Development of Optical Biosensor Using Protein A-Conjugated Chitosan–Gold Nanoparticles for Diagnosis of Cystic Echinococcosis

Human echinococcosis is a serious parasitic diseasethat still affects millions of people in many parts of the world. Since it can offer a critical threat to people’s health, it is important to discover a rapid, convenient, and economical method for detection. Herein, we propose a novel point of care assay, namely, an enhanced immuno-dot-blot assay for diagnosis of cystic echinococcosis (hydatidosis). This method is based on the formation of a sandwich complex between a goldnanoprobe (chitosan–gold nanoparticleprotein A) and hydatid cyst antigen (Ag B), which holds anti-Ag B antibodies. Briefly, protein A was conjugated to chitosan–gold nanoparticles via glutaraldehyde chemistry. Then, Ag B was immobilized on the surface of a nitrocellulose membrane, which was followed by the addition of the sera sample and gold nanoprobes. The positive signals were easily detectable by naked eye. The signal intensity of this biosensor was proportional to the concentration of active anti-Echinococcus granulosus antibodies on the surface of the nanoparticles, titer of antibodies in the sera samples, and concentration of Ag B coated on the nitrocellulose membrane. The minimum concentration to use the protein A for conjugation to detect titer of anti-Echinococcus IgGand the concentration of Ag B coated in nitrocellulose membrane were 0.5 and 0.3 mg/mL, respectively. This enhanced immuno-dot-blot assay offers a simple diagnostic technique withoutthe need for expensive equipment for diagnosis of echinococcosis.

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Specific Ligand Binding Attributable to Individual Epitopes of Gonococcal Transferrin Binding Protein A

Infection and Immunity ◽

10.1128/iai.70.2.732-740.2002 ◽

2002 ◽

Vol 70 (2) ◽

pp. 732-740 ◽

Cited By ~ 22

Author(s):

Heather P. Masri ◽

Cynthia Nau Cornelissen

Keyword(s):

Ligand Binding ◽

Fusion Proteins ◽

Iron Uptake ◽

Solid Phase ◽

Protein A ◽

Dot Blot ◽

Affinity Tags ◽

Amino Terminal ◽

Dot Blot Assay ◽

Specific Ligand

ABSTRACT The gonococcal transferrin receptor complex comprises two iron-regulated proteins, TbpA and TbpB. TbpA is essential for transferrin-iron uptake and is a TonB-dependent integral outer membrane protein. TbpB is thought to increase the efficiency of iron uptake from transferrin and is lipid modified and surface exposed. To evaluate the structure-function relationships in one of the components of the receptor, TbpA, we created constructs that fused individual putative loops of TbpA with amino-terminal affinity tags. The recombinant proteins were then overexpressed in Escherichia coli, and the fusions were recovered predominately from inclusion bodies. Inclusion body proteins were solubilized, and the epitope fusions were renatured by slow dialysis. To assess transferrin binding capabilities, the constructs were tested in a solid-phase dot blot assay followed by confirmatory quantitative chemiluminescent enzyme-linked immunosorbent assays. The constructs with only loop 5 and with loops 4 and 5 demonstrated dose-dependent specific ligand binding in spite of being out of the context of the intact receptor. The immunogenicities of individual TbpA-specific epitopes were investigated by generating rabbit polyclonal antisera against the fusion proteins. Most of the fusion proteins were immunogenic under these conditions, and the resulting sera recognized full-length TbpA in immunoblots. These results suggest that individual epitopes of TbpA are both immunogenic and functional with respect to ligand binding capabilities, and the vaccine implications of these findings are discussed.

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An advanced dual labeled gold nanoparticles probe to detect Cryptosporidium parvum using rapid immuno-dot blot assay

Biosensors and Bioelectronics ◽

10.1016/j.bios.2011.05.006 ◽

2011 ◽

Vol 26 (11) ◽

pp. 4624-4627 ◽

Cited By ~ 21

Author(s):

Chinnasamy Thiruppathiraja ◽

Senthilkumar Kamatchiammal ◽

Periyakaruppan Adaikkappan ◽

Muthukaruppan Alagar

Keyword(s):

Gold Nanoparticles ◽

Cryptosporidium Parvum ◽

Dot Blot ◽

Dot Blot Assay

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Rapid quantitation of uncoupling protein in brown adipose tissue mitochondria by a dot immunobinding ("dot blot") procedure: application to the measurement of uncoupling protein in Richardson's ground squirrel, rats, and mice

Biochemistry and Cell Biology ◽

10.1139/o90-143 ◽

1990 ◽

Vol 68 (6) ◽

pp. 973-979 ◽

Cited By ~ 6

Author(s):

