Abstract
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.
Introduction
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.
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Protocol
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.
- Capture and peroxidase-conjugated mAbs
- 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 °C.
- Blocking buffer (BB)
- 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.
- Add 0.02 g of phenol red as an indicator and adjust the pH to 7.4 with HCl. Store BB at 4 °C for up to one week, or aliquot into 50 mL for long-term storage at -20 °C.
- Positive controls
- To reconstitute the positive controls, rehydrate the lyophilized proteins by adding 1,000 μL of BB. Make aliquots of the stock positive control solutions as needed, and store them at -20 °C.
- For serial dilution, further dilute each positive control to the final working concentration in BB as follows: Pf, 2 pg/µL; Pv (VK210), 182 (pg/µL); Pv (VK247), 89 pg/µL.
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/μL for Pf; 182, 91, 46, 23, 11, 5.7 pg/μL for PV210; and 89, 45, 22, 11, 5.6, 2.8 pg/μL PV247.
- 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.- With BB as the negative control, use the 2-fold absorbance threshold for reliable positive readouts.
2. Mosquito sample preparation
- 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. - Add 50 μL of Grinding Buffer (GB) into each tube and homogenize the sample with a clean pestle (washed with soap). Rinse the used pestle with 250 μL of GB into the tube containing the homogenized mosquito(es) to a final volume of ~300 μL.
- Keep the sample in a freezer (-20 °C) until use or proceed immediately to perform ELISA.
3. Sporozoite ELISA
- Fill out the sporozoite ELISA worksheet (see Supplemental Material 1). Prepare one ELISA plate for each CSP (Pf, Pv-210, or Pv-247).
- Prepare the capture mAb working solution by dissolving the antibody in PBS: 4 µg/mL Pf; 2 µg/mL Pv-210; 2 µg/mL of Pv-247. Calculate the volumes required based on the addition of 5 mL per plate. Vortex the solution gently.
- Pipette 50 μL 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.
- 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. - Add 200 μL 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.
- Load the mosquito homogenate and the control on the plate as follows.
- Add 50 μL of the positive control to wells H1 and H2.
- Add 50 μL of BB to wells in columns 1 and 2 from row C to G. Then, add 50 μL 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 μL. - Add 50 μL of BB (negative control) to wells A1, A2, B1, and B2.
- Add 50 μL of each homogenate sample to an Unknown (Unk) well.
- Cover the plate and incubate for 2 h at room temperature.
- 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. - 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 µg/mL.
- Calculate the required volumes based on the addition of 5 mL of working conjugate mAb solution per plate.
- Test peroxidase activity by mixing 5 μL of the peroxidase-labeled mAb made in step 3.8 with 100 μL 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.
- Aspirate or dump the well contents and tap the plate upside down on paper towels 5 times to remove all liquid.
- Wash the wells twice with 200 μL of PBS-Tween, aspirate the well contents, and tap the plate 5 times each.
- Add 50 μL 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.
- Wash the wells 3 times with 200 μL of PBS-Tween, aspirate the well contents, and tap the plate 5 times each.
- Add 100 μL 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.
- After 30 min, read the absorbance at 405-414 nm using an ELISA plate reader.
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.
4. Analysis
- Detecting positive samples.
- Label samples with absorbance values above the cut-off (twice the mean absorbance value of the negative samples) as positive.
- Quantifying CSP
- Estimate the CSP concentration in the sample using the standard curve constructed from the control dilution series as follows.
- Create the standard curve by plotting the absorbance values (y-axis) of the serially diluted controls against their concentrations (x-axis).
- Perform linear regression to determine the best fit using y = A + Bx, where A and B are free parameters.
- Determine the CSP concentration for each positive sample by solving the equation for a given absorbance value.
- Estimate the CSP concentration in the sample using the standard curve constructed from the control dilution series as follows.
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Representative Results
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/μL (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.
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. Please click here to view a larger version of this figure.
