We thank Mr. Kirakorn Kiatibutr, MVRU, for training and guidance. We also thank Mrs. Pinyapat Kongngen, MVRU, for her technical assistance in preparing Figure 2. This work was supported by a grant from the National Institute for Allergy and Infectious Diseases and the National Institute of Health (U19 AI089672).

1. Preparation of reagents
NOTE: Refer to the Table of Materials for a list of equipment, reagents, and other consumables used in this protocol and to Table 1 for a list of solutions and their composition.
<ol>
	<li>Capture and peroxidase-conjugated mAbs
	<ol>
		<li>To reconstitute the mAb, resuspend the lyophilized mAb in a 1:1 mixture of distilled water:glycerol at 0.5 mg/mL. Make aliquots as needed to avoid repeated freeze-thawing, and store them at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Blocking buffer (BB)
	<ol>
		<li>Prepare the blocking buffer by dissolving 5 g of ELISA-grade casein in 100 mL of 0.1 N NaOH. Add 900 mL of phosphate-buffered saline (PBS) (see Table 1) to bring the final volume to 1.0 L.</li>
		<li>Add 0.02 g of phenol red as an indicator and adjust the pH to 7.4 with HCl. Store BB at 4 &#176;C for up to one week, or aliquot into 50 mL for long-term storage at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Positive controls
	<ol>
		<li>To reconstitute the positive controls, rehydrate the lyophilized proteins by adding 1,000 &#956;L of BB. Make aliquots of the stock positive control solutions as needed, and store them at -20 &#176;C.</li>
		<li>For serial dilution, further dilute each positive control to the final working concentration in BB as follows: Pf, 2 pg/&#181;L; Pv (VK210), 182 (pg/&#181;L); Pv (VK247), 89 pg/&#181;L. 
		&#8203;NOTE: The exact of concentrations of the positive controls may vary from one lot to the next. Consult the product information sheet for the exact concentration needed. The positive control concentrations, starting from the working concentration above, are 2, 1, 0.5, 0.25, 0.13, 0.06 pg/&#956;L for Pf; 182, 91, 46, 23, 11, 5.7 pg/&#956;L for PV210; and 89, 45, 22, 11, 5.6, 2.8 pg/&#956;L PV247.</li>
	</ol>
	</li>
	<li>Negative controls 
	NOTE: The ideal negative control is the head-thorax homogenate of female Anopheles mosquitoes prepared identically as the test samples. However, BB can also be used as a negative control.
	<ol>
		<li>With BB as the negative control, use the 2-fold absorbance threshold for reliable positive readouts.</li>
	</ol>
	</li>
</ol>
2. Mosquito sample preparation
<ol>
	<li>Separate the head and the thorax of each collected adult mosquito from the abdomen with a sterile razor blade. Place the head and thorax in a prelabeled 1.5 mL centrifuge grinding tube. Pool heads and thoraces of up to 10 mosquitoes if desired. 
	NOTE: For sample preparation, typically, the salivary gland from an infected mosquito will be dissected and subjected to CS-ELISA. However, the head and thorax of collected mosquitoes can also be used to perform CS-ELISA directly (without dissecting for salivary glands)12,13,19.</li>
	<li>Add 50 &#956;L of Grinding Buffer (GB) into each tube and homogenize the sample with a clean pestle (washed with soap). Rinse the used pestle with 250 &#956;L of GB into the tube containing the homogenized mosquito(es) to a final volume of ~300 &#956;L.</li>
	<li>Keep the sample in a freezer (-20 &#176;C) until use or proceed immediately to perform ELISA.</li>
</ol>
3. Sporozoite ELISA
<ol>
	<li>Fill out the sporozoite ELISA worksheet (see Supplemental Material 1). Prepare one ELISA plate for each CSP (Pf, Pv-210, or Pv-247).</li>
	<li>Prepare the capture mAb working solution by dissolving the antibody in PBS: 4 &#181;g/mL Pf; 2 &#181;g/mL Pv-210; 2 &#181;g/mL of Pv-247. Calculate the volumes required based on the addition of 5 mL per plate. Vortex the solution gently.</li>
	<li>Pipette 50 &#956;L of each working mAb solution from step 3.2 into each well of the ELISA plate. Cover the plate with a plastic lid and incubate for 30 min or overnight at room temperature.</li>
	<li>Aspirate the well contents and tap the plate upside down on paper towels at least 5 times to remove all liquid. 
	NOTE: If an aspiration system (multichannel vacuum suction connected to clean tips) is not available, dump out the antibodies into the sink and then tap the plate on paper towels.</li>
	<li>Add 200 &#956;L of BB to fill all wells in the plate. Cover the plate with a plastic lid. Incubate the plate for 1 h at room temperature. Aspirate or dump out the well contents. Tap the plate upside down on paper towels 5 times to remove all liquid.</li>
	<li>Load the mosquito homogenate and the control on the plate as follows.
	<ol>
		<li>Add 50 &#956;L of the positive control to wells H1 and H2.</li>
		<li>Add 50 &#956;L of BB to wells in columns 1 and 2 from row C to G. Then, add 50 &#956;L of the positive control into wells G1 and G2. Make a serial dilution of the positive control starting from G1 and G2 followed by F1 and F2 until C1 and C2. 
		NOTE: All positive control wells should contain 50 &#956;L.</li>
		<li>Add 50 &#956;L of BB (negative control) to wells A1, A2, B1, and B2.</li>
		<li>Add 50 &#956;L of each homogenate sample to an Unknown (Unk) well.</li>
		<li>Cover the plate and incubate for 2 h at room temperature.</li>
	</ol>
	</li>
	<li>After approximately 2 h, start preparing the substrates. For the ABTS substrate 2-component kit, mix substrate A and substrate B in a 1:1 ratio. 
	NOTE: A full 96-well plate requires 5 mL of substrate A and 5 mL of substrate B.</li>
	<li>Prepare the working solutions of peroxidase-labeled mAbs for Pf, Pv-210, and Pv-247 by adding BB to the reconstituted conjugate mAb to obtain a working concentration of 1 &#181;g/mL.
	<ol>
		<li>Calculate the required volumes based on the addition of 5 mL of working conjugate mAb solution per plate.</li>
		<li>Test peroxidase activity by mixing 5 &#956;L of the peroxidase-labeled mAb made in step 3.8 with 100 &#956;L of the substrate made in step 3.7 in a separate 1.5 mL tube. Vortex gently. 
		NOTE: There should be a rapid color change from clear to green, indicating that the peroxidase enzyme and the substrates are working.</li>
	</ol>
	</li>
	<li>Aspirate or dump the well contents and tap the plate upside down on paper towels 5 times to remove all liquid.</li>
	<li>Wash the wells twice with 200 &#956;L of PBS-Tween, aspirate the well contents, and tap the plate 5 times each.</li>
	<li>Add 50 &#956;L of peroxidase-labeled mAb made in step 3.8 to each well. Cover the plate and incubate for 1 h at room temperature in the dark. Aspirate or dump the well contents and tap the plate upside down on a paper towel 5 times.</li>
	<li>Wash the wells 3 times with 200 &#956;L of PBS-Tween, aspirate the well contents, and tap the plate 5 times each.</li>
	<li>Add 100 &#956;L of the substrate solution prepared in step 3.7 to each well. Cover the plate and incubate for 30 min at room temperature in the dark.</li>
	<li>After 30 min, read the absorbance at 405-414 nm using an ELISA plate reader. 
	&#8203;NOTE: Follow the specific instructions for the ELISA plate reader used. For details on the instructions for the software used for this protocol, refer to Supplemental Material S2. There should be a noticeable color change from clear to green in the positive control wells.</li>
</ol>
4. Analysis
<ol>
	<li>Detecting positive samples.
	<ol>
		<li>Label samples with absorbance values above the cut-off (twice the mean absorbance value of the negative samples) as positive.</li>
	</ol>
	</li>
	<li>Quantifying CSP
	<ol>
		<li>Estimate the CSP concentration in the sample using the standard curve constructed from the control dilution series as follows.
		<ol>
			<li>Create the standard curve by plotting the absorbance values (y-axis) of the serially diluted controls against their concentrations (x-axis).</li>
			<li>Perform linear regression to determine the best fit using y = A + Bx, where A and B are free parameters.</li>
			<li>Determine the CSP concentration for each positive sample by solving the equation for a given absorbance value.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

