Modified ELISA for Ultrasensitive Diagnosis

Tsurusawa, Naoko; Chang, Jyunhao; Namba, Mayuri; Makioka, Daiki; Yamura, Sou; Iha, Kanako; Kyosei, Yuta; Watabe, Shigeo; Yoshimura, Teizo; Ito, Etsuro

doi:10.3390/jcm10215197

Cited by 29 publications

(16 citation statements)

References 90 publications

(104 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The double antibody sandwich method to detect antigenic proteins employs a microtiter plate with wells coated with a high-a nity biotinylated antibody for detection of the sample or standard. The presence of the target protein in the sample is detected by a colorimetric reaction with a substrate solution that is terminated by the addition of a termination solution [23]. However, this method does not allow effective detection at the early stage of disease, thus delaying prognosis and threatening the health of the patient.…”

Section: Discussionmentioning

confidence: 99%

Recombinase Polymerase Amplification Assay with Lateral Flow Strips for Rapid Diagnosis of Candidiasis due to Candida parapsilosis

Wang

Fan

et al. 2022

Preprint

View full text Add to dashboard Cite

Background Candida parapsilosis is a common cause of candidiasis among hospitalized patients, often surpassing Candida albicans. Due to the recent increase in C. parapsilosis infections, there is an urgent need for rapid, sensitive, and real-time on-site detection of nucleic acids for timely diagnosis of candidiasis. Methods We developed an assay for detection of C. parapsilosis by combining recombinase polymerase amplification (RPA) with a lateral flow strip (LFS). The RPA-LFS assay was used to amplify the beta-1,3-glucan synthase catalytic subunit 2 (FSK2) gene of C. parapsilosis with a primer-probe set optimized by introducing base mismatches (4 bases modified by the probe and one by the reverse primer) to achieve specific and sensitive detection of clinical samples. Results The RPA assays can rapidly amplify and visualize a target gene within 30 min, while the entire process can be completed within 40 min by pre-processing the sample.The sensitivity and specificity of the RPA-LFS assay were determined by analysis of 35 common clinical pathogens and 281 clinical samples against quantitative PCR. Conclusions The results confirmed that the proposed RPA-LFS assay is a reliable molecular diagnostic method for the detection of C. parapsilosis to meet the urgent need for rapid, specific, sensitive, and portable field testing.

show abstract

Section: Discussionmentioning

confidence: 99%

Recombinase Polymerase Amplification Assay with Lateral Flow Strips for Rapid Diagnosis of Candidiasis due to Candida parapsilosis

Wang

Fan

et al. 2022

Preprint

View full text Add to dashboard Cite

show abstract

“…Measurements of GRP78 in exosomes were performed with the ultrasensitive thio-NAD cycling ELISA [ 19 , 20 ]. This ELISA is coupled with a thio-NAD cycling reaction, and thus the signal obtained from a sandwich ELISA using a primary and secondary antibody is amplified by the thio-NAD cycling reaction [ 21 , 22 ] ( Figure 1 A). For ultrasensitive detection of GRP78, we used a GRP78 ELISA kit (Human HSPA5/GRP78/BiP (sandwich ELISA) ELISA Kit; LS-F11578, LifeSpan Biosciences, Seattle, WA, USA) and thio-NAD cycling.…”

Section: Methodsmentioning

confidence: 99%

Ultrasensitive Detection of GRP78 in Exosomes and Observation of Migration and Proliferation of Cancer Cells by Application of GRP78-Containing Exosomes

Tsurusawa

Iha

Sato

et al. 2022

Cancers

Self Cite

View full text Add to dashboard Cite

Cancer cells communicate with each other via exosomes in the tumor microenvironment. However, measuring trace amounts of proteins in exosomes is difficult, and thus the cancer stemness-promoting mechanisms of exosomal proteins have not been elucidated. In the present study, we attempted to quantify trace amounts of 78-kDa glucose-regulated protein (GRP78), which is involved in cancer progression, in exosomes released from cultured gastric cancer cells using an ultrasensitive ELISA combined with thio-NAD cycling. We also evaluated the cancer stemness-promoting effects by the application of high-GRP78-containing exosomes to cultured gastric cancer cells. The ultrasensitive ELISA enabled the detection of GRP78 at a limit of detection of 0.16 pg/mL. The stemness of cancer cultured cells incubated with high-GRP78-containing exosomes obtained from GRP78-overexpressed cells was increased on the basis of both an MTT assay and a wound healing assay. Our results demonstrated that the ultrasensitive ELISA has strong potential to measure trace amounts of proteins in exosomes. Further, exosomes with a high concentration of GRP78 promote the cancer stemness of surrounding cells. The technique for quantifying proteins in exosomes described here will advance our understanding of cancer stemness progression via exosomes.

show abstract

“…This "instrumentalfree" approach is cost-effective and user-friendly, which can be applied in basic clinical laboratories. The most widely adopted colorimetric technique is ELISA, which is a standard method in the diagnosis of various microbial or viral infections [6,7]. Specific antibodies are immobilized on the surface of solid substrate to capture target antigens, followed by complexation with another antibody tagged enzymes.…”

Section: Colorimetric Aptasensorsmentioning

confidence: 99%

“…The immunological method relies on the specific binding of antibodies to the antigens corresponding to specific pathogens [5]. It is a traditional clinical pathogen detection method including enzyme-linked immunosorbent assay (ELISA) [6,7], fluorescence and luminescence immunoassay [8,9], and immunoblotting [10]. Immunoassay-based technique is sensitive and easy to perform in a basic clinical laboratory but demands particular antibody-related production, storage, and handling procedures.…”

Section: Introductionmentioning

confidence: 99%

Aptasensors for the detection of infectious pathogens: design strategies and point-of-care testing

2022

View full text Add to dashboard Cite

The epidemic of infectious diseases caused by contagious pathogens is a life-threatening hazard to the entire human population worldwide. A timely and accurate diagnosis is the critical link in the fight against infectious diseases. Aptamer-based biosensors, the so-called aptasensors, employ nucleic acid aptamers as bio-receptors for the recognition of target pathogens of interest. This review focuses on the design strategies as well as state-of-the-art technologies of aptasensor-based diagnostics for infectious pathogens (mainly bacteria and viruses), covering the utilization of three major signal transducers, the employment of aptamers as recognition moieties, the construction of versatile biosensing platforms (mostly micro and nanomaterial-based), innovated reporting mechanisms, and signal enhancement approaches. Advanced point-of-care testing (POCT) for infectious disease diagnostics are also discussed highlighting some representative ready-to-use devices to address the urgent needs of currently prevalent coronavirus disease 2019 (COVID-19). Pressing issues in aptamer-based technology and some future perspectives of aptasensors are provided for the implementation of aptasensor-based diagnostics into practical application.

show abstract

Modified ELISA for Ultrasensitive Diagnosis

Cited by 29 publications

References 90 publications

Recombinase Polymerase Amplification Assay with Lateral Flow Strips for Rapid Diagnosis of Candidiasis due to Candida parapsilosis

Recombinase Polymerase Amplification Assay with Lateral Flow Strips for Rapid Diagnosis of Candidiasis due to Candida parapsilosis

Ultrasensitive Detection of GRP78 in Exosomes and Observation of Migration and Proliferation of Cancer Cells by Application of GRP78-Containing Exosomes

Aptasensors for the detection of infectious pathogens: design strategies and point-of-care testing

Contact Info

Product

Resources

About