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Immunochromatographic test strip for diagnosing kala-azar based on detection of circulating antigens

An immune chromatography and kala-azar technology, applied in the field of bioengineering, can solve the problems of low accuracy, time-consuming and labor-intensive diagnosis of kala-azar, and achieve the effects of easy industrial production, broad application prospects, high sensitivity and specificity

Inactive Publication Date: 2014-11-12
STATION OF VIRUS PREVENTION & CONTROL CHINA DISEASES PREVENTION & CONTROL CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The object of the present invention is to propose a kind of immunochromatographic test strip for diagnosing kala-azar based on detection of circulating antigens. The immunochromatographic test strip for diagnosing kala-azar will solve the low accuracy rate of diagnosing kala-azar in the prior art. and time-consuming technical issues

Method used

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  • Immunochromatographic test strip for diagnosing kala-azar based on detection of circulating antigens
  • Immunochromatographic test strip for diagnosing kala-azar based on detection of circulating antigens

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Preparation of Leishmania donovani Amastigote Body Antigen

[0031] The amastigote antigen of Leishmania donovani was prepared as follows: take the proflagellates of Leishmania donovani 801 (MHOM / CN / / 801 / XJ) cultured in NNN medium at 22°C The body was inoculated in 199 medium (pH7.2-7.4) and expanded at 22°C. After the promastigotes reached a certain density, they were collected by centrifugation, and transferred to 199 medium (PH5.4) at 37°C for amastigote transformation. Centrifuge the transformed amastigotes at 3000g at 40C for 15 minutes to collect the amastigotes, discard the supernatant, and wash the pellet with PBS three times in the same way. In a water bath at 37°C, freeze and thaw repeatedly 5 times, then ultrasonically pulverize 3 times in an ice bath, centrifuge at 18000g, 4°C for 20min, and the supernatant is the soluble antigen. BALB / c mice were immunized with the antigen prepared by the above method, splenocytes from the immunized mice were fu...

Embodiment 2

[0034] Example 2 Preparation of monoclonal antibody against Leishmania donovani amastigote antigen

[0035] Immunization: Each BALB / c mouse was injected intraperitoneally with 100 μg of the suspension of the above-mentioned purified Leishmania donovani amastigote soluble antigen + Freund’s complete adjuvant for the first time, and then injected with 100 μg of purified Leishmania donovani every 1 month Protozoan amastigote soluble antigen + Freund's incomplete adjuvant suspension once, a total of 2 times, and 3 days before the mouse was killed and the spleen was taken for cell fusion, the antigen was directly injected through the tail vein to boost the immunization once.

[0036] Production and screening of hybridoma cells: fusion of SP2 / 0 tumor cells and splenocytes of immunized mice and cloning of hybridoma cells were carried out according to conventional methods in the art. The above-mentioned purified Leishmania donovani amastigote soluble antigen and glutathione-S-transfer...

Embodiment 3

[0044] Example 3 Preparation of immunochromatographic test strips for diagnosis of kala-azar

[0045] 1. The monoclonal antibody against the soluble antigen of Leishmania donovani was prepared from Example 2.

[0046] 2. Goat anti-mouse IgG was purchased.

[0047] 3. Preparation of immunocolloidal gold probes and gold standard pads

[0048] The monoclonal antibody colloidal gold probe and the gold standard pad of the soluble antigen of Leishmania donovani were prepared by the following method:

[0049] 1) Prepare colloidal gold particles by citrate reduction method, the specific method is: HAuCl 4 (Shanghai trial brand was purchased from Shanghai Sinopharm Chemical Reagent Co., Ltd.) to prepare 0.01% aqueous solution, take 100mL and heat to boiling, accurately add 1.6mL of 1% trisodium citrate aqueous solution under stirring, until the color of the liquid stabilizes into wine red, A colloidal gold solution is obtained.

[0050] 2) Determine the saturation concentration of ...

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Abstract

An immunochromatographic test strip for diagnosing kala-azar based on detection of circulating antigens comprises a sample mat, a gold labeling mat containing colloidal gold labeling probes, a cellulose membrane and a water absorption mat. A quality control line is arranged at the end, away from the gold labeling mat, of the cellulose membrane, a detection line is arranged on the portion, between the quality control line and the gold labeling mat, of the cellulose membrane, the detection line is composed of monoclonal antibodies of specific binding Leishmania donovani amastigote polypide antigens and generated by hybridomas, with the preservation number of CGMCC No.9240, of mice, the colloidal gold labeling probes are monoclonal antibodies of specific binding Leishmania donovani amastigote polypide antigens with different epitopes and generated by hybridomas, with the preservation number of CGMCC No.9239, of mice, and the quality control line is composed of secondary antibodies of the specific binding colloidal gold labeling probes or streptococcus G protein or staphylococcus aureus A protein. The immunochromatographic test strip is high in sensitivity and specificity, the overall sensitivity can reach 83%, and the specificity is 95%.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to an immunochromatographic test strip, in particular to an immunochromatographic test strip for diagnosing kala-azar based on detection of circulating antigens. Background technique [0002] Visceral leishmaniasis, or kala-azar, is parasitic on humans by protozoa of Leishmania donovanicomplex or L.infantum / L.chagasi A parasitic disease caused by the lympho-macrophage system of the sputum, which is transmitted by the bite of the vector sandfly. The disease is widespread worldwide, including 61 countries in Asia, Europe, Africa, and Central and South America, with about 500,000 new cases each year. Before the 1960s, our country was also deeply affected by it. The number of cases reached 600,000 and the death rate was extremely high. Accurate infection detection and diagnosis are critical for surveillance and treatment, so research on diagnostic techniques for kala-azar has always been...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/569
CPCG01N33/558G01N2800/00Y02A50/30
Inventor 汪俊云杨玥涛石锋高春花杨益周晓农
Owner STATION OF VIRUS PREVENTION & CONTROL CHINA DISEASES PREVENTION & CONTROL CENT
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