<ol><li>Bray, R. S., Garnham, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+life-cycle+of+primate+malaria+parasites%5BTitle%5D)+AND+%22British+Medical+Bulletin%22%5BJournal%5D)">The life-cycle of primate malaria parasites.</a> British Medical Bulletin. 38 (2), 117-122 (1982).</li>
<li>Held, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Primate+malaria%5BTitle%5D)+AND+%22Annals+of+the+New+York+Academy+of+Sciences%22%5BJournal%5D)">Primate malaria.</a> Annals of the New York Academy of Sciences. 162 (1), 587-593 (1969).</li>
<li>World Health Organization. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Division+of+Malaria+and+Other+Parasitic+Diseases.+Manual+on+practical+entomology+in+Malaria%2C+Part+I+and+Part+II%5BTitle%5D)+AND+%22World+Health+Organization%22%5BJournal%5D)">Division of Malaria and Other Parasitic Diseases. Manual on practical entomology in Malaria, Part I and Part II.</a> World Health Organization. , Geneva. (1975).</li>
<li>Robert, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Study+of+the+distribution+of+circumsporozoite+antigen+in+Anopheles+gambiae+infected+with+Plasmodium+falciparum%2C+using+the+enzyme-linked+immunosorbent+assay%5BTitle%5D)+AND+%22Transactions+of+the+Royal+Society+of+Tropical+Medicine+and+Hygiene%22%5BJournal%5D)">Study of the distribution of circumsporozoite antigen in Anopheles gambiae infected with Plasmodium falciparum, using the enzyme-linked immunosorbent assay.</a> Transactions of the Royal Society of Tropical Medicine and Hygiene. 82 (3), 389-391 (1988).</li>
<li>Fontenille, D., Meunier, J. Y., Nkondjio, C. A., Tchuinkam, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Use+of+circumsporozoite+protein+enzyme-linked+immunosorbent+assay+compared+with+microscopic+examination+of+salivary+glands+for+calculation+of+malaria+infectivity+rates+in+mosquitoes+%28Diptera%3A+Culicidae%29+from+Cameroon%5BTitle%5D)+AND+%22Journal+of+Medical+Entomology%22%5BJournal%5D)">Use of circumsporozoite protein enzyme-linked immunosorbent assay compared with microscopic examination of salivary glands for calculation of malaria infectivity rates in mosquitoes (Diptera: Culicidae) from Cameroon.</a> Journal of Medical Entomology. 38 (3), 451-454 (2001).</li>
<li>Echeverry, D. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fast+and+robust+single+PCR+for+Plasmodium+sporozoite+detection+in+mosquitoes+using+the+cytochrome+oxidase+I+gene%5BTitle%5D)+AND+%22Malaria+Journal%22%5BJournal%5D)">Fast and robust single PCR for Plasmodium sporozoite detection in mosquitoes using the cytochrome oxidase I gene.</a> Malaria Journal. 16 (1), 230(2017).</li>
<li>Singh, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+genus-+and+species-specific+nested+polymerase+chain+reaction+malaria+detection+assay+for+epidemiologic+studies%5BTitle%5D)+AND+%22American+Journal+of+Tropical+Medicine+and+Hygiene%22%5BJournal%5D)">A genus- and species-specific nested polymerase chain reaction malaria detection assay for epidemiologic studies.</a> American Journal of Tropical Medicine and Hygiene. 60 (4), 687-692 (1999).</li>
<li>Snounou, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genotyping+of+Plasmodium+spp.+Nested+PCR%5BTitle%5D)+AND+%22Methods+in+Molecular+Medicine%22%5BJournal%5D)">Genotyping of Plasmodium spp. Nested PCR.</a> Methods in Molecular Medicine. 72, 103-116 (2002).</li>
<li>Snounou, G., Singh, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nested+PCR+analysis+of+Plasmodium+parasites%5BTitle%5D)+AND+%22Methods+in+Molecular+Medicine%22%5BJournal%5D)">Nested PCR analysis of Plasmodium parasites.</a> Methods in Molecular Medicine. 72, 189-203 (2002).</li>
<li>Snounou, G., Viriyakosol, S., Jarra, W., Thaithong, S., Brown, K. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+of+the+four+human+malaria+parasite+species+in+field+samples+by+the+polymerase+chain+reaction+and+detection+of+a+high+prevalence+of+mixed+infections%5BTitle%5D)+AND+%22Molecular+and+Biochemical+Parasitology%22%5BJournal%5D)">Identification of the four human malaria parasite species in field samples by the polymerase chain reaction and detection of a high prevalence of mixed infections.</a> Molecular and Biochemical Parasitology. 58 (2), 283-292 (1993).</li>
<li>Wirtz, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+of+Plasmodium+vivax+sporozoites+in+mosquitoes+using+an+enzyme-linked+immunosorbent+assay%5BTitle%5D)+AND+%22American+Journal+of+Tropical+Medicine+and+Hygiene%22%5BJournal%5D)">Identification of Plasmodium vivax sporozoites in mosquitoes using an enzyme-linked immunosorbent assay.</a> American Journal of Tropical Medicine and Hygiene. 34 (6), 1048-1054 (1985).</li>
<li>Wirtz, R. A., Burkot, T. R., Graves, P. M., Andre, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Field+evaluation+of+enzyme-linked+immunosorbent+assays+for+Plasmodium+falciparum+and+Plasmodium+vivax+sporozoites+in+mosquitoes+%28Diptera%3A+Culicidae%29+from+Papua+New+Guinea%5BTitle%5D)+AND+%22Journal+of+Medical+Entomology%22%5BJournal%5D)">Field evaluation of enzyme-linked immunosorbent assays for Plasmodium falciparum and Plasmodium vivax sporozoites in mosquitoes (Diptera: Culicidae) from Papua New Guinea.</a> Journal of Medical Entomology. 24 (4), 433-437 (1987).</li>
<li>Wirtz, R. A., Sattabongkot, J., Hall, T., Burkot, T. R., Rosenberg, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+and+evaluation+of+an+enzyme-linked+immunosorbent+assay+for+Plasmodium+vivax-VK247+sporozoites%5BTitle%5D)+AND+%22Journal+of+Medical+Entomology%22%5BJournal%5D)">Development and evaluation of an enzyme-linked immunosorbent assay for Plasmodium vivax-VK247 sporozoites.</a> Journal of Medical Entomology. 29 (5), 854-857 (1992).</li>
<li>Burkot, T. R., Williams, J. L., Schneider, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+of+Plasmodium+falciparum-infected+mosquitoes+by+a+double+antibody+enzyme-linked+immunosorbent+assay%5BTitle%5D)+AND+%22American+Journal+of+Tropical+Medicine+and+Hygiene%22%5BJournal%5D)">Identification of Plasmodium falciparum-infected mosquitoes by a double antibody enzyme-linked immunosorbent assay.</a> American Journal of Tropical Medicine and Hygiene. 33 (5), 783-788 (1984).</li>
<li>Rosenberg, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Circumsporozoite+protein+heterogeneity+in+the+human+malaria+parasite+Plasmodium+vivax%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Circumsporozoite protein heterogeneity in the human malaria parasite Plasmodium vivax.</a> Science. 245 (4921), 973-976 (1989).</li>
<li>Marie, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluation+of+a+real-time+quantitative+PCR+to+measure+the+wild+Plasmodium+falciparum+infectivity+rate+in+salivary+glands+of+Anopheles+gambiae%5BTitle%5D)+AND+%22Malaria+Journal%22%5BJournal%5D)">Evaluation of a real-time quantitative PCR to measure the wild Plasmodium falciparum infectivity rate in salivary glands of Anopheles gambiae.</a> Malaria Journal. 12, 224(2013).</li>
<li>Arevalo-Herrera, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immunoreactivity+of+sera+from+low+to+moderate+malaria-eEndemic+areas+against+Plasmodium+vivax+rPvs48%2F45+proteins+produced+in+Escherichia+coli+and+chinese+hamster+ovary+systems%5BTitle%5D)+AND+%22Frontiers+in+Immunology%22%5BJournal%5D)">Immunoreactivity of sera from low to moderate malaria-eEndemic areas against Plasmodium vivax rPvs48/45 proteins produced in Escherichia coli and chinese hamster ovary systems.</a> Frontiers in Immunology. 12, 634738(2021).</li>
<li>Balkew, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+update+on+the+distribution%2C+bionomics%2C+and+insecticide+susceptibility+of+Anopheles+stephensi+in+Ethiopia%2C+2018-2020%5BTitle%5D)+AND+%22Malaria+Journal%22%5BJournal%5D)">An update on the distribution, bionomics, and insecticide susceptibility of Anopheles stephensi in Ethiopia, 2018-2020.</a> Malaria Journal. 20 (1), 263(2021).</li>
<li>Wirtz, R. A., Avery, M., Benedict, M., Sutcliffe, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Plasmodium+sporozoite+ELISA%5BTitle%5D)+AND+%22Methods+in+Anopheles+Research%22%5BJournal%5D)">Plasmodium sporozoite ELISA.</a> Methods in Anopheles Research. , 333-343 (2016).</li>
<li>Durnez, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((False+positive+circumsporozoite+protein+ELISA%3A+a+challenge+for+the+estimation+of+the+entomological+inoculation+rate+of+malaria+and+for+vector+incrimination%5BTitle%5D)+AND+%22Malaria+Journal%22%5BJournal%5D)">False positive circumsporozoite protein ELISA: a challenge for the estimation of the entomological inoculation rate of malaria and for vector incrimination.</a> Malaria Journal. 10, 195(2011).</li>
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The authors have no conflicts of interest to declare.

