,000 mol wt. Small amounts of C3c are also released at relatively high concentrations of H. On a molar basis, the efficiency of CR1 in the generation of C3c and C3d is 10(4)-10(5) greater than H. An additional observation was that C3c could be released by treating EAC1423bi with CR1 + I and that this reaction was also inhibited by monoclonal antibodies to CR1. Therefore, it is likely that CR1 has binding affinity for iC3b and that the degradation of C3b* proceeds as follows: C3b (formula, see text) C3c + C3d*. Taken together, our findings argue that the processing of C3b* in vivo occurs in solid phase, that is, on the surface of cells bearing CR1.

Serotypes of Neisseria meningitidis associated with an increased incidence of meningitis cases in the Hamilton area, Ontario, during 1978 and 1979

1983 ◽  
Vol 29 (1) ◽  
pp. 129-136 ◽  
Author(s):  
Fraser E. Ashton ◽  
J. Alan Ryan ◽  
Colina Jones ◽  
Bernard R. Brodeur ◽  
Benito B. Diena
Keyword(s):  
Neisseria Meningitidis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Age Groups ◽  
Double Diffusion ◽  
Sds Page ◽  
Class 1 ◽  
Group B

The distribution of serotypes among strains of Neisseria meningitidis responsible for a marked increase of meningitis cases in the Hamilton area, Ontario, in 1978 and 1979 was determined. Twenty-six serogroup B and two serogroup W135 strains isolated from cerebrospinal fluid, blood, and skin of 28 patients were serotyped by agar gel double diffusion. Twenty-one (81 %) of the group B strains were serotype 2b as judged by the formation of characteristic serotype precipitin bands with the specific anti-2996 (type 2b) serum. Fourteen of the serotype 2b strains also reacted with anti-77252 serum, which suggested that one strain or several closely related strains were mainly responsible for the increase in meningitis during the 2-year period. Examination of the outer membrane complexes (OMC) of the strains by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS–PAGE) revealed that all 21 of the serotype 2b strains contained the class 2 protein (molecular weight 41 500) which is known to be the site of the serotype 2b determinant. Further characterization of the serotype 2b,77252 strains by enzyme-linked immunosorbent assays (ELISA) and SDS–PAGE suggested that the 77252 determinant was present in the class 1 proteins of these strains. The serotype 2b containing strains were isolated from 77.7 and 70% of males and females, respectively, from 81.8% of children less than 5 years of age, and from 75.0% of patients of all age groups. The study indicates the important role of serotype 2b meningococci in causing the increased incidence of meningitis and further substantiates the important association of the serotype 2b determinant with group B serotype 2 meningococcal disease in Canada.

scholarly journals Role of Immunoglobulin A Monoclonal Antibodies against P23 in Controlling Murine Cryptosporidium parvum Infection

1998 ◽  
Vol 66 (9) ◽  
pp. 4469-4473 ◽  
Author(s):  
F. Javier Enriquez ◽  
Michael W. Riggs
Keyword(s):  
Monoclonal Antibodies ◽  
Cryptosporidium Parvum ◽  
Matched Control ◽  
Intestinal Parasites ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Immunoglobulin A ◽  
Passive Immunization ◽  
Immunofluorescence Assay

ABSTRACT Cryptosporidium parvum is an important diarrhea-causing protozoan parasite of immunocompetent and immunocompromised hosts. Immunoglobulin A (IgA) has been implicated in resistance to mucosal infections with bacteria, viruses, and parasites, but little is known about the role of IgA in the control of C. parvuminfection. We assessed the role of IgA during C. parvum infection in neonatal mice. IgA-secreting hybridomas were developed by using Peyer’s patch lymphocytes from BALB/c mice which had been orally inoculated with viable C. parvumoocysts. Six monoclonal antibodies (MAbs) were selected for further study based on indirect immunofluorescence assay reactivity with sporozoite and merozoite pellicles and the antigen (Ag) deposited on glass substrate by gliding sporozoites. Each MAb was secreted in dimeric form and recognized a 23-kDa sporozoite Ag in Western immunoblots. The Ag recognized comigrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with P23, a previously defined neutralization-sensitive zoite pellicle Ag. MAbs were evaluated for prophylactic or therapeutic efficacy against C. parvum, singly and in combinations, in neonatal BALB/c mice. A combination of two MAbs given prophylactically prior to and 12 h following oocyst challenge reduced the number of intestinal parasites scored histologically by 21.1% compared to the numbers in mice given an isotype-matched control MAb (P < 0.01). Individual MAbs given therapeutically in nine doses over a 96-h period following oocyst challenge increased efficacy against C. parvuminfection. Four MAbs given therapeutically each reduced intestinal infection 34.4 to 42.2% compared to isotype-matched control MAb-treated mice (P < 0.05). One MAb reduced infection 63.3 and 72.7% in replicate experiments compared to isotype-matched control MAb-treated mice (P < 0.0001). We conclude that IgA MAbs directed to neutralization-sensitive P23 epitopes may have utility in passive immunization against murineC. parvum infection.