Rachel E. Milner ◽

Paul Trayhurn

Keyword(s):

Adipose Tissue ◽

Brown Adipose Tissue ◽

Ground Squirrel ◽

Uncoupling Protein ◽

Protein A ◽

Sodium Dodecyl ◽

Enzyme Linked Immunosorbent Assay ◽

Nitrocellulose Membrane ◽

Dot Blot ◽

Brown Adipose

A dot immunobinding ("dot blot") method for measuring uncoupling protein in brown adipose tissue mitochondria is described. Mitochondrial proteins were solubilized in sodium dodecyl sulfate and applied directly to a nitrocellulose membrane housed in a 96-well microfiltration manifold. Spare binding sites on the nitrocellulose membrane were blocked with bovine serum albumin and then anti-(uncoupling protein) serum was applied. The antigen–antibody complex was detected by the addition of 125I-labelled protein A. Each nitrocellulose "dot" was cut out and its radioactivity was counted. A calibration curve was constructed from purified uncoupling protein standards, taken through the entire procedure. The dot immunobinding method is sensitive (nanogram quantities of uncoupling protein), and in contrast to conventional radioimmunoassay and enzyme-linked immunosorbent assay procedures, it is also rapid and appears to be very robust. The method has been successfully applied to the measurement of uncoupling protein in brown adipose tissue mitochondria of Richardson's ground squirrel, rats, and mice.Key words: brown adipose tissue, uncoupling protein, dot immunobinding, immunoassay, thermogenesis.

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Dot Blot Assay for Detection of Antidiacyltrehalose Antibodies in Tuberculous Patients

Clinical and Diagnostic Laboratory Immunology ◽

10.1128/cdli.6.5.686-689.1999 ◽

1999 ◽

Vol 6 (5) ◽

pp. 686-689 ◽

Cited By ~ 17

Author(s):

Lucio Vera-Cabrera ◽

Adrian Rendon ◽

Manuel Diaz-Rodriguez ◽

Vera Handzel ◽

Adalbert Laszlo

Keyword(s):

Mycobacterium Tuberculosis ◽

Colloidal Gold ◽

Protein A ◽

Gold Complex ◽

Rabbit Serum ◽

Enzyme Linked Immunosorbent Assay ◽

Dot Blot ◽

Human Immunoglobulins ◽

Dot Blot Assay ◽

Antigen Antibody

ABSTRACT A simple dot blot test with diacyltrehalose (DAT) as the antigen was developed to detect anti-DAT antibodies in tuberculous patients. To enhance antigen-antibody reaction detection, rabbit serum raised against human immunoglobulins was used prior to incubation with a protein A-colloidal gold complex. With the dot blot system, it was possible to obtain a sensitivity similar to that of enzyme-linked immunosorbent assay (ELISA) and a specificity of 97.14%, versus a specificity of 94.29% by the ELISA. We conclude that this simple and fast assay could be used in places where ELISA equipment is not easy available and that it might also be applicable with otherMycobacterium tuberculosis immunogenic antigens.

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An enhanced immuno-dot blot assay for the detection of white spot syndrome virus in shrimp using antibody conjugated gold nanoparticles probe

Aquaculture ◽

10.1016/j.aquaculture.2011.06.008 ◽

2011 ◽

Vol 318 (3-4) ◽

pp. 262-267 ◽

Cited By ~ 13

Author(s):

Chinnasamy Thiruppathiraja ◽

Subramani Kumar ◽

Vidhyapriya Murugan ◽

Periyakaruppan Adaikkappan ◽

Krishnan Sankaran ◽

...

Keyword(s):

Gold Nanoparticles ◽

White Spot Syndrome Virus ◽

White Spot ◽

Dot Blot ◽

Dot Blot Assay ◽

White Spot Syndrome

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APPLICATION OF REAL-TIME PRC TECHNIQUE AND REVERSE DOT-BLOT FOR DETECTION THE HIGH RISK TYPES OF HUMAN PAPILLOMAVIRUS CAUSING CERVICAL CANCER

Journal of Medicine and Pharmacy ◽

10.34071/jmp.2017.3.15 ◽

2017 ◽

pp. 99-103

Author(s):

Van Bao Thang Phan ◽

Hoang Bach Nguyen ◽

Van Thanh Nguyen ◽

Thi Nhu Hoa Tran ◽

Viet Quynh Tram Ngo

Keyword(s):