The CSP-ELISA provides a highly specific and cost-effective method to detect Plasmodium CSP. It allows discrimination between P. falciparum and P. vivax sporozoites as well as between the two subtypes, VK210 and VK247, of P vivax11,13,14,15. Certain critical points should be considered to obtain reliable and reproducible results. All solutions should be kept in the refrigerator for less than 1 week to prevent microbial growth. The mAb should be kept in diluent containing 50% glycerol and aliquoted as needed to prevent multiple freeze-thawing. The positive controls should be aliquoted for single use. The ELISA plate should be covered with the lid during the incubation period to prevent evaporation. Steps involving peroxidase-labeled mAb incubation should be carried out in the dark.
All steps involving solution change should be performed quickly to prevent drying out, which can lead to high background. The working substrate solution should be kept in the dark by wrapping it with aluminum foil and added to the plate immediately after preparation. When working with frozen mosquito homogenates, the samples should be tested on the same day of thawing. The pH of the reaction should be maintained in the range of 7-7.4 as the reaction is inhibited at pH values outside this range. Washing should be done carefully to avoid false positives. The inclusion of the non-ionic detergent, Tween-20, in the washing solution can minimize signal from the background.
This protocol was modified from the protocol described by Wirtz et al.19. One difference is the lower number of negative controls to allow for the six-point standard curve. In addition, the protein standards are serially diluted in BB without the mosquito lysate. Therefore, their background composition differs from that of the test samples. These standards are used to provide consistent CSP quantification of the test samples across different plates. If more accurate quantification is needed, the standards can be prepared using the lysate of uninfected mosquitoes processed identically to the test samples but with a known amount of protein added. Lastly, as with most diagnostic assays, the CSP ELISA is not error-free20. All positive samples should be confirmed by repeating the assay with heated homogenate (100 &#176;C, 10 min) or by Plasmodium species-specific PCR, using the remaining homogenate as the source of the DNA template20.
When performed correctly, this CSP ELISA method can be highly reliable. It has been, and likely will continue to be, used in several studies of malaria transmission, with the goals to determine the seasonal dynamics of mosquito infection and identify the species of the major malaria vectors12,13,17,18. Compared to direct microscopic examination of sporozoites, this assay has a much greater throughput and is more suitable for research involving a large number of mosquitoes. Compared to the PCR detection of sporozoites, the ELISA procedure takes more time but costs less16. Overall, its simplicity, high throughput, and relatively low cost permit large-scale testing in a standard laboratory.

