研究目的
To develop a signal-generation mechanism for the detection of anti-Leishmania sp. IgG antibodies with the naked eye using plasmonic ELISA (pELISA) as an additional tool for canine leishmaniasis (CanL) diagnosis.
研究成果
The developed indirect pELISA was effective in detecting anti-Leishmania sp. IgG antibodies with the naked eye, offering a practical tool for CanL diagnosis, especially in resource-constrained areas. The method's simplicity and lack of need for sophisticated equipment make it particularly useful for widespread application.
研究不足
The study acknowledges that environmental factors like humidity and temperature can affect the stability of nanoparticles, suggesting the need for systematic experiments to test ideal conditions. The method's sensitivity and specificity in field conditions and its comparison with other diagnostic methods were not extensively evaluated.
1：Experimental Design and Method Selection:
The study adapted the analytical principle of the pELISA to an indirect format for detecting anti-Leishmania sp. IgG antibodies. The method involves the growth of gold nanoparticles that produce blue or red colored solutions in the presence or absence of the antibodies.
2：Sample Selection and Data Sources:
Canine serum samples positive and negative for CanL were used. Serial dilutions of serum and anti-dog IgG biotinylated antibodies were analyzed.
3：List of Experimental Equipment and Materials:
Included reagents like recombinant antigen rK28, streptavidin, catalase, gold (III) chloride trihydrate, and equipment such as polystyrene microtiter plates and a Tecan microplate reader.
4：Experimental Procedures and Operational Workflow:
The assay involved sensitizing microtiter plates with recombinant antigen, incubating with serum samples, adding biotinylated secondary antibody and streptavidin-catalase conjugate, and then hydrogen peroxide and gold solution to observe color change.
5：Data Analysis Methods:
The growth of gold nanoparticles was monitored by measuring absorbance at 550 nm and visually assessing color changes.
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bovine serum albumin
cat. no. A7030
Sigma-Aldrich
Used as a blocking agent in the assay.
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Tween 20
cat. no. P9416
Sigma-Aldrich
Used in the washing buffer for the assay.
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gold (III) chloride trihydrate
cat. no. 520918
Sigma-Aldrich
Used for the growth of gold nanoparticles in the assay.
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hydrogen peroxide
cat. no. H1009
Sigma-Aldrich
Used in the assay to control the growth of gold nanoparticles.
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streptavidin
cat. no. S4762
Sigma-Aldrich
Used in the conjugation process with catalase for the pELISA assay.
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catalase from bovine liver
cat. no. C1345
Sigma-Aldrich
Used in the conjugation process with streptavidin for the pELISA assay.
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polystyrene microtiter plates
cat. no. 655081
Greiner Bio-one
Used as the platform for the pELISA assay.
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Tecan microplate reader
Sunrise model ref. 16039400
Tecan
Used to measure absorbance values at 550 nm.
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