Cervical Cancer ◽

High Risk ◽

Real Time ◽

Real Time Pcr ◽

Dot Blot ◽

The Real ◽

Dot Blot Assay ◽

Hpv Types ◽

High Risk Hpv ◽

Reverse Dot Blot

Introduction: Infection with HPV is the main cause of cervical cancer. Determining HPV infection and the types of HPV plays an important role in diagnosis, treatment and prognosis of cervicitis/cervical cancer. Aims: Determining proportion of high-risk HPV types and the occurrence of coinfection with multiple HPV types. Methods: 177 women with cervicitis or abnormal Pap smear result were enrolled in the study. Performing the real-time PCR for detecting HPV and the reverse DOT-BLOT assay for determining type of HPV in cases of positive PCR. Results: 7 types of high-risk HPV was dectected, the majority of these types were HPV type 18 (74.6%) and HPV type 16 (37.6%); the proportion of infection with only one type of HPV was 30.4% and coinfection with multiple HPV types was higher (69.6%), the coinfected cases with 2 and 3 types were dominated (32.2% and 20.3%, respectively) and the coinfected cases with 4 and 5 types were rare. Conclusion: Use of the real-time PCR and reverse DOT-BLOT assay can determine the high-risk HPV types and the occurrence of coinfection with multiple HPV types. Key words: HPV type, Reverse DOT-BLOT, real-time PCR,PCR, cervical cancer

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An Effort to Making a Colorimitric Nano-Biosensor for Vibrio cholera Detection

Current Nanoscience ◽

10.2174/1573413716666191230154316 ◽

2020 ◽

Vol 16 (5) ◽

pp. 793-804

Author(s):

Naimeh Mahheidari ◽

Jamal Rashidiani ◽

Hamid Kooshki ◽

Khadijeh Eskandari

Keyword(s):

Gold Nanoparticles ◽

Colorimetric Assay ◽

Au Nanoparticle ◽

Bacteria Detection ◽

Great Promise ◽

Bacterial Cells ◽

Vibrio Cholera ◽

Minimum Concentration ◽

Vis Spectroscopy ◽

Fast Procedure

Background: Today, nanoparticles hold great promise in biomedical researches and applications including bacteria detection. The rapid and sensitive outcomes of bacteria detection strategies using nanoparticle conjugates become determinative, especially in bacterial outbreaks. In the current research, we focused on detecting V. cholera bacteria and its toxin using a thiocyanate/Au nanoparticle. Thiocyanate adsorbed strongly on the surface of gold nanoparticles and changed the surface by enhancing surface plasmon resonance of gold nanoparticles. Objective: This method is tried to introduce a simple and fast procedure to assay vibrio cholera. So, it is observed by the naked eyes as well. Methods: We used two antibodies (Ab) for V. cholera detection: a) a primary antibody conjugated to magnetic nanoparticles (MNPs) for trapping V. cholera bacterial cells, and b) a secondary Abconjugated thiocyanate-GNPs as a colorimetric detector. Then, an immuno-magnetic separation system connected to a colorimetric assay was designed based on the GNPs. The results were measured by ultraviolet-visible (UV-Vis) spectroscopy. Results: The results showed that gold nanoparticles are an appropriate optical assay for detecting biological samples in a minimum concentration and also it can be easily seen by the naked eyes. The linear range of this biosensor is 3.2×104 to 28×104 cells per ml. Conclusion: In this research, a colorimetric immune assay based on gold nanoparticles was designed to improve the sensitivity of V. cholera detection. Also, this method can be used for the detection of other biological agents.

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Use of monoclonal and polyclonal antibodies to detect prostatic acid phosphatase by immunoblotting.

Clinical Chemistry ◽

10.1093/clinchem/32.10.1832 ◽

1986 ◽

Vol 32 (10) ◽

pp. 1832-1835 ◽

Cited By ~ 2

Author(s):

P C Patel ◽

L Aubin ◽

J Côte

Keyword(s):

Monoclonal Antibodies ◽

Acid Phosphatase ◽

Western Blot ◽

Polyclonal Antibodies ◽

Prostatic Acid Phosphatase ◽

The Other ◽

Dot Blot ◽

Western Blots ◽

Dot Blot Assay ◽

Polyclonal Antisera

Abstract We investigated two techniques of immunoblotting--the Western blot and the dot blot--for use in detecting prostatic acid phosphatase (PAP, EC 3.1.3.2). We used polyclonal antisera to human PAP, produced in rabbits by hyperimmunization with purified PAP, and PAP-specific monoclonal antibodies in the immunoenzymatic protocols. We conclude that PAP can be readily detected by Western blots with use of polyclonal antisera, but not with monoclonal antibodies. On the other hand, using a dot blot assay, we could easily detect PAP with both polyclonal and monoclonal antibodies.

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