Plasmodium sporozoites are the infectious stage of the malaria parasites in the mosquitoes. The sporozoites are delivered to humans via mosquito bites. In the mosquito, the sporozoites first form inside the oocysts on the midgut wall. Once ready, they are released into the hemocoel and travel to the mosquito salivary glands. There, they mature and become ready for transmission to humans during blood feeding. In humans, the sporozoites are deposited in the dermis. Then, they enter the blood vessel and travel along the blood circulation to reach the liver to establish infection in the hepatocytes1,2.
Three different methods have been used to determine sporozoite infection of the mosquito salivary glands. The first method is the dissection of the salivary glands followed by direct examination of sporozoites under a light microscope. This method is the gold standard to detect and quantify sporozoites in Anopheles mosquito salivary glands3. However, it requires a technician well trained in both dissection and microscopic examination. Moreover, it cannot be used to determine Plasmodium species and CSP subtyping (for P. vivax)4,5. The second method uses polymerase chain reaction (PCR) to detect Plasmodium DNA in the upper part of the mosquito body6. Given the specificity of PCR, both species and subtyping of the parasite are possible7,8,9,10. Although PCR is increasingly used, it requires relatively expensive equipment and well-trained staff. The last method, the ELISA to detect the Plasmodium specific circumsporozoite protein (CSP), has been the mainstay for three decades11,12,13. CSP is present in both oocyst sporozoites and salivary gland sporozoites12,14. Using specific antibodies, this method allows Plasmodium species identification and CSP subtyping of P. vivax sporozoites. The rationale for this assay is the requirement of a simple high-throughput assay to examine a large number of wild mosquitoes to understand malaria transmission (i.e., determine the sporozoite infection rate).
The ELISA method has two key advantages over microscopic examination. First, it allows researchers to keep mosquito samples until they are ready for sample processing. Second, the ELISA method can be used to differentiate Plasmodium species through species-specific monoclonal antibodies. In addition, ELISA can accommodate a larger number of mosquito specimens, permitting a much higher throughput15. Compared to PCR, which detects sporozoite DNA, the ELISA procedure takes more time but costs less16. The ELISA assay described here was developed to determine the mosquito infectivity and separately detect CSP of P. falciparum and each of the two CSP variants of P. vivax, VK210 and VK247. This ELISA method has been used in many studies to determine the seasonal dynamics of mosquito infection and identify the species of the major malaria vectors in the field12,13,17,18. To perform this assay, a standard laboratory equipped with an ELISA plate reader is sufficient.
The overall approach is summarized in Figure 1. In this sandwich ELISA, the primary (capture) monoclonal antibody (mAb) specific for each Plasmodium species/subtype is first used to coat the ELISA plate. Each plate is coated with a single capture mAb. The function of the mAb is to capture the corresponding CSP antigen in the mosquito homogenates. After antigen capture and plate washes, a second CSP-specific antibody labeled with peroxidase is used to detect the presence of CSP bound to the capture mAb. The chemical reaction catalyzed by peroxidase results in color development in wells positive for CSP.

