Evaluation of in vitro bactericidal activity of human serum against Salmonella Typhi in relation to serogroups (original) (raw)
Related papers
Clinical & Experimental Immunology, 2008
We have investigated the cross-reactivity of various species in neoepitope-specific methods for quantification of human complement activation products. In contrast to most other species examined, baboon showed a substantial cross-reactivity supporting a high degree of homology between human and baboon complement. An assay for C3b, iC3b and C3c (MoAb bH6) showed moderately good reactivity, in contrast to a C3a assay which did not cross-react. Excellent reactivity was found for C5a using MoAbs C17/5 and G25/2. The reactivity of an established TCC assay (MoAb aElI to a C9 neoepitope and polyclonal antibody to C5) was improved substantially by replacing the anti-C5 antibody with a new MoAb to C6 particularly selected on the basis of baboon cross-reactivity. Plasma samples from baboons receiving 2-5 x 109 and 1 0 x 1010 live Escherichia coli bacteria/kg were examined with the assays described. In vivo complement activation with the lowest dose was moderate and kept under control, in contrast to the highest dose, where an uncontrolled increase in all activation products continued throughout the infusion period. These results support the hypothesis that sufficiently high amounts of endotoxin lead to uncontrolled activation of complement as seen in irreversible septic shock. The results are discussed with particular emphasis on activation of the terminal complement pathway.
Measuring the 50% Haemolytic Complement (CH 50 ) Activity of Serum
Journal of Visualized Experiments, 2010
The complement system is a group of proteins that when activated lead to target cell lysis and facilitates phagocytosis through opsonisation. Individual complement components can be quantified however this does not provide any information as to the activity of the pathway. The CH50 is a screening assay for the activation of the classical complement pathway and it is sensitive to the reduction, absence and/or inactivity of any component of the pathway. The CH50 tests the functional capability of serum complement components of the classical pathway to lyse sheep red blood cells (SRBC) pre-coated with rabbit anti-sheep red blood cell antibody (haemolysin). When antibody-coated SRBC are incubated with test serum, the classical pathway of complement is activated and haemolysis results. If a complement component is absent, the CH50 level will be zero; if one or more components of the classical pathway are decreased, the CH50 will be decreased. A fixed volume of optimally sensitised SRBC is added to each serum dilution. After incubation, the mixture is centrifuged and the degree of haemolysis is quantified by measuring the absorbance of the haemoglobin released into the supernatant at 540nm. The amount of complement activity is determined by examining the capacity of various dilutions of test serum to lyse antibody coated SRBC. This video outlines the experimental steps involved in analysing the level of complement activity of the classical complement pathway.
Mechanism of bacterial resistance to complement-mediated killing
Molecular Immunology, 1982
The interaction of C3 and terminal complement components with three isogenic strains of Escherichia coli 0111B4 varying in outer membrane and capsule composition was examined. Strains CL99 and 1-1, which possess 0-antigen capsule and 74 to 77% coverage of lipid A core oligosaccharide, were sensitive to killing in pooled normal human serum (PNHS) or magnesium ethylene glycoltetraacetic acid PNHS in the presence but not the absence of antibody, although 1-1 contained 35% more lipopolysaccharide than CL99 and was slightly less sensitive to alternative pathway killing. In contrast, strain 1-2 lacks 0-antigen capsule but contains 84% coverage and resists serum killing in the presence and absence of antibody in both PNHS and magnesium ethylene glycoltetraacetic acid PNHS. All three strains consumed C3 and C9 when incubated in PNHS, but consumgtion was most rapid with 1-2, which also bound the largest number of C3 molecules per CFU. Between 15 x 10 and 24 x 103 molecules of C9 per CFU bound to CL99 and 1-1 during incubation in 10% PNHS or 10% magnesium ethylene glycoltetraacetic acid PNHS, and binding was relatively stable. Binding and release of 3 x 10' to 8 x 103 molecules of C9 per CFU was observed for strain 1-2. The majority of C9 bound to CL99 and 1-1 in the presence of antibody distributed with the outer membrane after lysis of the organisms in a French press, whereas only 16,1 to 20.1% of C9 was deposited on these organisms in the absence of antibody, and 31.5 to 39.8% of C9 on strain 1-2 with or without antibody sedimented with the outer membrane. Between 4.6 x 103 and 5.5 x 10 molecules of C9 per CFU remained bound in a salt-and trypsin-resistant form to the outer membrane of organisms that were killed, whereas fewer than 1.4 x 103 molecules of C9 per CFU were bound to the outer membrane of organisms not killed by serum. These results indicate that C5b-9 that is bound to the outer membrane of E. coli 0111B4 in a form resistant to salt or protease elution correlates with bacterial killing.
