Author:
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- Range of detection of pLDH and HRP in in vitro–cultivated P. falciparum material.
- Construction of standard graphs for pLDH and HRP-2.
- Comparison of pLDH and HRP levels in graded whole culture material.
- Comparison of microscopy and ELISA (pLDH and HRPs) from the same patient whole blood sample.
- Measurement of pLDH and HRP in malaria-infected human plasma samples.
- DISCUSSION
ProCite
RefWorks
Reference Manager
BibTeX
Zotero
EndNote
-
1
Hanscheid T, 1999. Diagnosis of malaria: a review of alternatives to conventional microscopy. Clin Lab Haematol 21 :235–245.
-
2
Leke RF, Djokam RR, Mbu R, Leke RJ, Fogako J, Megnekou R, Metenou S, Sama G, Zhou Y, Cadigan T, Parra M, Taylor DW, 1999. Detection of the Plasmodium falciparum antigen histidine-rich protein2 in blood of pregnant women: implications for diagnosing placental malaria. J Clin Microbiol 37 :2992–2996.
-
3
Oaks SC, Mitchell VS, Pearson GW, Carpenter CJ (eds.), 1991. Malaria: Obstacles and Opportunities. Washington, DC: National Academy Press.
-
4
Dondorp AM, Desakorn V, Pongtavornpinyo W, Sahassananda D, Silamut K, 2005. Estimation of the total parasite biomass in acute falciparum malaria from plasma PfHRP2. PLoS Med 2 :788–797.
-
5
Druilhe P, Moreno L, Blanc C, Brasseur PH, Jacquier P, 2001. A calorimetric in vitro drug sensitivity assay for Plasmodium falciparum based on a highly sensitive double-site lactate dehydrogenase antigen-capture enzyme-linked immunosorbent assay. Am J Trop Med Hyg 64 :233–241.
-
6
Makler MT, Hinrichs DJ, 1993. Measurement of the lactate dehydrogenase activity of Plasmodium falciparum as an assessment of parasitaemia. Am J Trop Med Hyg 48 :205–210.
-
7
Piper R, Lebras J, Wentworth L, Hunt-Cooke A, Houzé S, Chiodini P, Makler M, 1999. Immunocapture diagnostic assays for malaria utilizing Plasmodium lactate dehydrogenase (pLDH). Am J Trop Med Hyg 60 :109–118.
-
8
Trager W, Jensen JB, 1976. Human malarial parasites in continuous culture. Science 193 :673–675.
-
9
Wellems TE, Walker-Jonah A, Panton LJ, 1991. Genetic mapping of the chloroquine-resistance locus on Plasmodium falciparum chromosome 7. Proc Natl Acad Sci USA 88 :3382–3386.
-
10
Sasaki D, Mitchell RA, 2007. How to obtain reproducible quantitative ELISA results. Available at: www.oxfordbiomed.com/artqelis.html.
-
11
TrochimWMK, 2006. Research methods knowledge base.Available at: www.socialresearchmethods.net/kb/satcorr.php.
-
12
Kilejian A, 1974. A unique histidine-rich polypeptide from the malaria parasite, Plasmodium lophurae. J Biol Chem 249 :4650–4655.
-
13
Kilejian A, Jensen JB, 1977. A histidine-rich protein from Plasmodium falciparum and its interaction with membranes. Bull World Health Organ 55 :191–197.
-
14
Noedl H, Wernsdorfer WH, Miller RS, Wongsrichanalai C, 2002. Histidine-rich protein-II: a novel approach to malaria drug sensitivity testing. Antimicrob Agents Chemother 46 :1658–1664.
-
15
Taylor DW, Voller A, 1993. The development and validation of a simple detection ELISA for Plasmodium falciparum malaria. Trans R Soc Trop Med Hyg 87 :29–31.
-
16
Beadle C, Long GW, Weiss WR, McElroy PD, Maret SM, Oloo AJ, Hoffman SL, 1994. Diagnosis of malaria by detection of Plasmodium falciparum HRP-2 antigen with a rapid dipstick antigen-capture assay. Lancet 343 :564–568.
-
17
Bell DR, Wilson DW, Martin LB, 2005. False positive results of a Plasmodium falciparum histidine-rich protein-2 detecting malaria rapid diagnostic test due to high sensitivity in a community with fluctuating low parasite density. Am J Trop Med Hyg 73 :199–203.
-
18
Vander Jagt DL, Hunsaker LA, Heidrich JE, 1981. Partial purification and characterization of lactate dehydrogenase from Plasmodium falciparum. Mol Biochem Parasitol 4 :255–264.
-
19
Vander Jagt DL, Hunsaker LA, Campos NM, Baack BR, 1990. D-lactate production in erythrocytes infected with Plasmodium falciparum. Mol Biochem Parasitol 42 :277–284.