<table><tbody><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>ELISA plate reader</td><td>BioTek Instrument, Inc.</td><td>Synergy H1</td><td>Absorbance is measured using UV-VIS absorbance detection mode</td></tr><tr><td>Grinder pestle</td><td>Axygen</td><td>PES-15-B-SI</td><td>For homogenizing the mosquito head/thorax</td></tr><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>ABTS substrate 2-component</td><td>KPL</td><td>50-62-00</td><td>For peroxidase driven detection</td></tr><tr><td>Blocking buffer (BB)</td><td></td><td></td><td>Note: solution</td></tr><tr><td>Capture and peroxidase-conjugated monoclonal antibodies (mAbs)</td><td></td><td></td><td>Note: mAbs can be obtained in the lyophilized form as part of the Sporozoite ELISA Reagent Kits (MRA-890 for P. falciparum and MRA-1028K for P. vivax) from BEI Resources (https://www.beiresources.org.)</td></tr><tr><td>Casein</td><td>Sigma aldrich</td><td>9000-71-9</td><td>For blocking buffer preparation</td></tr><tr><td>Grinding buffer (GB)</td><td></td><td></td><td>Note: solution</td></tr><tr><td>Igepal CA-630</td><td>Sigma aldrich</td><td>9002-93-1</td><td>For grinding buffer preparation</td></tr><tr><td>KCl</td><td>Sigma aldrich</td><td>7447-40-7</td><td>For phosphate buffer saline preparation</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma aldrich</td><td>7778-77-0</td><td>For phosphate buffer saline preparation</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma aldrich</td><td>7558-79-4</td><td>For phosphate buffer saline preparation</td></tr><tr><td>NaCl</td><td>Sigma aldrich</td><td>7647-14-5</td><td>For phosphate buffer saline preparation</td></tr><tr><td>NaOH</td><td>Sigma aldrich</td><td>1310-73-2</td><td>For blocking buffer preparation</td></tr><tr><td>PBS-Tween</td><td></td><td></td><td></td></tr><tr><td>Pf capture mAb</td><td>CDC</td><td>Pf-CAP</td><td>For capturing Pf circumsporozoite protein</td></tr><tr><td>Pf peroxidase mAb</td><td>CDC</td><td>Pf-HRP</td><td>For detecting Pf circumsporozoite protein</td></tr><tr><td>Pf positive control</td><td>CDC</td><td>Pf-PC</td><td>Pf positive control</td></tr><tr><td>Phenol red</td><td>Sigma aldrich</td><td>143-74-8</td><td>For blocking buffer preparation</td></tr><tr><td>Phosphate buffered saline (PBS)</td><td></td><td></td><td>Note: solution</td></tr><tr><td>Positive controls</td><td></td><td></td><td>Note: solution</td></tr><tr><td>Pv210 capture mAb</td><td>CDC</td><td>Pv210-CAP</td><td>For capturing Pv210 circumsporozoite protein</td></tr><tr><td>Pv210 peroxidase mAb</td><td>CDC</td><td>Pv210-HRP</td><td>For detecting Pv210 circumsporozoite protein</td></tr><tr><td>Pv210 positive control</td><td>CDC</td><td>Pv210-PC</td><td>Pv210 positive control</td></tr><tr><td>Pv247 capture mAb</td><td>CDC</td><td>Pv247-CAP</td><td>For capturing Pv247 circumsporozoite protein</td></tr><tr><td>Pv247 peroxidase mAb</td><td>CDC</td><td>Pv247-HRP</td><td>For detecting Pv247 circumsporozoite protein</td></tr><tr><td>Pv247 positive control</td><td>CDC</td><td>Pv247-PC</td><td>Pv247 positive control</td></tr><tr><td>Tween20</td><td>Sigma aldrich</td><td>9005-64-5</td><td>For PBS-Tween preparation</td></tr><tr><td>&lt;strong&gt;Consumables&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Disposable pipetting reservoirs</td><td>Generic</td><td></td><td>For working reagents</td></tr><tr><td>ELISA plates: 96 well clear round bottom PVS</td><td>Corning Life Science</td><td>2797</td><td>For ELISA</td></tr><tr><td>Gloves: disposable gloves and freezer gloves</td><td>Generic</td><td></td><td>For personal protection</td></tr><tr><td>Lab gown</td><td>Generic</td><td></td><td>for personal protection</td></tr><tr><td>Multichannel Pipette P30-300</td><td>Generic</td><td></td><td>For solution transfer</td></tr><tr><td>Pipette set, P2, P20, P200, and P1000</td><td>Generic</td><td></td><td>For solution transfer</td></tr><tr><td>Pipette tips 10 &amp;micro;L, 200 &amp;micro;L, and 1000 &amp;micro;L</td><td>Generic</td><td></td><td>For solution transfer</td></tr><tr><td>Serological pipettes 5 mL, 10 mL</td><td>Generic</td><td></td><td>For solution transfer</td></tr><tr><td>Transfer pipettes</td><td>Generic</td><td></td><td>For solution transfer</td></tr></tbody></table>

detection of plasmodium sporozoites in anopheles mosquitoes using an enzyme-linked immunosorbent assay

Plasmodium sporozoites are the infective stage of malaria parasites that infect humans. The sporozoites residing in the salivary glands of female Anopheles mosquitoes are transmitted to humans via mosquito bites during blood feeding. The presence of sporozoites in the mosquito salivary glands thus defines mosquito infectiousness. To determine whether an Anopheles mosquito carries Plasmodium sporozoites, the enzyme-linked immunosorbent assay (ELISA) method has been the standard tool to detect the Plasmodium circumsporozoite protein (CSP), the major surface protein of the sporozoites. In this method, the head along with the thorax of each mosquito is separated from the abdomen, homogenized, and subjected to a sandwich ELISA to detect the presence of CSP specific to Plasmodium falciparum and each of the two subtypes, VK210 and VK247, of Plasmodium vivax.This method has been used to study malaria transmission, including the seasonal dynamics of mosquito infection and the species of the major malaria vectors in the study sites.

