Development and optimization of high-throughput methods to measure Plasmodium falciparum-specific growth inhibitory antibodies

Abstract

Antibodies that inhibit replication of Plasmodium falciparum in erythrocytes are thought to be important both in acquired immunity to malaria and as mediators of immunity generated by candidate blood-stage vaccines. However, several constraints have limited the study of these functional antibodies in population studies and vaccine trials. We report the development and optimization of high-throughput growth inhibition assays with improved sensitivity that use minimal volumes of test serum. The major inhibitory activity of serum from exposed donors was antibody mediated, but nonspecific inhibitory factors were found in untreated serum. Culture volumes could be effectively reduced to 25 microl to limit amounts of test serum or inhibitors used in assays. Performing inhibition assays over two cycles of parasite replication gave greater sensitivity than single-cycle assays, and a simple two-cycle inhibition assay was developed that yielded highly reproducible results. Determination of parasite growth by flow cytometry was most suitable for high-throughput assays using small culture volumes and was more sensitive than parasite lactate dehydrogenase assays and less prone to error and variation than microscopy. We evaluated and optimized methods to remove antimalarials and nonspecific inhibitory factors from serum that are suitable for use with small volumes of samples that are typically obtained from clinical studies. Both microdialysis and immunoglobulin purification by ammonium sulfate precipitation were effective and practical. These methods should facilitate evaluation of vaccine trials and clinical studies of immunity and are also suitable for testing drugs and other compounds for antimalarial activity.

FIG. 1.

Measurement of parasite LDH activity for evaluation of P. falciparum growth. (A) Sera from adult donors were tested in a growth inhibition assay over one cycle of parasite replication, and parasitemia was measured using microscopy or pLDH activity. Growth inhibition determined by the two methods was significantly correlated (r = 0.794, P = 0.001). Results are expressed as percentages of parasite growth among nonexposed serum controls. (B) Sera were tested in repeat growth inhibition assays over one cycle of replication, with evaluation of parasitemia by pLDH activity. Results were significantly correlated (r = 0.804, P < 0.001). Data are expressed as percentages of parasite growth among nonexposed serum controls. (C) Parasite LDH activity was measured among ring-stage parasites (rings), mature pigmented trophozoites, pigmented trophozoites (trophs) with spent culture medium (CM), or spent culture medium alone. LDH activity was severalfold higher among pigmented trophozoites compared to rings, and the majority of activity was present in parasitized erythrocytes rather than in the parasite culture medium. The parasite line CS2 was used. Results represent means + standard errors of the means (samples tested in triplicate).

FIG. 2.

Comparison of P. falciparum growth inhibition assays performed over one or two life cycles. The starting parasitemia (using parasite line 3D7) for the two-cycle assay was one-fourth of the parasitemia for the single-cycle assay. The degree of inhibition by sera or antibodies was greater in the two-cycle assay. Results are expressed as percentages of PBS controls (contr) for each assay. Sercontr, serum from normal Australian donor; AMA, rabbit polyclonal antibody against AMA1, used at 0.5 or 0.25 mg/ml; PNG 1 to 6, dialyzed sera from Papua New Guinean adults. All samples were used in duplicate, and error bars indicate ranges.

FIG. 3.

Evaluation of parasitemia using flow cytometry. 3D7 parasites were used in a growth inhibition assay performed over two life cycles; the assay was stopped after approximately 80 h. Parasitized erythrocytes were labeled with ethidium bromide, and cells were processed using a FACSCalibur flow cytometer. Data were analyzed and plotted using FlowJo software. The population of intact erythrocytes was first gated and then plotted as a histogram. The y axis shows the number of events, and the x axis shows fluorescence in channel 2 (for ethidium bromide). The total parasitemia in this example was 10.4%.

FIG. 4.

Two-cycle growth inhibition assays. (A) Comparison of flow cytometry versus microscopy for evaluation of parasite growth using dialyzed serum from exposed and nonexposed donors. (B) Comparison of results from growth inhibition assays performed with or without intermittent agitation, using dialyzed serum samples. Results are expressed as a percentage of parasite growth among nonexposed serum controls. S1 to 4, sera from nonexposed Australian donors; 1 to 24, sera from adults living in Papua New Guinea. (C) Dialyzed sera were tested in repeat growth inhibition assays over two cycles of replication, with evaluation of parasitemia by flow cytometry. Results were significantly correlated (r = 0.872, P < 0.001). Data are expressed as percentages of parasite growth among nonexposed serum controls.

FIG. 5.

Evaluation of two-cycle growth inhibition assays with measurement of parasitemia (3D7 parasites) using pLDH activity (LDH) versus flow cytometry (Facs). (A) Different concentrations of polyclonal rabbit antibody against AMA1 were tested for inhibition of parasite growth. Final concentrations (mg/ml) are as follows: 1, 0.5; 2, 0.25; 3, 0.12; 4, 0.06; 5, 0.03. (B) The effect of using different starting parasitemias on parasite replication over two cycles was evaluated. Starting parasitemias are as follows: 1, 0.8%; 2, 0.6%; 3, 0.4%; 4, 0.2%; 5, 0.1%. Results are expressed as percentages of parasite growth among nonexposed serum controls. All samples were tested in duplicate, and error bars indicate ranges.

FIG. 6.

(A) Dialysis of serum samples effectively removes antimalarial drugs added to serum. Quinine in different concentrations was added to serum from a nonexposed Australian donor. The serum was dialyzed and used in a growth inhibition assay performed over one cycle of replication with measurement of parasitemia by flow cytometry. Untr ser, untreated serum; Q, quinine; Dial, dialyzed. (B) Comparison of dialyzed serum samples, Ig purified from the same sera, and untreated serum tested for growth inhibitory activity using an assay performed over two cycles of parasite replication. S1 and S2, sera from nonexposed Australian donors; A to K, sera from adult residents of PNG. (C) Different dilutions of serum or plasma were tested in a growth inhibition assay performed over two life cycles. AMA, rabbit polyclonal antibody against AMA1; PNG, plasma from an adult from PNG; Ser, serum from a nonimmune Swedish donor. All samples were used in duplicate with the 3D7 parasite line, and error bars indicate ranges.

FIG. 7.

SDS-PAGE of serum and immunoglobulins stained with Coomassie. Lanes 1 to 3, serum from the same nonimmune Australian donor; lane 1, dialyzed serum; lane 2, Ig purified from serum by ammonium sulfate precipitation; lane 3, untreated serum; lane 4, purified human IgG (from Pierce).