New developments in malaria diagnostics

Figures & data

Figure 1 Detection of native antigens in a crude parasite extract dilution from P. falciparum culture with selected monoclonal antibodies. (A) HRP2 antibodies (C1–13 and PTL3, triangles) compared with strongest HDP (marked with H, solid lines) and DHFR-TS (marked with D, open squares) antibodies, (B) DHFR-TS antibodies, (C) HDP antibodies and (D) GLURP antibodies.

Table 1 Antibody response of mouse sera to the recombinant antigen in ELISA at different days before each (booster) immunization during the immunization procedure

Antigen	Mouse	Before immunization (Day 0)	Day 29	Day 42	Day 48	#Cells * 10^6 harvested/ml
GLURP R0	A	0	1:12,000	≥1:15,36,000	1:3,20,000	60
	B§	0	1:3,000	≥1:15,36,000	≥1:102,40,000	60
GLURP R2	A§	0	1:3,000	≥1:15,36,000	1:40,000	30
	B	0	1:24,000	≥1:15,36,000	1:12,80,000	70
DHFR-TS	A	0	1:24,000	≥1:15,36,000	≥1:102,40,000	50
	B§	0	1:24,000	≥1:15,36,000	1:25,60,000	70
DHFR-1&2	A	0	1:12,000	≥1:15,36,000	≥1:102,40,000	20
	B§	0	1:24,000	≥1:15,36,000	≥1:102,40,000	50
HDP	A§	1:3,000	1:12,000*	1:48,000**	≥1:61,44,000***	220
	B	0	1:24,000*	1:96,000**	1:15,36,000***	390

Mouse A of HDP already had a background titer at Day 0.

§ This mouse was used for the fusion to myeloma cells; *day 28; **day 35; ***day 42.

Table 2 Overview and characteristics of hybridoma clones during immunization and selection process

Percentage of clones highly reactive in ELISA
Antigen target	Nr of wells started with	With recombinant antigen	With NF54	#selected clones based on ELISA	Proportion IgG	Proportion IgM
DHFR-TS insert 1 and 2	1008	8%	9%	16	1/16	15/16
TS	960	8%	6%	12	2/12	10/12
GLURP R0	1504	5%	5%	4	1/4	3/4
GLURP R2	576	9%	11%	10	7/10	3/10
HDP	2112	10%	5%	33	9/33	24/33

Table 3 Dissociation constants (K_D) of monoclonal antibodies as determined by ELISA

Target	mAb	Isotype	KD (nM)	SE	Target	mAb	Isotype	KD (nM)	SE	Target	mAb	Isotype	KD (nM)	SE
DHFR	D5	IgG3	0.10	0.014	HDP	H1	IgG1	1.5	0.64	GLURP	G2	IgG3	1.42	0.27
	D6	IgM	0.068	0.015		H2	IgM	0.93	0.11		G4	IgG1	3.4	0.45
	D7	IgM	0.36	0.039		H3	IgM	0.23	0.046		G12	IgM	0.54	0.089
	D10	IgM	0.66	0.19		H4	IgG2b	0.23	0.057		G13	IgM	0.45	0.079
	D13	IgM	0.41	0.20		H5	IgG3	2.1	0.82		G14	IgG2a	0.53	0.64
	D15		0.80	0.49		H6	IgM	0.70	0.16		G15	IgG2a	0.54	0.11
	D16	IgM	0.16	0.062		H7	IgM	0.32	0.030		G16	IgM	0.94	0.26
	D19	IgG1	0.88	0.32		H8	IgM	0.56	0.081		G18	IgG1	0.70	0.12
	D20	IgM	0.21	0.10		H12	IgG2a	0.83	0.25		G19	IgG3	0.56	0.10
	D24	IgM	2.4	1.3		H16	IgM	0.10	0.022		G22	IgG1	0.33	0.093
HRP2	PTL3	IgM	0.16	0.13		H18	IgM	0.21	0.022		G23	IgM	0.11	0.028
	C1–13	IgG	1.0	0.049							G24	IgM	0.34	0.14
											G25	IgM	1.4	0.33

Table 4 Detection limit of crude parasite antigen in ELISA by selected antibodies compared with HRP2 antibodies

	mAb		Detection limit of crude antigen
Antigen		ng	ng
HRP2	C1–13	250	8
HRP2	C1–13	25	8
DHFR	D6	250	25
DHFR	D15	25	25
HDP	H4	25	8
HDP	H12	25	8
HDP	H12	250	3
HDP	H15	250	3
HDP	H16	250	25
HRP2	PTL3	25	8
HRP2	PTL3	250	3

Table 5 Primer sequences for recombinant antigens and encoding nucleotide range

Fragment name	Nucleotide range		Primer
DHFR-TS insert 1 and 2	46–327 in PFD0830w	Forward primer	GGT GGT GCT AGC GCA TGT TGT AAG GTT GAA AGC
		Reverse primer	GGT GGT TGC TCT TCC GCA CCA GCT TGT TCT TCC CAT AAC
TS	1024–1824 in PFD0830w	Forward primer	GGT GGT GCT AGC CAA AGT GAT CGA ACG GGA GTA
		Reverse primer	GGT GGT TGC TCT TCC GCA AGC AGC CAT ATC CAT TGA AAT
GLURP R0	9–1281 in PF10_0344	Forward primer	GGT GGA CAT ATG AAT AAA CGA ATC GGG GGT CCT
		Reverse primer	GGT GGT TGC TCT TCC GCA TTT AGG GGA AGG CAC ATC
GLURP R2	2497–3204 in PF10_0344	Forward primer	GGT GGT CAT ATG GAA GTG GCT CAT CCA GAA
		Reverse primer	GGT GGT TGC TCT TCC GCA TTC ATT GTT ATT TGT TTG TGA TGG