-
20
Makler MT, Palmer CJ, Ager AL, 1998. A review of practical techniques for the diagnosis of malaria. Ann Trop Med Parasitol 92 :419–433.
-
21
Rajasekariah GR, Kay GE, Russell NV, Smithyman AM, 2003. Assessment of assay sensitivity and precision in a malaria antibody ELISA. J Immunoassay Immunochem 24 :89–112.
| Past two years | Past Year | Past 30 Days | |
|---|---|---|---|
| Abstract Views | 26 | 26 | 11 |
| Full Text Views | 250 | 83 | 0 |
| PDF Downloads | 62 | 14 | 0 |
Unified Parasite Lactate Dehydrogenase and Histidine-Rich Protein ELISA for Quantification of Plasmodium falciparum
Samuel K. Martin
Samuel K. Martin
United States Army Medical Research Unit, Nairobi, Kenya; Biofirm Pty, Sydney, Australia
Search for other papers by Samuel K. Martin in
Current site
Google Scholar
PubMed
Search for other papers by Samuel K. Martin in
Current site
Google Scholar
PubMed
G-Halli Rajasekariah
G-Halli Rajasekariah
United States Army Medical Research Unit, Nairobi, Kenya; Biofirm Pty, Sydney, Australia
Search for other papers by G-Halli Rajasekariah in
Current site
Google Scholar
PubMed
Search for other papers by G-Halli Rajasekariah in
Current site
Google Scholar
PubMed
George Awinda
George Awinda
United States Army Medical Research Unit, Nairobi, Kenya; Biofirm Pty, Sydney, Australia
Search for other papers by George Awinda in
Current site
Google Scholar
PubMed
Search for other papers by George Awinda in
Current site
Google Scholar
PubMed
John Waitumbi
John Waitumbi
United States Army Medical Research Unit, Nairobi, Kenya; Biofirm Pty, Sydney, Australia
Search for other papers by John Waitumbi in
Current site
Google Scholar
PubMed
Search for other papers by John Waitumbi in
Current site
Google Scholar
PubMed
Carolyne Kifude
Carolyne Kifude
United States Army Medical Research Unit, Nairobi, Kenya; Biofirm Pty, Sydney, Australia
Search for other papers by Carolyne Kifude in
Current site
Google Scholar
PubMed
Search for other papers by Carolyne Kifude in
Current site
Google Scholar
PubMed
There is a need for more objective and quantitative tools to replace microscopy in malaria diagnosis. Emphasis has recently been placed on alternative methods such as immunochromatography-based rapid tests. However, these tests provide only qualitative results. Two bio-molecules, parasite lactate dehydrogenase (pLDH) and histidine-rich proteins (HRPs), that are released by the intra-erythrocytic stages of the parasite offer certain specific characteristics that could potentially improve malaria diagnosis. In this paper, we describe a protocol for a unified sandwich ELISA that allows for the separate but concurrent measurement of pLDH and HRP biomolecules in aliquots taken from the same samples. Freshly drawn blood from a healthy unexposed adult male was used to serially dilute in vitro cultivated and synchronized ring stage Plasmodium falciparum parasites. Commercially available ELISA formats were modified to allow for the measurement of pLDH and HRP from aliquots of the same samples. The pLDH and HRP levels in the samples spiked with known numbers of infected red blood cells (iRBCs) were measured, and the values were used to generate standard graphs. The standard graphs were used to estimate the numbers of iRBCs in test samples. Serially diluted recombinant proteins were similarly used to generate a calibration curve, allowing for the expression of test results in nanograms of their respective recombinant protein. Levels of pLDH and HRPs were determined by using 1) P. falciparum culture material (cells and medium) 2) P. falciparum infected human blood (N = 6) samples, and 3) plasma from P. falciparum–infected patient (N = 22) samples. The parasite density of all culture and infected patient samples was also estimated by microscopy. Both pLDH and HRP levels correlated positively with the parasite density assessed by microscopy: Pearson correlation coefficient pLDH (r = 0.754, P < 0.0001, 95% CI: 0.47–0.89); HRP (r = 0.552, P < 0.007, 95% CI: 0.16–0.79). The HRPs seem to be released in larger quantities than pLDH (in a ratio of ~1 pLDH:~6 HRP), making the detection of HRP in culture material, blood, and plasma easier. The modified ELISA assay with quantitative measurement of pLDH and HRPs may provide a valuable tool for malaria research and patient management.
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- Range of detection of pLDH and HRP in in vitro–cultivated P. falciparum material.
- Construction of standard graphs for pLDH and HRP-2.
- Comparison of pLDH and HRP levels in graded whole culture material.
- Comparison of microscopy and ELISA (pLDH and HRPs) from the same patient whole blood sample.
- Measurement of pLDH and HRP in malaria-infected human plasma samples.
- DISCUSSION
ProCite
RefWorks
Reference Manager
BibTeX
Zotero
EndNote
-
1
Hanscheid T, 1999. Diagnosis of malaria: a review of alternatives to conventional microscopy. Clin Lab Haematol 21 :235–245.
