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One-step antibody immobilization-based rapid and highly-sensitive sandwich ELISA procedure for potential in vitro diagnostics.

ABSTRACT: An improved enzyme-linked immunosorbent (ELISA) assay using one-step antibody immobilization has been developed for the detection of human fetuin A (HFA), a specific biomarker for atherosclerosis and hepatocellular carcinoma. The anti-HFA formed a stable complex with 3-aminopropyltriethoxysilane (APTES) by ionic and hydrophobic interactions. The complex adsorbed on microtiter plates exhibited a detection range of 4.9?pg mL(-1) to 20?ng mL(-1) HFA, with a limit of detection of 7?pg mL(-1). Furthermore, an analytical sensitivity of 10?pg mL(-1) was achieved, representing a 51-fold increase in sensitivity over the commercial sandwich ELISA kit. The results obtained for HFA spiked in diluted human whole blood and plasma showed the same precision as the commercial kit. When stored at 4°C in 0.1?M phosphate-buffered saline (PBS, pH 7.4), the anti-HFA bound microtiter plates displayed no significant decrease in their functional activity after two months. The new ELISA procedure was extended for the detection of C-reactive protein, human albumin and human lipocalin-2 with excellent analytical performance.

SUBMITTER: Vashist SK

PROVIDER: S-EPMC3957147 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature

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One-step antibody immobilization-based rapid and highly-sensitive sandwich ELISA procedure for potential in vitro diagnostics.

Vashist Sandeep Kumar SK Marion Schneider E E Lam Edmond E Hrapovic Sabahudin S Luong John H T JH

Scientific reports 20140318

An improved enzyme-linked immunosorbent (ELISA) assay using one-step antibody immobilization has been developed for the detection of human fetuin A (HFA), a specific biomarker for atherosclerosis and hepatocellular carcinoma. The anti-HFA formed a stable complex with 3-aminopropyltriethoxysilane (APTES) by ionic and hydrophobic interactions. The complex adsorbed on microtiter plates exhibited a detection range of 4.9 pg mL(-1) to 20 ng mL(-1) HFA, with a limit of detection of 7 pg mL(-1). Furthe ...[more]

PMID: 24638258

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Project description:IntroductionChikungunya is caused by an alpha virus transmitted to humans by an infected mosquito. Infection is generally considered to be self-limiting and non-critical. Chikungunya infection may be diagnosed by severe joint pain with fever, but it is difficult to diagnose because the symptoms of chikungunya are common to many pathogens, including dengue fever. Diagnosis mainly depends on viral culture, reverse transcriptase polymerase chain reaction (RT-PCR), and IgM ELISA. Early and accurate diagnosis of the virus can be achieved by the application of PCR methods, but the high cost and the need for a thermal cycler restrict the use of such methods. On the other hand, antibody-based IgM ELISA is considered to be inexpensive, but antibodies against chikungunya virus (CHIKV) only develop after 4 days of infection, so it has limited application in the earlier diagnosis of viral infection and the management of patients. Because of these challenges, a simple antigen-based sensitive, specific, and rapid detection method is required for the early and accurate clinical diagnosis of chikungunya.MethodsThe amino acid sequence of CHIKV ectodomain E1 and E2 proteins was analyzed using bioinformatics tools to determine the antigenic residues, particularly the B-cell epitopes and their characteristics. Recombinant E2-E1 CHIKV antigen was used for the development of polyclonal antibodies in hamsters and IgG was purified. Serological tests of 96 CHIKV patients were conducted by antigen-capture ELISA using primary antibodies raised against rCHIKV E2-E1 in hamsters and human anti-CHIKV antibodies.ResultsWe observed high specificity and sensitivity, of 100% and 95.8%, respectively, and these values demonstrate the efficiency of the test as a clinical diagnostic tool. There was no cross-reactivity with samples taken from dengue patients.DiscussionOur simple and sensitive sandwich ELISA for the early-phase detection of CHIKV infection may be used to improve the diagnosis of chikungunya.

Project description:African swine fever (ASF) is one of the most serious transnational swine diseases in the world. The case fatality rate of susceptible pigs is up to 100%. Currently, no commercial vaccine is available, so the prevention and control of ASF mainly relies on early diagnosis and culling of infected pigs. As the ASF virus continues to evolve, develop, and diversify, nucleic acid testing becomes less efficient. Here, we developed a method for the rapid and direct optical measurement of African swine fever virus (ASFV) antibody in vitro. This one-step procedure requires nearly no sample preparation and involves p30 protein-specific label-free integration into standard 96-well plates. Using a nanoplasmonic biosensor with extraordinary optical transmission (EOT) effect, one-step sample addition, ASFV antibody was detected within 20 min. The positive antibody showed a satisfactory sensitivity and linear relationship in the dilution ratio of 1:100-1:16000. It was used for the detection of clinical serum samples with a coincidence rate of 96.6%. The measurement results can be automatically analyzed and displayed on a conventional microplate meter computer and connected device. Our detection method can be widely applied in point-of-care testing (POCT) of ASFV antibody in pig farms. IMPORTANCE African swine fever (ASF) is a serious transnational disease caused by the African swine fever virus (ASFV), which is highly contagious in wild boars and domestic pigs. There is currently no available vaccine for ASF; therefore, development efforts are a key priority as ASFV continues to evolve and diversify. The ASF antibody rapid detection platform comprising the nanoplasmonic biosensor with extraordinary optical transmission effect can greatly reduce the detection time and improve detection flux while maintaining detection sensitivity and specificity. The one-step sample addition can effectively avoid cross contamination of samples in the detection process. The detection method provides a solution for the rapid and accurate real-time monitoring of ASF in pig farms.

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One-step antibody immobilization-based rapid and highly-sensitive sandwich ELISA procedure for potential in vitro diagnostics.

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One-step antibody immobilization-based rapid and highly-sensitive sandwich ELISA procedure for potential in vitro diagnostics.

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