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Dried blood spots as a source of anti-malarial antibodies for epidemiological studies.

Corran PH, Cook J, Lynch C, Leendertse H, Manjurano A, Griffin J, Cox J, Abeku T, Bousema T, Ghani AC, Drakeley C, Riley E - Malar. J. (2008)

Bottom Line: Eluates of these spots were assayed for antibodies against two Plasmodium falciparum antigens, MSP-119 and MSP2, and calculated titres used to fit an exponential (first order kinetic) decay model.When desiccated, recoveries of antibodies that are predominantly of IgG1 or IgG3 subclasses were similar.This study has demonstrated the suitability of filter paper blood spots paper for collection of serum antibodies, and provided clear guidelines for the treatment and storage of filter papers which emphasize the importance of desiccation and minimisation of time spent at ambient temperatures.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, London WC1E 7HT, UK. patrick.corran@lshtm.ac.uk

ABSTRACT

Background: Blood spots collected onto filter paper are an established and convenient source of antibodies for serological diagnosis and epidemiological surveys. Although recommendations for the storage and analysis of small molecule analytes in blood spots exist, there are no published systematic studies of the stability of antibodies under different storage conditions.

Methods: Blood spots, on filter paper or glass fibre mats and containing malaria-endemic plasma, were desiccated and stored at various temperatures for different times. Eluates of these spots were assayed for antibodies against two Plasmodium falciparum antigens, MSP-119 and MSP2, and calculated titres used to fit an exponential (first order kinetic) decay model. The first order rate constants (k) for each spot storage temperature were used to fit an Arrhenius equation, in order to estimate the thermal and temporal stability of antibodies in dried blood spots. The utility of blood spots for serological assays was confirmed by comparing antibodies eluted from blood spots with the equivalent plasma values in a series of samples from North Eastern Tanzania and by using blood spot-derived antibodies to estimate malaria transmission intensity in this site and for two localities in Uganda.

Results: Antibodies in spots on filter paper and glass fibre paper had similar stabilities but blood was more easily absorbed onto filter papers than glass fibre, spots were more regular and spot size was more closely correlated with blood volume for filter paper spots. Desiccated spots could be stored at or below 4 degrees C for extended periods, but were stable for only very limited periods at ambient temperature. When desiccated, recoveries of antibodies that are predominantly of IgG1 or IgG3 subclasses were similar. Recoveries of antibodies from paired samples of serum and of blood spots from Tanzania which had been suitably stored showed similar recoveries of antibodies, but spots which had been stored for extended periods at ambient humidity and temperature showed severe loss of recoveries. Estimates of malaria transmission intensity obtained from serum and from blood spots were similar, and values obtained using blood spots agreed well with entomologically determined values.

Conclusion: This study has demonstrated the suitability of filter paper blood spots paper for collection of serum antibodies, and provided clear guidelines for the treatment and storage of filter papers which emphasize the importance of desiccation and minimisation of time spent at ambient temperatures. A recommended protocol for collecting, storing and assaying blood spots is provided.

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Kinetics of loss of antibodies at different storage temperatures. Blood spots were stored at + 23, 35, 47, 57 or 65°C for up to 130 days and then removed to -20°C until analysis. ODs were converted to antibody concentrations by reference to a standard curve. Lines represent the best-fit first order rate constants for each temperature.(a) MSP-119 antibodies. (b) MSP-2 antibodies.
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Figure 4: Kinetics of loss of antibodies at different storage temperatures. Blood spots were stored at + 23, 35, 47, 57 or 65°C for up to 130 days and then removed to -20°C until analysis. ODs were converted to antibody concentrations by reference to a standard curve. Lines represent the best-fit first order rate constants for each temperature.(a) MSP-119 antibodies. (b) MSP-2 antibodies.

Mentions: Eluates were prepared from sets of blood spots from 3 MM and Glass Fibre paper that had been stored for differing lengths of time at temperatures between 23°C and 65°C and tested by ELISA for reactivity with MSP-119 and MSP-2. Antibody concentrations were calculated by reference to the standard curve. The rate of decay of the antibody concentrations fitted a classical first-order kinetic plot of ln(ct/c0) (where ct is the concentration of antibody remaining at time t days at elevated temperature and c0 is the original concentration) (Figure 4). When used to construct an Arrhenius plot (Figure 5), the fitted values, weighted by the log of the reciprocal of the variance of the fit, could be used to predict rates of loss of antibody titre at other temperatures (Table 1). From this, we extrapolated the storage time and temperature at which 5, 10 or 20% (t0.95, t0.9 or t0.8 respectively) of the original antibody activity would be lost or, conversely, the length of time for which spots could be stored at varying temperatures in order to recover 80, 90 or 95% of the original antibody reactivity (Table 2). Antibodies to both MSP-119 (which are predominantly of the IgG1 subclass[36]) and MSP-2 (predominantly IgG3[36]) decayed at the same rate (Figure 3 and Table 1).