-
2
Leke RF, Djokam RR, Mbu R, Leke RJ, Fogako J, Megnekou R, Metenou S, Sama G, Zhou Y, Cadigan T, Parra M, Taylor DW, 1999. Detection of the Plasmodium falciparum antigen histidine-rich protein2 in blood of pregnant women: implications for diagnosing placental malaria. J Clin Microbiol 37 :2992–2996.
-
3
Oaks SC, Mitchell VS, Pearson GW, Carpenter CJ (eds.), 1991. Malaria: Obstacles and Opportunities. Washington, DC: National Academy Press.
-
4
Dondorp AM, Desakorn V, Pongtavornpinyo W, Sahassananda D, Silamut K, 2005. Estimation of the total parasite biomass in acute falciparum malaria from plasma PfHRP2. PLoS Med 2 :788–797.
-
5
Druilhe P, Moreno L, Blanc C, Brasseur PH, Jacquier P, 2001. A calorimetric in vitro drug sensitivity assay for Plasmodium falciparum based on a highly sensitive double-site lactate dehydrogenase antigen-capture enzyme-linked immunosorbent assay. Am J Trop Med Hyg 64 :233–241.
-
6
Makler MT, Hinrichs DJ, 1993. Measurement of the lactate dehydrogenase activity of Plasmodium falciparum as an assessment of parasitaemia. Am J Trop Med Hyg 48 :205–210.
-
7
Piper R, Lebras J, Wentworth L, Hunt-Cooke A, Houzé S, Chiodini P, Makler M, 1999. Immunocapture diagnostic assays for malaria utilizing Plasmodium lactate dehydrogenase (pLDH). Am J Trop Med Hyg 60 :109–118.
-
8
Trager W, Jensen JB, 1976. Human malarial parasites in continuous culture. Science 193 :673–675.
-
9
Wellems TE, Walker-Jonah A, Panton LJ, 1991. Genetic mapping of the chloroquine-resistance locus on Plasmodium falciparum chromosome 7. Proc Natl Acad Sci USA 88 :3382–3386.
-
10
Sasaki D, Mitchell RA, 2007. How to obtain reproducible quantitative ELISA results. Available at: www.oxfordbiomed.com/artqelis.html.
-
11
TrochimWMK, 2006. Research methods knowledge base.Available at: www.socialresearchmethods.net/kb/satcorr.php.
-
12
Kilejian A, 1974. A unique histidine-rich polypeptide from the malaria parasite, Plasmodium lophurae. J Biol Chem 249 :4650–4655.
-
13
Kilejian A, Jensen JB, 1977. A histidine-rich protein from Plasmodium falciparum and its interaction with membranes. Bull World Health Organ 55 :191–197.
-
14
Noedl H, Wernsdorfer WH, Miller RS, Wongsrichanalai C, 2002. Histidine-rich protein-II: a novel approach to malaria drug sensitivity testing. Antimicrob Agents Chemother 46 :1658–1664.
-
15
Taylor DW, Voller A, 1993. The development and validation of a simple detection ELISA for Plasmodium falciparum malaria. Trans R Soc Trop Med Hyg 87 :29–31.
-
16
Beadle C, Long GW, Weiss WR, McElroy PD, Maret SM, Oloo AJ, Hoffman SL, 1994. Diagnosis of malaria by detection of Plasmodium falciparum HRP-2 antigen with a rapid dipstick antigen-capture assay. Lancet 343 :564–568.
-
17
Bell DR, Wilson DW, Martin LB, 2005. False positive results of a Plasmodium falciparum histidine-rich protein-2 detecting malaria rapid diagnostic test due to high sensitivity in a community with fluctuating low parasite density. Am J Trop Med Hyg 73 :199–203.
-
18
Vander Jagt DL, Hunsaker LA, Heidrich JE, 1981. Partial purification and characterization of lactate dehydrogenase from Plasmodium falciparum. Mol Biochem Parasitol 4 :255–264.
-
19
Vander Jagt DL, Hunsaker LA, Campos NM, Baack BR, 1990. D-lactate production in erythrocytes infected with Plasmodium falciparum. Mol Biochem Parasitol 42 :277–284.
-
20
Makler MT, Palmer CJ, Ager AL, 1998. A review of practical techniques for the diagnosis of malaria. Ann Trop Med Parasitol 92 :419–433.
-
21
Rajasekariah GR, Kay GE, Russell NV, Smithyman AM, 2003. Assessment of assay sensitivity and precision in a malaria antibody ELISA. J Immunoassay Immunochem 24 :89–112.
| Past two years | Past Year | Past 30 Days | |
|---|---|---|---|
| Abstract Views | 26 | 26 | 11 |
| Full Text Views | 250 | 83 | 0 |
| PDF Downloads | 62 | 14 | 0 |