Cyclic Nucleotide (cAMP and cGMP) Assays and Capture ELISA for Quantitative Analysis of Plasmodium falciparum Blood-stage Egress

Materials and Reagents

1. Plasmodium falciparum schizonts
   
2. DetectX Direct cGMP or cAMP immunoassay kit (Arbor Assays, catalog number: K020-H1 or K019-H1 )
   
3. Protein-free RPMI 1640 (Life Technologies, Invitrogen^TM)
   
4. Albumax
   
5. Zaprinast (Sigma-Aldrich, catalog number: Z0878 )
   Note: Make up as a 50 mM stock solution in DMSO and stored at -20 °C.
   
6. PKG inhibitor compound 1 {4-[2-(4-fluorophenyl)-5-(1-methylpiperidine-4-yl)-1H-pyrrol-3-yl] pyridine} (Merck KGaA)
   Note: Make up as a 10 mM stock solution in DMSO and stored at -20 °C.
   
7. Anti-AMA1 monoclonal antibody (clone 4G2dc1) (Kocken et al., 1998)
   
8. Complete RPMI 1640 culture medium (Blackman, 1994)
   
9. Tween 20
   
10. Rabbit polyclonal anti-AMA1 serum (Collins et al., 2009)
    
11. Phosphatase-conjugated goat anti-rabbit IgG (whole molecule) (Sigma-Aldrich, catalog number: A3687 )
    
12. Phosphatase substrate (Sigma-Aldrich, catalog number: S0942 )
    
13. Phosphate-buffered saline (PBS)
    
14. Sodium azide [10% (w/v) stock solution in water]
    
15. Dry ice-ethanol mix for freezing
    
16. Gelatine-tween stock solution (blocking buffer) (see Recipes)
    
17. 0.1 M Sodium carbonate/bicarbonate (pH 9.6) (see Recipes)
    
18. Diethanolamine buffer (pH 9.8) (see Recipes)
    

Equipment

1. Centrifuge
   
2. 96-well Immulon plates (Nunc^®)
   
3. ELISA reader
   
4. Incubator
   

Procedure

1. Assay of cGMP levels in the malaria parasite
   1. For measurement of intracellular cyclic nucleotide levels, Percoll-enriched schizonts were suspended at a 5% haematocrit in gassed, protein-free RPMI 1640 at 37 °C. Standard procedures were used to culture the parasites and enrich schizonts (Blackman, 1994; Yeoh et al., 2007). The suspension was divided into aliquots each containing 2 x 10⁸ schizonts (2 x 10⁸ schizonts corresponds to ~20 μl packed cells).
      
   2. Duplicate zero time samples (each containing 2 x 10⁸ schizonts) were pelleted by centrifugation (1 min, 13,000 x g) and snap-frozen in a dry ice-ethanol mix.
      
   3. Further duplicate samples were then taken at various time intervals following addition of 75 µM zaprinast, zaprinast plus the 2.5 µM PKG inhibitor compound 1, or vehicle only (DMSO), and similarly processed. Zaprinast is an inhibitor of parasite phosphodiesterase (which degrades cGMP and cAMP), and so results in an increase in cGMP levels, inducing egress.
      
   4. All samples were stored at -70 °C until required for assay.
      
   5. cGMP and cAMP levels in schizont extracts were measured using a DetectX Direct cGMP or cAMP immunoassay kit , precisely as directed by the manufacturer.
      
      
2. Capture ELISA for quantitative analysis of P. falciparum blood-stage egress
   1. Coat 96-well Immulon plates with 100 μl/well of a stock solution of purified monoclonal antibody 4G2dc1 made up at 2.5 μg/ml in carbonate/bicarbonate buffer (pH 9.6).
      
   2. Seal the plates and incubate overnight in a humidified atmosphere at 4 °C. This and all subsequent incubations are done whilst stationary.
      
   3. Wash the plates once with PBS 0.5% (v/v) Tween 20 (PBS/T), then block by adding 250 μl/well of a 1:4 dilution of gelatine-Tween stock solution, and incubating for 2 h at room temperature.
      
   4. Prepare serial 2-fold dilutions of test culture supernatant samples in a separate 96-well plate. Dilute the samples into RPMI 1640 culture medium containing 0.5% Albumax. For negative control wells, use RPMI Albumax medium only. Add samples to the washed ELISA plates, then seal and incubate the plates for 1 h at room temperature.
      