Representative ELISA results are shown in Figure 2. In this experiment, the P. falciparum ELISA detected sporozoite infection in well A7. The positive well could be visually detected by its faint green color (Figure 2A). The absorbance value of this well was above the cut-off threshold (twice the mean value of the four negative control wells) (Figure 2B). The distribution of the absorbance values of all 80 unknown wells is depicted in Figure 2C. CSP quantification of well A7 by the in-plate standard curve after background (negative control) subtraction suggests a CSP concentration of 0.35 pg/&#956;L (Figure 2D). The P. vivax assays for VK210 and VK247 were both negative for this sample set (data not shown), indicating that the sporozoite infection in A7 was mono-species P. falciparum.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63158/63158fig01.jpg" /> 
Figure 1: Overview of CSP sandwich ELISA. (A) Specific capture monoclonal antibody (capture mAb) is used to coat the surface of each well. CSP antigen in the mosquito homogenate binds to the mAb-coated wells. (B) HRP-labeled mAb is used to detect the captured Ag. Abbreviations: CSP = circumsporozoite protein; ELISA = enzyme-linked immunosorbent assay; Ag = antigen; mAb = monoclonal antibody; HRP = horseradish peroxidase; OD = optical density; BB = blocking buffer. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63158/63158fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63158/63158fig02.jpg" /> 
Figure 2: Representative results for Plasmodium falciparum CSP detection. (A) The image of the ELISA plate after 30 min incubation with ABTS. The red arrow and red circle represent the positive unknown well (A7). (B) The absorbance values read by the ELISA plate reader. The four upper left wells (A1, A2 and B1, B2) were the negative controls. Diluted positive controls were run in duplicates: C1/C2 (1:32), D1/D2 (1:16), E1/E2 (1:8), F1/F2 (1:4), and G1/G2 (1:2). The undiluted positive controls were H1/H2. (C) The absorbance distribution of the 80 unknown wells. The solid line represents the mean absorbance of the negative control wells. The dashed line represents the positivity threshold. (D) The standard curve constructed from the two-fold serial dilution of the positive control. The highest concentration of the positive control was 2 pg/&#956;L. Linear regression of the data estimated the CSP concentration in A7 as 0.35 pg/&#956;L. Abbreviations: CSP = circumsporozoite protein; ABTS = 2,2&#39;-azino-di-(3-ethylbenzthiazoline-6-sulfonate); ELISA = enzyme-linked immunosorbent assay; Abs = absorbance. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63158/63158fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Recipes. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63158/JoVE_Table1_Recipes.xlsx" target="_blank">Please click here to download this Table.</a>
Supplemental Material S1: Sporozoite ELISA worksheet. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63158/ELISA WORKSHEET template_Jove.docx" target="_blank">Please click here to download this File.</a>
Supplemental Material S2: Guidelines for ELISA plate reader software. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63158/Guileline for GEN5 program use for ELISA plate reading.pdf" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Detection of Plasmodium Sporozoites in Anopheles Mosquitoes using an Enzyme-linked Immunosorbent Assay at JoVE.com

Detection of Plasmodium Sporozoites in Anopheles Mosquitoes using an Enzyme-linked Immunosorbent Assay

This protocol describes a sandwich enzyme-linked immunosorbent assay to detect salivary gland sporozoites in mosquitoes. Using easily available monoclonal antibodies, the method enables cost-effective, high-throughput detection of mosquitoes carrying Plasmodium falciparum or Plasmodium vivax. The method is suitable for malaria transmission research, including vector surveys.

Detection of Plasmodium Sporozoites in Anopheles Mosquitoes using an Enzyme-linked Immunosorbent Assay

Plasmodium sporozoites are the infective stage of malaria parasites that infect humans. The sporozoites residing in the salivary glands of female ...

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Nanyang Technological University

Research

JoVE Journal

Biology

2.2K Views.  Mahidol University. De ELISA-techniek is erg nuttig voor het detecteren van malariaparasiet in de muggenvector met hoge gevoeligheid en specificiteit, en in staat om tegelijkertijd veel monsters te verwerken. Deze techniek stelt onderzoekers in staat om muggenmonsters te bewaren totdat ze klaar zijn voor monsterverwerking, en het kan worden uitgevoerd om Plasmodium-soorten te differentiëren met behulp van soortspecifieke monoklonale antilichamen. Bereid het monster voor door het hoofd en de borstkas van de mugg...

Video: Detection of Plasmodium Sporozoites in Anopheles Mosquitoes using an Enzyme-linked Immunosorbent Assay

Protocol for Plasmodium falciparum Infections in Mosquitoes and Infection Phenotype Determination

Once a gene is identified as potentially refractory for malaria, it must be evaluated for its role in preventing Plasmodium infections within the mosquito.    This protocol illustrates how the extent of plasmodium infections of mosquitoes can be assayed.  The techniques for preparing the gametocyte culture, membrane feeding mosquitoes human blood, and assaying viral titers in the mosquito midgut are demonstrated.

Once a gene is identified as potentially refractory for malaria, it must be evaluated for its role in preventing Plasmodium infections within the mosquito. This protocol illustrates how the extent of plasmodium infections of mosquitoes can be assayed. The techniques for preparing the gametocyte culture, membrane feeding mosquitoes human blood, and assaying viral titers in the mosquito midgut are demonstrated.

Once a gene is identified as potentially refractory for malaria, it must be evaluated for its role in preventing Plasmodium infections within the ...

Detection and Quantification of Plasmodium falciparum in Aqueous Red Blood Cells by Attenuated Total Reflection Infrared Spectroscopy and Multivariate Data Analysis

We demonstrate a method of quantification and detection of parasites in aqueous red blood cells (RBCs) by using a simple benchtop Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) spectrometer in conjunction with Multivariate Data Analysis (MVDA). 3D7 P. falciparum were cultured to 10% parasitemia ring stage parasites and used to spike fresh donor isolated RBCs to create a dilution series between 0&#8211;1%. 10 &#181;L of each sample were placed onto the center of the ATR diamond window to acquire the spectrum. The sample data was treated to improve the signal to noise ratio and to remove the contribution of water, and then the second derivative was applied to resolve spectral features. The data were then analyzed using two types of MVDA: first Principal Component Analysis (PCA) to determine any outliers and then Partial Least Squares Regression (PLS-R) to build the quantification model.

Detection and Quantification of Plasmodium falciparum in Aqueous Red Blood Cells by Attenuated Total Reflection Infrared Spectroscopy and Multivariate Data Analysis

Here, we present a protocol for the detection and quantification of Plasmodium falciparum in infected aqueous red blood cells using an attenuated total reflection infrared spectrometer and multivariate data analysis.

