Detection of Plasmodium Sporozoites in Anopheles Mosquitoes using an Enzyme-linked Immunosorbent Assay
Summary
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.
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.
Protocol
Representative Results
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 …
Declarações
The authors have nothing to disclose.
Acknowledgements
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
| 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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