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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+life-cycle+of+primate+malaria+parasites.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primate+malaria.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Division+of+Malaria+and+Other+Parasitic+Diseases.+Manual+on+practical+entomology+in+Malaria,+Part+I+and+Part+II.">Division of Malaria and Other Parasitic Diseases. Manual on practical entomology in Malaria, Part I and Part II.</a> World Health Organization. , (1975).</li><li>Robert, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+of+the+distribution+of+circumsporozoite+antigen+in+Anopheles+gambiae+infected+with+Plasmodium+falciparum,+using+the+enzyme-linked+immunosorbent+assay.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+and+robust+single+PCR+for+Plasmodium+sporozoite+detection+in+mosquitoes+using+the+cytochrome+oxidase+I+gene.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genus-+and+species-specific+nested+polymerase+chain+reaction+malaria+detection+assay+for+epidemiologic+studies.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genotyping+of+Plasmodium+spp.+Nested+PCR.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nested+PCR+analysis+of+Plasmodium+parasites.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&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.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+Plasmodium+vivax+sporozoites+in+mosquitoes+using+an+enzyme-linked+immunosorbent+assay.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Field+evaluation+of+enzyme-linked+immunosorbent+assays+for+Plasmodium+falciparum+and+Plasmodium+vivax+sporozoites+in+mosquitoes+(Diptera:+Culicidae)+from+Papua+New+Guinea.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+evaluation+of+an+enzyme-linked+immunosorbent+assay+for+Plasmodium+vivax-VK247+sporozoites.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+Plasmodium+falciparum-infected+mosquitoes+by+a+double+antibody+enzyme-linked+immunosorbent+assay.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circumsporozoite+protein+heterogeneity+in+the+human+malaria+parasite+Plasmodium+vivax.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+a+real-time+quantitative+PCR+to+measure+the+wild+Plasmodium+falciparum+infectivity+rate+in+salivary+glands+of+Anopheles+gambiae.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+update+on+the+distribution,+bionomics,+and+insecticide+susceptibility+of+Anopheles+stephensi+in+Ethiopia,+2018-2020.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmodium+sporozoite+ELISA.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=False+positive+circumsporozoite+protein+ELISA:+a+challenge+for+the+estimation+of+the+entomological+inoculation+rate+of+malaria+and+for+vector+incrimination.">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></ol>

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 ...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Equipment</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>Reagents</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>KH2PO4</td>
			<td>Sigma aldrich</td>
			<td>7778-77-0</td>
			<td>For phosphate buffer saline preparation</td>
		</tr>
		<tr>
			<td>Na2HPO4</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>Consumables</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 &#181;L, 200 &#181;L, and 1000 &#181;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 ...

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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1.8K Views.  Mahidol University. 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.

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

Injection of An. stephensi Embryos to Generate Malaria-resistant Mosquitoes

The introduction of exogenous genes into the genomes of mosquitoes requires microinjection techniques tailored to the specific species of interest. This video protocol demonstrates a method used by the James laboratory to microinject DNA constructs into Anopheles stephensi embryos for the generation of transformed mosquitoes. Techniques for preparing microinjection needles, collecting and preparing embryos and performing the microinjection are illustrated.

Anopheles stephensi mosquitoes are vectors for malaria inhabiting India and throughout Asia. This video demonstrates the technique for performing microinjections of this species with transgenes that will confer resistance to the malaria to the mosquito. Much of the methodology demonstrated in this video is applicable to microinjection techniques of other mosquito species.

The introduction of exogenous genes into the genomes of mosquitoes requires microinjection techniques tailored to the specific species of interest. This ...

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 ...

Titration of Human Coronaviruses Using an Immunoperoxidase Assay

Determination of infectious viral titers is a basic and essential experimental approach for virologists. Classical plaque assays cannot be used for viruses that do not cause significant cytopathic effects, which is the case for prototype strains 229E and OC43 of human coronavirus (HCoV). Therefore, an alternative indirect immunoperoxidase assay (IPA) was developed for the detection and titration of these viruses and is described herein. Susceptible cells are inoculated with serial logarithmic dilutions of virus-containing samples in a 96-well plate format. After viral growth, viral detection by IPA yields the infectious virus titer, expressed as 'Tissue Culture Infectious Dose 50 percent' (TCID50). This represents the dilution of a virus-containing sample at which half of a series of laboratory wells contain infectious replicating virus. This technique provides a reliable method for the titration of HCoV-229E and HCoV-OC43 in biological samples such as cells, tissues and fluids. This article is based on work first reported in Methods in Molecular Biology (2008) volume 454, pages 93-102.

In this video, we demonstrate an alternative method for detection and titering of viruses using an enzymatic antigen detection technique known as an immunoperoxidase assay. Here, we will show you how to collect your viral samples, prepare the cells for testing, and finally the immunoperoxidase assay using serial dilutions to determine the viral titer.