Dried blood spots as a source of anti-malarial antibodies for epidemiological studies.

Corran PH, Cook J, Lynch C, Leendertse H, Manjurano A, Griffin J, Cox J, Abeku T, Bousema T, Ghani AC, Drakeley C, Riley E - Malar. J. (2008)

Kinetics of loss of antibodies at different storage temperatures. Blood spots were stored at + 23, 35, 47, 57 or 65°C for up to 130 days and then removed to -20°C until analysis. ODs were converted to antibody concentrations by reference to a standard curve. Lines represent the best-fit first order rate constants for each temperature.(a) MSP-119 antibodies. (b) MSP-2 antibodies.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2567984&req=5

Figure 4: Kinetics of loss of antibodies at different storage temperatures. Blood spots were stored at + 23, 35, 47, 57 or 65°C for up to 130 days and then removed to -20°C until analysis. ODs were converted to antibody concentrations by reference to a standard curve. Lines represent the best-fit first order rate constants for each temperature.(a) MSP-119 antibodies. (b) MSP-2 antibodies.
Mentions: Eluates were prepared from sets of blood spots from 3 MM and Glass Fibre paper that had been stored for differing lengths of time at temperatures between 23°C and 65°C and tested by ELISA for reactivity with MSP-119 and MSP-2. Antibody concentrations were calculated by reference to the standard curve. The rate of decay of the antibody concentrations fitted a classical first-order kinetic plot of ln(ct/c0) (where ct is the concentration of antibody remaining at time t days at elevated temperature and c0 is the original concentration) (Figure 4). When used to construct an Arrhenius plot (Figure 5), the fitted values, weighted by the log of the reciprocal of the variance of the fit, could be used to predict rates of loss of antibody titre at other temperatures (Table 1). From this, we extrapolated the storage time and temperature at which 5, 10 or 20% (t0.95, t0.9 or t0.8 respectively) of the original antibody activity would be lost or, conversely, the length of time for which spots could be stored at varying temperatures in order to recover 80, 90 or 95% of the original antibody reactivity (Table 2). Antibodies to both MSP-119 (which are predominantly of the IgG1 subclass[36]) and MSP-2 (predominantly IgG3[36]) decayed at the same rate (Figure 3 and Table 1).

Bottom Line: Eluates of these spots were assayed for antibodies against two Plasmodium falciparum antigens, MSP-119 and MSP2, and calculated titres used to fit an exponential (first order kinetic) decay model.When desiccated, recoveries of antibodies that are predominantly of IgG1 or IgG3 subclasses were similar.This study has demonstrated the suitability of filter paper blood spots paper for collection of serum antibodies, and provided clear guidelines for the treatment and storage of filter papers which emphasize the importance of desiccation and minimisation of time spent at ambient temperatures.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, London WC1E 7HT, UK. patrick.corran@lshtm.ac.uk

ABSTRACT

Background: Blood spots collected onto filter paper are an established and convenient source of antibodies for serological diagnosis and epidemiological surveys. Although recommendations for the storage and analysis of small molecule analytes in blood spots exist, there are no published systematic studies of the stability of antibodies under different storage conditions.

Methods: Blood spots, on filter paper or glass fibre mats and containing malaria-endemic plasma, were desiccated and stored at various temperatures for different times. Eluates of these spots were assayed for antibodies against two Plasmodium falciparum antigens, MSP-119 and MSP2, and calculated titres used to fit an exponential (first order kinetic) decay model. The first order rate constants (k) for each spot storage temperature were used to fit an Arrhenius equation, in order to estimate the thermal and temporal stability of antibodies in dried blood spots. The utility of blood spots for serological assays was confirmed by comparing antibodies eluted from blood spots with the equivalent plasma values in a series of samples from North Eastern Tanzania and by using blood spot-derived antibodies to estimate malaria transmission intensity in this site and for two localities in Uganda.

Results: Antibodies in spots on filter paper and glass fibre paper had similar stabilities but blood was more easily absorbed onto filter papers than glass fibre, spots were more regular and spot size was more closely correlated with blood volume for filter paper spots. Desiccated spots could be stored at or below 4 degrees C for extended periods, but were stable for only very limited periods at ambient temperature. When desiccated, recoveries of antibodies that are predominantly of IgG1 or IgG3 subclasses were similar. Recoveries of antibodies from paired samples of serum and of blood spots from Tanzania which had been suitably stored showed similar recoveries of antibodies, but spots which had been stored for extended periods at ambient humidity and temperature showed severe loss of recoveries. Estimates of malaria transmission intensity obtained from serum and from blood spots were similar, and values obtained using blood spots agreed well with entomologically determined values.

Conclusion: This study has demonstrated the suitability of filter paper blood spots paper for collection of serum antibodies, and provided clear guidelines for the treatment and storage of filter papers which emphasize the importance of desiccation and minimisation of time spent at ambient temperatures. A recommended protocol for collecting, storing and assaying blood spots is provided.

Show MeSH
Related in: MedlinePlus