Infection and …, 1993
JP DE BOER,1 AA CREASEY,2 A. CHANG,3 D. ROEM,1 AJM EERENBERG,1 CE HACK,`* AND FB TAYLOR, JR.3 Central Laboratory of the Netherlands Red Cross Blood Transfiusion Service and Laboratory for Experimental and Clinical Immunology, University ofAmsterdam, ...
Journal of Experimental Medicine, 1982
Gram-positive cocci resist direct killing by serum. The mechanism of resistance was studied by measuring consumption of terminal complement components from serum and uptake of purified, radiolabeled C7 and C9 on rough and encapsulated type 7 Streptococcus pneumoniae. Extensive consumption of C5, C7, and C9 occurred when 5 x lo8 rough or type 7 pneumococci were incubated for 1 hr in 10% pooled normal human serum (PNHS). Approximately 10,OOO molecules of C7 and C9 bound per organism during the same period of incubation. Twenty to 30% of C7 and C9 was released from rough organisms. Release was not due to autolysis since it occurred with glutaraldehyde-fixed organisms as well as in S. pneumoniae that were rendered resistant to autolysis by growth in ethanolamine. Between 10 and 30% of bound 1251C9 counts were eluted from the rough and type 7 organisms by incubation in 1 M NaCl or 0.01 M EDTA, which suggests that bound C5b-9 was not attached by predominantly ionic interactions. Elution of 44 to 74% of lZ51C9 from live and glutaraldehyde-fixed organisms by 1% sodium deoxycholate suggests that hydrophobic bonds are involved in C5b-9 attachment. Trypsin cleaved 67 and 55% of l29C9 counts from live rough and type 7 S. pneumoniae, respectively which indicates that the bound complex is not protected by the cell wall from proteolytic attack. Serum resistance in S. pneumoniae does not represent a failure to form C5b-9 on the bacterial cell wall but apparently reflects a failure of the bound complex to penetrate the thick peptidoglycan layer.
A New Tool for Complement Research: In vitro Reconstituted Human Classical Complement Pathway
Frontiers in Immunology, 2018
The complement, as part of the innate immune system, represents the first line of defense against Gram-negative bacteria invading the bloodstream. The complement system is a zymogen cascade that ultimately assemble into the so-called membrane attack complex (MAC), which lyses Gram-negative bacteria upon insertion into the outer membrane. Traditionally, serum has been used as complement source, for example to study the bactericidal activity of monoclonal antibodies or antibodies raised upon vaccination. Due to the significant donor to donor variability, as well as susceptibility of complement factors to handling and storage conditions, assay reproducibility using human serum is low. Moreover, the presence of pre-existing antibodies and antimicrobial compounds are confounding factors. To remove antibodies from human serum, we applied κ/λ-light chain specific affinity chromatography, however the method severely reduced the complement activity due to the depletion of complement components. Therefore, we attempted to reconstitute human complement-namely the alternative (rAP) and the classical (rCP) pathways-from purified complement factors. We found that adding C1-inhibitor to the mixture was essential to maintain a stable and functional C1 and thus to generate an active rCP. We further confirmed the functionality of the rCP by testing the complement-dependent bactericidal activity of a human monoclonal antibody, A1124 against an E. coli clinical isolate belonging to the ST131 clonal complex, and that of a polyclonal IVIg against a laboratory E. coli strain (MG1655) not expressing LPS O-antigen and capsule. Although the alternative pathway did not have any bactericidal activity by itself, it enhanced MAC deposition induced by rCP and increased the overall bactericidal activity against the ST131 E. coli strain. In conclusion, we report for the first time the successful in vitro reconstitution of the classical pathway of the human complement to establish a serum-free, complement dependent bactericidal assay. This system offers high level of standardization and could support the study of the complement in different research fields.