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Evaluation of Dot Immunogold Filtration Assay (DIGFA) By Recombinant Protein EPC1 for Anti- Echinococcus granulosus IgG Antibody.

Kordafshari S, Hosseini SH, Jalousian F, Rajabibazl M, Jones M, Etebar F - Iran J Parasitol (2015 Jan-Mar)

Bottom Line: Colloidal gold was prepared by controlled reduction of a boiling solution of chloroauric acid (H [AuCl4]) with sodium citrate and labeled with recombinant EPC1.Gold labeled antigen, showed a dark purple dot with agglutination particles in positive sera and light purple dot without agglutination in negative sera.Among 30 serum samples, 23 were positive and 7 were negative with DIGFA and 24 were positive, 6 were negative with ELISA.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Parasitology, School of Veterinary Medicine, Tehran University, Tehran, Iran.

ABSTRACT

Background: Cystic echinococcosis is an important zoonosis caused by the Echinococcus granulosus with a substantial impact in human and animal health in endemic areas. The purpose of the present study was serodiagnosis optimizing of dog echinococcosis in order to achieve a rapid diagnostic method.

Methods: Eight dogs were challenged with protoscoleces in order to have positive echinococcosis serum and 2 two-month old puppies were used as uninfected controls. Colloidal gold was prepared by controlled reduction of a boiling solution of chloroauric acid (H [AuCl4]) with sodium citrate and labeled with recombinant EPC1. Dot immunogold filtration assay (DIGFA) was developed by coating rEPC1 labeled colloidal gold on nitrocellulose membrane. The canine sera, taken three times including, 15, 28 and 35 days post infection were tested. A total of 30 serum samples including 24 sera from 8 infected dogs and 6 sera from 2 puppies were comparatively detected with both DIGFA and ELISA.

Results: Gold labeled antigen, showed a dark purple dot with agglutination particles in positive sera and light purple dot without agglutination in negative sera. Among 30 serum samples, 23 were positive and 7 were negative with DIGFA and 24 were positive, 6 were negative with ELISA.

Conclusion: DIGFA as a rapid and simple procedure could be utilized in quickly diagnosis of echinococcosis.

No MeSH data available.


Related in: MedlinePlus

Lane 1: 100 bp DNA Ladder, Lane 2, 3 & 4 EPC1 gene PCR products
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Figure 1: Lane 1: 100 bp DNA Ladder, Lane 2, 3 & 4 EPC1 gene PCR products

Mentions: The protein was labeled with colloidal gold as follows: 250 μl of recombinant EPC1 was added to 12 ml of liquid contains Colloidal gold with the help of magnetic stirring. The pH of 1 ml colloidal gold adjusted to a pH of 8.8 using 0.1 mol/L K2CO3. After 10 min, bovine serum albumin (BSA) was added to get the concentration of 10 g/L. After that, the mixture was centrifuged at 13000 × g for 15 min. The supernatant was discarded and the precipitate was dissolved by 5 g/L BSA-PBS, thus forming the colloidal gold labeling reagent (Fig. 1).


Evaluation of Dot Immunogold Filtration Assay (DIGFA) By Recombinant Protein EPC1 for Anti- Echinococcus granulosus IgG Antibody.

Kordafshari S, Hosseini SH, Jalousian F, Rajabibazl M, Jones M, Etebar F - Iran J Parasitol (2015 Jan-Mar)

Lane 1: 100 bp DNA Ladder, Lane 2, 3 & 4 EPC1 gene PCR products
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4403537&req=5

Figure 1: Lane 1: 100 bp DNA Ladder, Lane 2, 3 & 4 EPC1 gene PCR products
Mentions: The protein was labeled with colloidal gold as follows: 250 μl of recombinant EPC1 was added to 12 ml of liquid contains Colloidal gold with the help of magnetic stirring. The pH of 1 ml colloidal gold adjusted to a pH of 8.8 using 0.1 mol/L K2CO3. After 10 min, bovine serum albumin (BSA) was added to get the concentration of 10 g/L. After that, the mixture was centrifuged at 13000 × g for 15 min. The supernatant was discarded and the precipitate was dissolved by 5 g/L BSA-PBS, thus forming the colloidal gold labeling reagent (Fig. 1).

Bottom Line: Colloidal gold was prepared by controlled reduction of a boiling solution of chloroauric acid (H [AuCl4]) with sodium citrate and labeled with recombinant EPC1.Gold labeled antigen, showed a dark purple dot with agglutination particles in positive sera and light purple dot without agglutination in negative sera.Among 30 serum samples, 23 were positive and 7 were negative with DIGFA and 24 were positive, 6 were negative with ELISA.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Parasitology, School of Veterinary Medicine, Tehran University, Tehran, Iran.

ABSTRACT

Background: Cystic echinococcosis is an important zoonosis caused by the Echinococcus granulosus with a substantial impact in human and animal health in endemic areas. The purpose of the present study was serodiagnosis optimizing of dog echinococcosis in order to achieve a rapid diagnostic method.

Methods: Eight dogs were challenged with protoscoleces in order to have positive echinococcosis serum and 2 two-month old puppies were used as uninfected controls. Colloidal gold was prepared by controlled reduction of a boiling solution of chloroauric acid (H [AuCl4]) with sodium citrate and labeled with recombinant EPC1. Dot immunogold filtration assay (DIGFA) was developed by coating rEPC1 labeled colloidal gold on nitrocellulose membrane. The canine sera, taken three times including, 15, 28 and 35 days post infection were tested. A total of 30 serum samples including 24 sera from 8 infected dogs and 6 sera from 2 puppies were comparatively detected with both DIGFA and ELISA.

Results: Gold labeled antigen, showed a dark purple dot with agglutination particles in positive sera and light purple dot without agglutination in negative sera. Among 30 serum samples, 23 were positive and 7 were negative with DIGFA and 24 were positive, 6 were negative with ELISA.

Conclusion: DIGFA as a rapid and simple procedure could be utilized in quickly diagnosis of echinococcosis.

No MeSH data available.


Related in: MedlinePlus