Determination of infectious viral titers is a basic and essential experimental approach for virologists. Classical plaque assays cannot be used for ...

ELIME (Enzyme Linked Immuno Magnetic Electrochemical) Method for Mycotoxin Detection

Immunoassays are a valid alternative to the more expensive and time consuming quantitative HPLC or GC1, 2 methods for the screening detection of hazardous mycotoxins in food commodities. In this protocol we show how to fabricate and interrogate an electrochemical competitive Enzyme linked immunomagnetic assay based on the use of magnetic beads as solid support for the immunochemical chain3 and screen printed electrodes as sensing platform.
Our method aims to determine the total amount of HT-2 and T-2 toxins, mycotoxins belonging to the trichothecenes family and of great concern for human health4. The use of an antibody clone with a cross reactivity of 100% towards HT-2 and T-2 allows to simultaneously detect both toxins with similar sensitivity5.
The first step of our assay is the coating step where we immobilize HT2-KLH conjugate toxin on the surface of magnetic beads. After a blocking step, necessary to avoid non-specific absorptions, the addition of a monoclonal antibody allows the competition between immobilized HT-2 and free HT-2 or T-2 present in the sample or dissolved in a standard solution.
At the end of the competition step, the amount of monoclonal antibody linked to the immobilized HT-2 will be inversely proportional to the amount of toxin in the sample solution.
A secondary antibody labeled with alkaline phosphatase (AP) is used to reveal the binding between the specific antibody and the immobilized HT-2. The final measurement step is performed by dropping an aliquot of magnetic bead suspension, corresponding to a specific sample/standard solution, on the surface of a screen-printed working electrode; magnetic beads are immobilized and concentrated by means of a magnet placed precisely under the screen-printed electrode. After two minutes of incubation between magnetic beads and a substrate for AP, the enzymatic product is detected by Differential Pulse Voltammetry (DPV) using a portable instrument (PalmSens) also able to initiate automatically eight measurements within an interval of few seconds.

ELIME Enzyme Linked Immuno Magnetic Electrochemical Method for Mycotoxin Detection

A protocol to detect trichothecenes (mycotoxins of concern for human health) using a newly developed screening method based on a competitive immunochemical method and a final electrochemical detection is demonstrated.

Immunoassays are a valid alternative to the more expensive and time consuming quantitative HPLC or GC1, 2 methods for the screening detection of hazardous ...

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or separation step. When the IL-8 target was present, a 'sandwich'-based assay attached magnetic beads with IL-8 capture antibody to streptavidin coupled fluorescent protein via the IL-8 target and a biotinylated IL-8 antibody. The magnetic beads are maneuvered into oscillatory motion by applying an alternating magnetic field gradient through two electromagnetic poles. The fluorescent proteins, which are attached to the magnetic beads are condensed into the detection area and their movement in and out of an orthogonal laser beam produces a periodic fluorescent signal that is demodulated using synchronous detection. The magnetic modulation biosensing system was previously used to detect the coding sequences of the non-structural Ibaraki virus protein 3 (NS3) complementary DNA (cDNA) [2]. The techniques that are demonstrated in this work for external manipulation and condensation of particles may be used for other applications, e.g. delivery of magnetically-coupled drugs in-vivo or enhancing the contrast for in-vivo imaging applications.

Magnetic modulation biosensing system is utilized to rapidly, sensitively and simply detect biological assays, such as DNA molecules and proteins.

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or ...

Detection of Signaling Effector-Complexes Downstream of BMP4 Using in situ PLA, a Proximity Ligation Assay

BMPs are responsible for a wide range of developmental and biological effects. BMP receptors activate (phosphorylate) the Smad1/5/8 effectors, which then, form a complex with Smad4 and translocate to the nucleus where they function as transcription factors to initiate BMP specific downstream effects 1. Traditional immuno-fluorescence techniques with antibodies against phospho-Smad peptides exhibit low sensitivity, high background and offer gross quantification as they rely on intensity of the antibody signal particularly if this is photosensitive fluorescent. In addition, phospho-Smads may not all be in complex with Smad4 and engaged in active transcription.
In situ PLA is a technology capable of detecting protein interactions with high specificity and sensitivity 2-4. This new technology couples antibody recognition with the amplification of DNA surrogate of the protein. It generates a localized, discrete signal in a form of spots revealing the exact position of the recognition event. The number of signals can be counted and compared providing a measurement. We applied in situ PLA, using the Duolink kit, with a combination of antibodies that allows the detection of the BMP signaling effectors phospho-Smad1/5/8 and Smad4 only when these are in proximity i.e. in a complex, which occurs only with signaling activation. This allowed for the first time, the visualization and measurement of endogenous BMP signaling with high specificity and sensitivity in a time course experiment under BMP4 stimulation.