scholarly journals The use of oligonucleotide probes for meningococcal serotype characterization

1998 ◽  
Vol 40 (2) ◽  
pp. 113-117 ◽  
Author(s):  
Claudio Tavares SACCHI ◽  
Ana Paula Silva de LEMOS ◽  
Anne M. Whitney ◽  
Carmo Elias A. MELLES ◽  
Claude André SOLARI ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Neisseria Meningitidis ◽  
Rapid Method ◽  
Gene Sequence ◽  
Prototype Strain ◽  
Oligonucleotide Probes ◽  
Type B ◽  
Variable Regions ◽  
Antigenic Diversity ◽  
Potential Use

In the present study we examine the potential use of oligonucleotide probes to characterize Neisseria meningitidis serotypes without the use of monoclonal antibodies (MAbs). Antigenic diversity on PorB protein forms the bases of serotyping method. However, the current panel of MAbs underestimated, by at least 50% the PorB variability, presumably because reagents for several PorB variable regions (VRs) are lacking, or because a number of VR variants are not recognized by serotype-defining MAbs12. We analyzed the use of oligonucleotide probes to characterize serotype 10 and serotype 19 of N. meningitidis. The porB gene sequence for the prototype strain of serotype 10 was determined, aligned with 7 other porB sequences from different serotypes, and analysis of individual VRs were performed. The results of DNA probes 21U (VR1-A) and 615U (VR3-B) used against 72 N. meningitidis strains confirm that VR1 type A and VR3 type B encode epitopes for serotype-defined MAbs 19 and 10, respectively. The use of probes for characterizing serotypes possible can type 100% of the PorB VR diversity. It is a simple and rapid method specially useful for analysis of large number of samples.

scholarly journals Identification of three antigens in human brain associated with similar antigens on human leukaemic cells

1985 ◽  
Vol 225 (2) ◽  
pp. 291-299 ◽  
Author(s):  
E J Quackenbush ◽  
T F Cruz ◽  
M A Moscarello ◽  
M Letarte
Keyword(s):  
Monoclonal Antibodies ◽  
White Matter ◽  
Human Brain ◽  
Sodium Dodecyl Sulphate ◽  
Grey Matter ◽  
Lymphocytic Leukaemia ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Acute Lymphocytic Leukaemia ◽  
Leukaemia Cell Line

Monoclonal antibodies prepared against a non-T and non-B acute-lymphocytic-leukaemia cell line were tested for reactivity against human brain tissue. Several of the monoclonal antibodies were found to react specifically with brain fractions. Three antigens, 44H4, 44D7 and 44D10, were identified in white matter. Although 44D10 was absent from grey matter, the levels of 44H4 and 44D7 antigens present in grey matter were 2- and 4-fold higher respectively than in white matter. Fractionation of white matter indicated that all three antigens were absent from the multilamellar compact myelin, but associated with a membrane fraction of higher density. All three antigens, which required detergent for solubilization from the membranes, were purified by affinity to monoclonal antibodies and/or were analysed by immunoblotting. The 44H4 and 44D10 antigens were single polypeptide chains with Mr 94000 and 80000 respectively when resolved by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Monoclonal antibody 44D7 reacted with a complex of a Mr greater than 120000 under non-reducing conditions in the presence of sodium dodecyl sulphate. This complex dissociated on reduction into four bands with Mr values of 80000, 57000, 47000 and 41000. The brain antigens are present on proteins similar to, or identical with, those isolated from acute-lymphocytic-leukaemia cells.

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