   5. Wash plates 3x in PBS/T.
      
   6. Make up a solution of rabbit polyclonal anti-AMA1 serum diluted 1:1,000 in PBS/T. Add 100 μl/well, then seal and incubate the plates a further 1 h at room temperature.
      
   7. Wash plates 3x with PBS.
      Note: No Tween at this stage, as it interferes with the alkaline phosphatase activity in the next step of the assay.
      
   8. Add 100 μl/well phosphatase-conjugated goat anti-rabbit IgG (whole molecule), diluted 1:30,000 in PBS. Seal the plates, then incubate 1 h at room temperature.
      
   9. Wash plates 5x with PBS (no tween) then add 50 μl/well substrate (1 tablet of phosphatase substrate, in 5 ml diethanolamine buffer). Incubate in the dark at 37 °C for at least 30 min. Determine OD₄₀₅ using an ELISA reader.
      
   10. Use the data to produce titration curves of antigen levels in the various culture supernatants.
       

Recipes

1. Gelatine-tween stock solution (blocking buffer)
   5 g Gelatine
   2.5 ml Tween-20
   150 μl 10% (w/v) sodium azide
   500 ml PBS
   Warm at 37 °C to dissolve the gelatine
   Use at 1: 4 dilution in PBS
   Stored stock at 4 °C
   May require warming before coaxing out of the bottle
   
2. 0.1 M Sodium carbonate/bicarbonate (pH 9.6)
   Make up the two component buffers first
   These are
   7.155 g Na₂CO₃^.10H₂O made up to 250 ml water
   2.1 g NaHCO₃ made up to 250 ml water
   Mix the above buffers to obtain a buffer with a pH of 9.6
   As an approximate measure, this requires ~37 ml 0.1 M Na₂CO₃^.10H₂O plus 73 ml 0.1 M NaHCO₃
   
3. Diethanolamine buffer (pH 9.8)
   48.5 ml Diethanolamine
   982 μl1 M MgCl₂
   2.5 ml10% (w/v) sodium azide
   Add 400 ml water then adjust pH to 9.8 with 1 M HCl before making up to a total of 500 ml with water
   Stored in the dark at 4 °C
   

Acknowledgments

This method is adapted from an original method described in Collins et al. (2013). The authors are grateful to Alan Thomas (Biomedical Primate Research Centre, Rijswijk, The Netherlands) for the gift of monoclonal antibody 4G2dc1. This work was supported by the UK Medical Research Council (U117532063), and received funding from the European Community's Seventh Framework Programme (FP7/2007-2013) under grant agreement N° 242095 (EviMalAR).

References

1. Blackman, M. J. (1994). Purification of Plasmodium falciparum merozoites for analysis of the processing of merozoite surface protein-1. Methods Cell Biol 45: 213-220. 
2. Collins, C. R., Withers-Martinez, C., Hackett, F. and Blackman, M. J. (2009). An inhibitory antibody blocks interactions between components of the malarial invasion machinery. PLoS Pathog 5(1): e1000273.
   
3. Collins, C. R., Hackett, F., Strath, M., Penzo, M., Withers-Martinez, C., Baker, D. A. and Blackman, M. J. (2013). Malaria parasite cGMP-dependent protein kinase regulates blood stage merozoite secretory organelle discharge and egress. PLoS Pathog 9(5): e1003344.
   
4. Kocken, C. H., van der Wel, A. M., Dubbeld, M. A., Narum, D. L., van de Rijke, F. M., van Gemert, G. J., van der Linde, X., Bannister, L. H., Janse, C., Waters, A. P. and Thomas, A. W. (1998). Precise timing of expression of a Plasmodium falciparum-derived transgene in Plasmodium berghei is a critical determinant of subsequent subcellular localization. J Biol Chem 273(24): 15119-15124. 
   
5. Yeoh, S., O'Donnell, R. A., Koussis, K., Dluzewski, A. R., Ansell, K. H., Osborne, S. A., Hackett, F., Withers-Martinez, C., Mitchell, G. H., Bannister, L. H., Bryans, J. S., Kettleborough, C. A. and Blackman, M. J. (2007). Subcellular discharge of a serine protease mediates release of invasive malaria parasites from host erythrocytes. Cell 131(6): 1072-1083.