Lipoteichoic acid enzyme-linked immunosorbent assay (ELISA) detection kit and preparative technique thereof

A detection kit and lipoteichoic acid technology, applied in the field of biomedicine, can solve problems such as low efficiency, time-consuming and labor-intensive efficiency, unfavorable anti-infection treatment, etc., and achieve the effects of strong specificity, high sensitivity, easy operation and interpretation

Inactive Publication Date: 2013-01-23
TIANJIN LANRY BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the automated blood culture system has been widely used in clinical microbiology laboratories. Usually, when a positive alarm bottle appears, the bacterial solution in the bottle is first transferred to a blood plate, and then identification and drug sensitivity tests are performed. The resul

Method used

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Experimental program
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Effect test

Embodiment 1

[0027] The present invention obtains highly specific lipoteichoic acid monoclonal antibody through antigen immunization, and establishes a competitive ELISA kit after separation and purification. Antibody separation and purification adopts ammonium sulfate fractional precipitation and affinity chromatography technology. The applied ammonium sulfate concentration was 50% and 33% respectively. After overnight at 4°C, centrifuge at 10000rpm and 4°C for 20min, and resuspend the pellet to its original volume in 0.01M PBS. After dialysis with pH 7.4 and 0.01M PBS for 48 hours, the protein G purification column was used to equilibrate with pH 7.0 and 0.02M PB balance solution, and the 0.1M and pH 2.7 Glycine-HCl eluent was eluted. The purified antibody is stored at -20°C for later use.

Embodiment 2

[0029] In the present invention, gradient dilution of antigen and gradient dilution of enzyme-labeled antibody are performed, and the checkerboard method is used for detection to establish the best working dilution. The serum samples were diluted, boiled, and compared with the treatment conditions of the EDTA treatment solution. After testing, it was determined that the pretreatment conditions were that the serum was diluted ten times and then boiled for 5 minutes. The test conditions of the kit were established through optimization: the serum sample was diluted tenfold and boiled for 5 minutes, incubated with enzyme-labeled antibody outside the wells at 37°C for 1 hour, then added to the lipoteichoic acid antigen-coated enzyme-labeled plate, and incubated at 37°C Wash the plate 5 times for 1 hour, 2 min each time, add the color developing solution and stop the reaction after 20 min, and measure the OD value by a microplate reader.

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Abstract

The invention relates to preparation of lipoteichoic acid monoclonal antibodies and an e-linked immunosorbent assay (ELISA) detection kit manufactured by using the preparation. An ELISA detection reagent is a high-technology product in the field of biological medicines, and is an in-vitro diagnostic reagent. Following a search, the ELISA detection reagent fills the domestic blank, the current situation of detecting staphylococcus aureus by using a time-wasting and labor-wasting blood culture method is changed, an enzyme linked immunosorbent assay which is high in sensitivity and specificity is adopted, and detection time is shortened. Balb/c female mice are immuned by lipoteichoic acid antigens extracted from cell walls of the staphylococcus aureus, then monoclonal antibodies L are obtained and are IgG immune globulin after being detected, and the valence of the antibodies is 1*106. The monoclonal antibodies are purified by a saturated ammonium sulfate precipitation and affinity chromatography technology. Lipoteichoic acids serve as coating antigens, the obtained monoclonal antibodies serve as enzyme-labeled antibodies, and then a competition-method ELISA kit is manufactured and is used for diagnosing staphylococcus aureus infection which serves as one of references for clinical diagnosis.

Description

Technical field: [0001] The invention relates to a lipoteichoic acid ELISA detection kit and its preparation technology, which are used for the rapid detection of clinical Staphylococcus aureus infection and belong to the field of biomedicine. Background technique: [0002] With the aging of the population, the widespread development of organ transplant operations, the use of immunosuppressants, and the influence of social factors, the incidence of Gram-positive bacterial infections has increased, and the pathogenic bacteria of bacteremia accounted for Gram-positive bacteria. 59.6%, of which Staphylococcus accounts for 38.6%. At present, automated blood culture systems have been widely used in clinical microbiology laboratories. Under normal circumstances, when a positive alarm bottle appears, the bacteria in the bottle are first transferred to the blood plate, and then the identification and drug sensitivity test are performed. The result is 48-72 hours after the positive alarm...

Claims

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Application Information

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IPC IPC(8): G01N33/577
Inventor 苑庆华何永胜
Owner TIANJIN LANRY BIO TECH
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