Detection of Signaling Effector-Complexes Downstream of BMP4 Using in situ PLA, a Proximity Ligation Assay

Here we show how to use Proximity Ligation Assay (PLA), with a combination of antibodies to visualize Bone Morphogenetic Protein (BMP) signaling in fixed cells. This technique allowed us to follow the nuclear accumulation of endogenous BMP activated effector-complexes and quantify their levels over time under BMP4 stimulation.

BMPs are responsible for a wide range of developmental and biological effects. BMP receptors activate (phosphorylate) the Smad1/5/8 effectors, which then, ...

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, ...

Detection of miRNA Targets in High-throughput Using the 3'LIFE Assay

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an array of hundreds of queried 3&#8217;UTRs. Target identification is based on the dual-luciferase assay, which detects binding at the mRNA level by measuring translational output, giving a functional readout of miRNA targeting. 3&#8217;LIFE uses non-proprietary buffers and reagents, and publically available reporter libraries, making genome-wide screens feasible and cost-effective. 3&#8217;LIFE can be performed either in a standard lab setting or scaled up using liquid handling robots and other high-throughput instrumentation. We illustrate the approach using a dataset of human 3&#8217;UTRs cloned in 96-well plates, and two test miRNAs, let-7c and miR-10b. We demonstrate how to perform DNA preparation, transfection, cell culture and luciferase assays in 96-well format, and provide tools for data analysis. In conclusion 3&#39;LIFE is highly reproducible, rapid, systematic, and identifies high confidence targets.

Detection of miRNA Targets in High-throughput Using the 3&#039;LIFE Assay

Luminescent identification of functional elements in 3&#8217; untranslated regions (3&#8217;UTRs) (3&#8217;LIFE) is a technique to identify functional regulation in 3&#8217;UTRs by miRNAs or other regulatory factors. This protocol utilizes high-throughput methodology such as 96-well transfection and luciferase assays to screen hundreds of putative interactions for functional repression.

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an ...

A Bacterial Oral Feeding Assay with Antibiotic-Treated Mosquitoes

The mosquito midgut harbors a highly dynamic microbiome that affects the host metabolism, reproduction, fitness, and vector competence. Studies have been conducted to investigate the effect of gut microbes as a whole; however, different microbes could exert distinct effects toward the host. This article provides the methodology to study the effect of each specific mosquito gut microbe and the potential mechanism.
This protocol contains two parts. The first part introduces how to dissect the mosquito midgut, isolate cultivable bacteria colonies, and identify bacteria species. The second part provides the procedure to generate antibiotic-treated mosquitoes and reintroduce one specific bacteria species.

This article presents a protocol to investigate the effect of individual mosquito gut bacteria, including isolation and identification of mosquito midgut cultivable microbes, antibiotic depletion of mosquito gut bacteria, and reintroduce one specific bacteria species.

The mosquito midgut harbors a highly dynamic microbiome that affects the host metabolism, reproduction, fitness, and vector competence. Studies have been ...

Dissection and Immunostaining of Larval Salivary Glands from Anopheles gambiae Mosquitoes

Mosquito salivary glands (SGs) are a requisite gateway organ for the transmission of insect-borne pathogens. Disease-causing agents, including viruses and the Plasmodium parasites that cause malaria, accumulate in the secretory cavities of SG cells. Here, they are poised for transmission to their vertebrate hosts during a subsequent blood meal. As adult glands form as an elaboration of larval SG duct bud remnants that persist beyond early pupal SG histolysis, the larval SG is an ideal target for interventions that limit disease transmission. Understanding larval SG development can help develop a better understanding of its morphology and functional adaptations and aid in the assessment of new interventions that target this organ. This video protocol demonstrates an efficient technique for isolating, fixing, and staining larval SGs from Anopheles gambiae mosquitoes. Glands dissected from larvae in a 25% ethanol solution are fixed in a methanol-glacial acetic acid mixture, followed by a cold acetone wash. After a few rinses in phosphate-buffered saline (PBS), SGs can be stained with a broad array of marker dyes and/or antisera against SG-expressed proteins. This method for larval SG isolation could also be used to collect tissue for in situ hybridization analysis, other transcriptomic applications, and proteomic studies.

Dissection and Immunostaining of Larval Salivary Glands from Anopheles gambiae Mosquitoes

The adult mosquito salivary gland (SG) is required for the transmission of all mosquito-borne pathogens to their human hosts, including viruses and parasites. This video demonstrates efficient isolation of the SGs from the larval (L4) stage Anopheles gambiae mosquitoes and preparation of the L4 SGs for further analysis.

Mosquito salivary glands (SGs) are a requisite gateway organ for the transmission of insect-borne pathogens. Disease-causing agents, including viruses and ...