Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012;7(11):e49147.
doi: 10.1371/journal.pone.0049147. Epub 2012 Nov 8.

The stability of complement-mediated bactericidal activity in human serum against Salmonella

Colette M O'Shaughnessy  1 , Adam F Cunningham, Calman A Maclennan
Affiliations

The stability of complement-mediated bactericidal activity in human serum against Salmonella

Colette M O'Shaughnessy et al. PLoS One. 2012.

Abstract

The complement cascade includes heat-labile proteins and care is required when handling serum in order to preserve its functional integrity. We have previously used a whole human serum bactericidal assay to show that antibody and an intact complement system are required in blood for killing of invasive isolates of Salmonella. The aim of the present study was to evaluate the conditions under which human serum can be stored and manipulated while maintaining complement integrity. Serum bactericidal activity against Salmonella was maintained for a minimum of 35 days when stored at 4°C, eight days at 22°C and 54 hours at 37°C. Up to three freeze-thaw cycles had no effect on the persistence of bactericidal activity and hemolytic complement assays confirmed no effect on complement function. Delay in the separation of serum for up to four days from clotted blood stored at 22°C did not affect bactericidal activity. Dilution of serum resulted in an increased rate of loss of bactericidal activity and so serum should be stored undiluted. These findings indicate that the current guidelines concerning manipulation and storage of human serum to preserve complement integrity and function leave a large margin for safety with regards to bactericidal activity against Salmonella. The study provides a scheme for determining the requirements for serum handling in relation to functional activity of complement in other systems.

PubMed Disclaimer

Conflict of interest statement

Competing Interests: C.A. MacLennan is an employee of the Novartis Vaccines Institute for Global Health and a recipient of a Clinical Research Fellowship from GlaxoSmithKline. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Percentage of human serum stored at 22°C required to kill S. Typhimurium D23580 and anti-S. Typhimurium antibody levels in study sera.
Killing of bacteria by different percentages of freshly thawed serum from A. subject 1 and B. subject 2 at 45, 90 and 180 minutes in serum bactericidal assay using 106 cfu/ml S. Typhimurium D23580∶10% (red circles), 20% (green squares), 30% (blue triangles), 40% (purple diamonds) and 50% (orange inverted triangles) serum diluted with PBS, 100% PBS (black crosses), 100% fresh serum (pink vertical crosses) and 100% heat-inactivated serum (blue circle and dashed lines). Initial concentration of bacteria 106 cfu/ml with numbers of viable bacteria plotted against duration of assay. Killing of bacteria by different percentages of serum from C. subject 1 and D. subject 2 at 180 minutes following storage at 22°C. Change in viable count (cfu/ml) plotted against duration of time serum stored at 22°C. E. Levels of anti-S. Typhimurium D23580 IgG, IgA and IgM antibodies in human sera used in study as assessed by flow cytometry. F. Killing of bacteria by fresh serum (solid lines) and heat-inactivated serum (dashed lines) from subject 1 (red circles), 2 (green squares), 3 (blue triangles) and 4 (purple diamonds) at 45, 90 and 180 minutes.
Figure 2
Figure 2. Effect of duration of storage of human serum at 4°C and freeze-thaw cycles on its ability to kill S. Typhimurium D23580.
Serum from four healthy adults (subjects 1–4 corresponding with panels A.–D.) following one (green squares), two (blue triangles), three (purple diamonds) or no (red circles) freeze-thaw cycles was maintained at 4°C and examined at intervals for ability to kill in the serum bactericidal assay. Normal killing designated as a reduction of 1.0 log10 cfu/ml viable bacteria. Numbers of viable Salmonellae after 180 minutes in the assay are plotted against number of days serum kept at 4°C. Negative values show a decrease in viable bacteria compared with initial concentration. Classical (E.) and alternative (F.) pathway hemolytic complement activity in sera from the four subjects (1– red circles, 2– green squares, 3– blue triangles, 4– purple diamonds) stored at 4°C.
Figure 3
Figure 3. Effect of duration of storage of human serum at 22°C and 37°C on ability to kill S. Typhimurium D23580.
Serum from the same four adults as in Fig. 1 (1– red circles, 2– green squares, 3– blue triangles, 4– purple diamonds) was thawed and maintained at 22°C (A.) or 37°C (B.) with serum bactericidal assays performed daily (for samples at 22°C) or six- to twelve-hourly (for samples at 37°C) using 106 cfu/ml. Viable bacteria at 180 minutes in each serum bactericidal assay are plotted against duration of serum storage.
Figure 4
Figure 4. Effect of delayed separation of serum from clotted human blood on ability to kill S. Typhimurium D23580.
Blood from four healthy adults (subjects 1–4 corresponding with panels A.–B., C.–D., E.–F. and G.–H. respectively) was kept at 4°C (A., C., E., G.) or 22°C (B., D., F., H.) post-venesection and serum separated the same day (red circles) or after one (green squares), two (blue triangles), three (purple diamonds ), or four (orange inverted triangles) days after venesection. Separated sera were then maintained at 4°C without freezing for a total of four days post venesection before being used in the serum bactericidal assays and compared with fresh serum from each donor (black circles). Changes in cfu/ml are plotted against the duration of assay.
Figure 5
Figure 5. Effect of delayed separation of serum from clotted human blood on classical and alternative pathway hemolytic complement activity.
Classical (A., C., E., G.) and alternative (B., D., F., H.) pathway hemolytic complement activity in sera from four subjects (corresponding with panels A–B, C–D, E–F, G–H) separated from clotted blood on the day of venesected (day 0) or stored at 4°C (red circles) or 22°C (green squares) and separated 1–4 days post-venesection. Once separated, all samples were maintained at −80°C until tested in the radial immunodiffusion assays.
Figure 6
Figure 6. Effect of presence of Factor B antibody on ability of human serum to kill S. Typhimurium D23580.
Killing of bacteria by 90% fresh serum from subject 1 and 10% polyclonal anti-Factor B antibody (final concentration 0.92 mg/ml) (red circles), compared with 90% fresh serum from subject 1 and 10% PBS (green squares) and 100% heat-inactivated serum (black triangles) at 45, 90 and 180 minutes in the serum bactericidal assay using 106 cfu/ml S. Typhimurium D23580.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Nuttall GHF (1888) Experimente uber die bacterienfeindlichen Einflusse des thierischen Korpers. Zeitschr f Hygiene iv: 353–394.
    1. Bordet J (1895) Les leucocytes et les propriétés actives du sérum chez les vaccinés. Ann Inst Pasteur 9: 462.
    1. MacLennan CA, Gondwe EN, Msefula CL, Kingsley RA, Thomson NR, et al. (2008) The neglected role of antibody in protection against bacteremia caused by nontyphoidal strains of Salmonella in African children. The Journal of clinical investigation 118: 1553–1562. - PMC - PubMed
    1. Gondwe EN, Molyneux ME, Goodall M, Graham SM, Mastroeni P, et al... (2010) Importance of antibody and complement for oxidative burst and killing of invasive nontyphoidal Salmonella by blood cells in Africans. in press. doi/10.1073/pnas.0910497107. - PMC - PubMed
    1. Noguchi H, Bronfenbrenner J (1911) Effects of mechanical agitation and of temperature upon complement. J Exp Med 13: 229–233. - PMC - PubMed

Publication types

Substances