We demonstrate a method of quantification and detection of parasites in aqueous red blood cells (RBCs) by using a simple benchtop Attenuated Total ...

Chemistry

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of free merozoites from the blood by phagocytic cells. However assessing the functional efficacy of merozoite specific opsonizing antibodies is challenging due to the short half-life of merozoites and the variability of primary phagocytic cells. Described in detail herein is a method for generating viable merozoites using the E64 protease inhibitor, and an assay of merozoite opsonin-dependent phagocytosis using the pro-monocytic cell line THP-1. E64 prevents schizont rupture while allowing the development of merozoites which are released by filtration of treated schizonts. &#160;Ethidium bromide labelled merozoites are opsonized with human plasma samples and added to THP-1 cells. Phagocytosis is assessed by a standardized high throughput protocol. Viable merozoites are a valuable resource for assessing numerous aspects of P. falciparum biology, including assessment of immune function. Antibody levels measured by this assay are associated with clinical immunity to malaria in naturally exposed individuals. The assay may also be of use for assessing vaccine induced antibodies. &#160;

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Measuring antibody function is key to understanding immunity to Plasmodium falciparum malaria. This method describes the purification of viable merozoites, and measurement of opsonization-dependent phagocytosis by flow cytometry.

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of ...

Immunology and Infection

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly motile parasite not only reaches the liver to invade hepatocytes and transform into erythrocyte-infective form. It also migrates into the skin and to the proximal lymph node draining the injection site, where it can be recognized and degraded by resident and/or recruited myeloid cells. Intravital imaging reported the early recruitment of brightly fluorescent Lys-GFP positive leukocytes in the skin and the interactions between sporozoites and CD11c+ cells in the draining lymph node. We present here an efficient procedure to recover, identify and enumerate the myeloid cell subsets that are recruited to the mouse skin and draining lymph node following intradermal injection of immunizing doses of sporozoites in a murine model. Phenotypic characterization using multi-parametric flow cytometry provides a reliable assay to assess early dynamic cellular changes during inflammatory response to Plasmodium infection.

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

We describe here a protocol for isolating myeloid cells from mouse skin and draining lymph node following intradermal injection of Plasmodium sporozoites. Flow cytometry of collected cells provides a reliable assay to characterize the skin and draining lymph node inflammatory response to the parasite.

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly ...

Phenotypic Analysis of Rodent Malaria Parasite Asexual and Sexual Blood Stages and Mosquito Stages

Recent advances in genetics and systems biology technologies have promoted our understanding of the biology of malaria parasites on the molecular level. However, effective malaria parasite targets for vaccine and chemotherapy development are still limited. This is largely due to the unavailability of relevant and practical in vivo infection models for human Plasmodium species, most notably for P.&#160;falciparum and P. vivax. Therefore, rodent malaria species have been extensively used as practical alternative in vivo models for malaria vaccine, drug targeting, immune response, and functional characterization studies of conserved Plasmodiumspp. genes. Indeed, rodent malaria models have proven to be invaluable, especially for exploring mosquito transmission and liver stage biology, and were indispensable for immunological studies. However, there are discrepancies in the methods used to evaluate the phenotypes of transgenic and wild-type asexual and sexual blood-stage parasites.&#160;Examples of these discrepancies are the choice of an intravenous vs. intraperitoneal infection of rodents with blood-stage parasites and the evaluation of male gamete exflagellation. Herein, we detail standardized experimental methods to evaluate the phenotypes of asexual and sexual blood stages in transgenic parasites expressing reporter-gene or wild-type rodent malaria parasite species. We also detail the methods to evaluate the phenotypes of malaria parasite mosquito stages (gametes, ookinetes, oocysts, and sporozoites) inside Anopheles mosquito vectors. These methods are detailed and simplified here for the lethal and non-lethal strains of P. berghei and P. yoelii but can also be applied with some adjustments to P. chabaudi and P. vinckei rodent malaria species.

Due to the striking similarities of the life cycle and biology of rodent malaria parasites to human malaria parasites, rodent malaria models have become indispensable for malaria research. Herein, we standardized some of the most important techniques used in the phenotypic analysis of wild-type and transgenic rodent malaria species.

Recent advances in genetics and systems biology technologies have promoted our understanding of the biology of malaria parasites on the molecular level. ...

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of cerebral malaria can be difficult. We present an experimental mouse model of cerebral malaria that shares multiple features of the human disease, including edema and microvascular pathology. Using magnetic resonance imaging (MRI), we can detect and track the blood-brain barrier disruption, edema development, and subsequent brain swelling. We describe multiple MRI techniques that can visualize these pertinent pathological changes. Thus, we show that MRI represents a valuable tool to visualize and track pathological changes, such as edema, brain swelling, and microvascular pathology, in vivo.

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

We describe a mouse model of experimental cerebral malaria and show how inflammatory and microvascular pathology can be tracked in vivo using magnetic resonance imaging.

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of ...

Medicine

Standard Membrane Feeding Assay for the Detection of Plasmodium falciparum Infection in Anopheles Mosquito Vectors

Malaria remains one of the most devastating diseases worldwide and, to date, the African region is still responsible for 94% of all cases worldwide. This parasitic disease requires a protozoan parasite, an Anopheles mosquito vector, and a vertebrate host. The Anopheles genus comprises more than 500 species, of which 60 are known as vectors of the parasite. The Plasmodium parasite genus consists of 250 species, and 48 of these are involved in disease transmission. Furthermore, the Plasmodium falciparum parasite has contributed toward an estimated 99.7% of malaria cases in sub-Saharan Africa in recent years. 
Gametocytes form part of the sexual stage of the parasite and are ingested by the female mosquito upon feeding on an infected human host. Further development of the parasite within the mosquito is enhanced by favorable environmental conditions in the midgut of the mosquito. Here, the fusion of the female and male gametes takes place, and the motile ookinetes originate. The ookinetes enter the midgut epithelium of the mosquito, and mature ookinetes form oocysts, which, in turn, produce motile sporozoites. These sporozoites migrate to the mosquito's salivary glands and are injected as a mosquito takes a blood meal. 
For drug discovery purposes, mosquitoes were artificially infected with gametocyte-infected blood in the standard membrane feeding assay (SMFA). To detect infection within the mosquito and/or to assess the efficacy of antimalarial compounds, the midguts of the female mosquitoes were removed post infection and were stained with mercurochrome. This method was used to enhance the visual detection of oocysts under the microscope for the accurate determination of oocyst prevalence and intensity.