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/μL. Linear regression of the data estimated the CSP concentration in A7 as 0.35 pg/μL. Abbreviations: CSP = circumsporozoite protein; ABTS = 2,2'-azino-di-(3-ethylbenzthiazoline-6-sulfonate); ELISA = enzyme-linked immunosorbent assay; Abs = absorbance. Please click here to view a larger version of this figure.
Table 1: Recipes. Please click here to download this Table.
Supplemental Material S1: Sporozoite ELISA worksheet. Please click here to download this File.
Supplemental Material S2: Guidelines for ELISA plate reader software. Please click here to download this File.
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Discussion
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 °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.
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Disclosures
The authors have no conflicts of interest to declare.
Acknowledgments
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).
Materials
| Name | Company | Catalog Number | Comments |
| Equipment | |||
| ELISA plate reader | BioTek Instrument, Inc. | Synergy H1 | Absorbance is measured using UV-VIS absorbance detection mode |
| Grinder pestle | Axygen | PES-15-B-SI | For homogenizing the mosquito head/thorax |
| Reagents | |||
| ABTS substrate 2-component | KPL | 50-62-00 | For peroxidase driven detection |
| Blocking buffer (BB) | Note: solution | ||
| Capture and peroxidase-conjugated monoclonal antibodies (mAbs) | 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.) | ||
| Casein | Sigma aldrich | 9000-71-9 | For blocking buffer preparation |
| Grinding buffer (GB) | Note: solution | ||
| Igepal CA-630 | Sigma aldrich | 9002-93-1 | For grinding buffer preparation |
| KCl | Sigma aldrich | 7447-40-7 | For phosphate buffer saline preparation |
| KH2PO4 | Sigma aldrich | 7778-77-0 | For phosphate buffer saline preparation |
| Na2HPO4 | Sigma aldrich | 7558-79-4 | For phosphate buffer saline preparation |
| NaCl | Sigma aldrich | 7647-14-5 | For phosphate buffer saline preparation |
| NaOH | Sigma aldrich | 1310-73-2 | For blocking buffer preparation |
| PBS-Tween | |||
| Pf capture mAb | CDC | Pf-CAP | For capturing Pf circumsporozoite protein |
| Pf peroxidase mAb | CDC | Pf-HRP | For detecting Pf circumsporozoite protein |
| Pf positive control | CDC | Pf-PC | Pf positive control |
| Phenol red | Sigma aldrich | 143-74-8 | For blocking buffer preparation |
| Phosphate buffered saline (PBS) | Note: solution | ||
| Positive controls | Note: solution | ||
| Pv210 capture mAb | CDC | Pv210-CAP | For capturing Pv210 circumsporozoite protein |
| Pv210 peroxidase mAb | CDC | Pv210-HRP | For detecting Pv210 circumsporozoite protein |
| Pv210 positive control | CDC | Pv210-PC | Pv210 positive control |
| Pv247 capture mAb | CDC | Pv247-CAP | For capturing Pv247 circumsporozoite protein |
| Pv247 peroxidase mAb | CDC | Pv247-HRP | For detecting Pv247 circumsporozoite protein |
| Pv247 positive control | CDC | Pv247-PC | Pv247 positive control |
| Tween20 | Sigma aldrich | 9005-64-5 | For PBS-Tween preparation |
| Consumables | |||
| Disposable pipetting reservoirs | Generic | For working reagents | |
| ELISA plates: 96 well clear round bottom PVS | Corning Life Science | 2797 | For ELISA |
| Gloves: disposable gloves and freezer gloves | Generic | For personal protection | |
| Lab gown | Generic | for personal protection | |
| Multichannel Pipette P30-300 | Generic | For solution transfer | |
| Pipette set, P2, P20, P200, and P1000 | Generic | For solution transfer | |
| Pipette tips 10 µL, 200 µL, and 1000 µL | Generic | For solution transfer | |
| Serological pipettes 5 mL, 10 mL | Generic | For solution transfer | |
| Transfer pipettes | Generic | For solution transfer |
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