Direct fluorescene immunoassay for viral antigens

A virus antigen and antigen technology, applied in the field of biological samples, can solve problems such as loss

Active Publication Date: 2012-05-30
DIAGNOSTIC HYBRIDS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Also, if the cells are not completely dry, they can be lost during processing steps, resulting in insufficient numbers of cells judged to be present.
Current DFA methods also require highly skilled technicians to prepare, read and interpret the results, as non-specifically stained mucus or debris can be seen in samples

Method used

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  • Direct fluorescene immunoassay for viral antigens
  • Direct fluorescene immunoassay for viral antigens
  • Direct fluorescene immunoassay for viral antigens

Examples

Experimental program
Comparison scheme
Effect test

Embodiment I

[0256] [Example 1: LDFA detection of influenza A virus]

[0257] will repeat R- sv / cs cell culture monolayers with a series of 4 10-fold serial dilutions of influenza A virus (A / H3N2) or herpes simplex virus (HSV-1) (i.e., designated as samples: 4+, 3+, 2 + and 1+) inoculations and comparison with negative control (NC). Infected cultures were grown on coverslips inside shell flasks to allow virus replication for approximately 22 hours.

[0258] For each virus group, media was aspirated from 4+, 1+ and NC shell bottles. Phosphate buffered saline (PBS; 200 μl) was then added to each shell flask; and the cell monolayers were scraped coverslips and transferred to labeled 1.5 ml Eppendorf centrifuge tubes. Acetone (100%; 800 μl) was added to each centrifuge tube to a final volume of 1 ml to create an 80% acetone / cell suspension solution, which was incubated at room temperature for about 10 minutes to permeabilize the cells.

[0259] Permeabilized cells were then harvested and ...

Embodiment II

[0261] [Example II: Duet MAb virus screening in clinical aspirates]

[0262] Nasal discharge samples were collected from patients. Aliquot each sample into Tube A / B; Tube R / M; or Tube P / Ad (A-Influenza A; B-Influenza B; R-Respiratory Virus; M-Pneumovirus; P-Parainfluenza Virus ; Ad-adenovirus).

[0263] 1. Add 70 μl of cell suspension to each tube

[0264] 2. Add 2 drops of the corresponding MAb

[0265] 3. Incubate at 35℃~37℃ for 5 minutes

[0266] 4. Wash with 1.5ml of PBS

[0267] 5. Centrifuge at 2000×g for 2 minutes

[0268] 6. Aspirate the supernatant with a transfer pipette

[0269] 7. Add 20 μl of resuspension buffer to each tube

[0270] 8. Loading Slides

Embodiment III

[0271] [Example III: MAb cross-reactivity: list of materials]

[0272] The following is a list of the materials with lot numbers used in the cross-reactivity described in the studies presented in Examples IV and V herein

[0273]

[0274]

[0275]

[0276]

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Abstract

The present invention describes a liquid direct fluorescence antibody assay that is rapid and sensitive to detect respiratory virus in infected cells. The assay includes centrifugation of the specimen, incubation of sample and reagents in solution, and detection of the absence or presence of respiratory virus. Sapogenin is used as a detergent to permeabilize the cells for entry of the monoclonal antibodies to react with intracellular antigens. The cells are stained with fluorescently labeled monoclonal antibodies against the viral antigens along with a background stain and a fluorescent nuclear stain. This counter staining decreases background and allows co-localization of antigen and nuclear structures for enhanced detection.

Description

【Technical field】 [0001] The present invention relates to the processing of biological samples in liquid form for direct virus detection. For example, a sample may originate from the respiratory system. Detection methods may use antibodies that bind directly to viral antigens thereby allowing identification as well as detection. In some examples, the antibody is a labeled monoclonal antibody. The method can be integrated with a device comprising an algorithm capable of discriminating between multiple fluorescent signals. 【Background technique】 [0002] Viral infections (ie, eg, influenza A and B viruses) are responsible for annual disease epidemics in children and adults. Diseases caused by influenza A and B viruses are clinically indistinguishable, and may circulate simultaneously, Van Voris et al., "Influenza viruses" pp. 267-297. In: R.B. Belshe (ed.), Textbook Of Human Virology. PSG Publishing Co., Littleton, Mass. (1984). Antiviral chemoprophylaxis and treatment ar...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/58
CPCG01N33/56983G01N33/5091G01N33/54326G01N2333/11G01N2333/075G01N2333/115G01N2333/135
Inventor D・R・肖勒J・L・布朗J・若里克R・洛拉尔
Owner DIAGNOSTIC HYBRIDS
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