Standard Membrane Feeding Assay for the Detection of Plasmodium falciparum Infection in Anopheles Mosquito Vectors

The standard membrane feeding assay (SMFA) is regarded as the gold standard for the assessment and identification of potential antimalarial compounds. This artificial feeding system is used to infect mosquitoes to further evaluate the effects of such compounds on the intensity and prevalence of the Plasmodium falciparum parasite.

Malaria remains one of the most devastating diseases worldwide and, to date, the African region is still responsible for 94% of all cases worldwide. This ...

Generating Genetically Modified Plasmodium berghei Sporozoites

Malaria is a deadly disease caused by the parasite Plasmodium and is transmitted through the bite of female Anopheles mosquitoes. The sporozoite stage of Plasmodium deposited by mosquitoes in the skin of vertebrate hosts undergoes a phase of mandatory development in the liver before initiating clinical malaria. We know little about the biology of Plasmodium development in the liver; access to the sporozoite stage and the ability to genetically modify such sporozoites are critical tools for studying the nature of Plasmodium infection and the resulting immune response in the liver. Here, we present a comprehensive protocol for the generation of transgenic Plasmodium berghei sporozoites. We genetically modify blood-stage P. berghei and use this form to infect Anopheles mosquitoes when they take a blood meal. After the transgenic parasites undergo development in the mosquitoes, we isolate the sporozoite stage of the parasite from the mosquito salivary glands for in vivo and in vitro experimentation. We demonstrate the validity of the protocol by generating sporozoites of a novel strain of P. berghei expressing the green fluorescent protein (GFP) subunit 11 (GFP11), and show how it could be used to investigate the biology of liver-stage malaria.

Generating Genetically Modified Plasmodium berghei Sporozoites

Malaria is transmitted through inoculation of the sporozoite stage of Plasmodium by infected mosquitoes. Transgenic Plasmodium has allowed us to understand the biology of malaria better and has contributed directly to malaria vaccine development efforts. Here, we describe a streamlined methodology to generate transgenic Plasmodium berghei sporozoites.

Malaria is a deadly disease caused by the parasite Plasmodium and is transmitted through the bite of female Anopheles mosquitoes. The sporozoite stage of ...

A Multi-detection Assay for Malaria Transmitting Mosquitoes

The Anopheles gambiae species complex includes the major malaria transmitting mosquitoes in Africa. Because these species are of such medical importance, several traits are typically characterized using molecular assays to aid in epidemiological studies. These traits include species identification, insecticide resistance, parasite infection status, and host preference. Since populations of the Anopheles gambiae complex are morphologically indistinguishable, a polymerase chain reaction (PCR) is traditionally used to identify species. Once the species is known, several downstream assays are routinely performed to elucidate further characteristics. For instance, mutations known as KDR in a para gene confer resistance against DDT and pyrethroid insecticides. Additionally, enzyme-linked immunosorbent assays (ELISAs) or Plasmodium parasite DNA detection PCR assays are used to detect parasites present in mosquito tissues. Lastly, a combination of PCR and restriction enzyme digests can be used to elucidate host preference (e.g., human vs. animal blood) by screening the mosquito bloodmeal for host-specific DNA. We have developed a multi-detection assay (MDA) that combines all of the aforementioned assays into a single multiplex reaction genotyping 33SNPs for 96 or 384 samples at a time. Because the MDA includes multiple markers for species, Plasmodium detection, and host blood identification, the likelihood of generating false positives or negatives is greatly reduced from previous assays that include only one marker per trait. This robust and simple assay can detect these key mosquito traits cost-effectively and in a fraction of the time of existing assays.

Malaria transmitting mosquitoes have a number of epidemiologically important characteristics that can only be detected using molecular techniques. Utilizing a MALDI-TOF based SNP genotyping platform, we developed an assay for simultaneously detecting multiple key traits (species, insecticide resistance, parasite infection and host choice) of malaria vectors.

The Anopheles gambiae species complex includes the major malaria transmitting mosquitoes in Africa. Because these species are of such medical importance, ...

Plasmodium falciparum Gametocyte Culture and Mosquito Infection Through Artificial Membrane Feeding

Malaria remains one of the most important public health problems, causing significant morbidity and mortality. Malaria is a mosquito borne disease transmitted through an infectious bite from the female Anopheles mosquito. Malaria control will eventually rely on a multitude of approaches, which includes ways to block transmission to, through and from mosquitoes. To study mosquito stages of malaria parasites in the laboratory, we have optimized a protocol to culture highly infectious Plasmodium falciparum gametocytes, a parasite stage required for transmission from the human host to the mosquito vector. P. falciparum gametocytes mature through five morphologically distinct steps, which takes approximately 1-2 weeks. Gametocyte culture described in this protocol is completed in 15 days and are infectious to mosquitoes from days 15-18. These protocols were developed to maintain a continuous cycle of infection competent gametocytes and to maintain uninterrupted supply of mosquito stages of the parasite. Here, we describe the methodology of gametocyte culture and how to infect mosquitoes with these parasites using glass membrane feeders.

Plasmodium falciparum Gametocyte Culture and Mosquito Infection Through Artificial Membrane Feeding

Detailed investigations on mosquito stages of malaria parasites are critical to design effective transmission blocking strategies. This protocol demonstrates how to effectively culture infectious gametocytes and then feed these gametocytes to mosquitoes to generate mosquito stages of P. falciparum.

Detection of Plasmodium Sporozoites in Anopheles Mosquitoes using an Enzyme-linked Immunosorbent Assay

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