Brucellosis CF-ELISA antibody detection kit

A technology for detection of brucellosis and antibodies, which is applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of missed detection of brucellosis antibodies, etc., and achieve the effects of improving detection sensitivity, easy use, and easy standardization of reagents

Active Publication Date: 2017-04-26
CHINA INST OF VETERINARY DRUG CONTROL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the ELISA detection method, the enzyme-labeled secondary antibody used only detects IgG-like brucellosis antibodies, and there are missed detections of IgM and other types of brucellosis antibodies

Method used

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  • Brucellosis CF-ELISA antibody detection kit
  • Brucellosis CF-ELISA antibody detection kit
  • Brucellosis CF-ELISA antibody detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1. Preparation and assembly of main components of the kit

[0029] 1. Preparation of main components of the kit

[0030] The main components of this kit: antigen coating plate, strong positive control serum, weak positive control serum, negative control serum, guinea pig complement, HRP-labeled guinea pig complement C1q-B monoclonal antibody 60G4, substrate color solution, stop solution, 20 times concentrated PBS-Tween washing solution.

[0031] 1.1 Preparation of LPS antigen for coating

[0032] Inoculate Brucella S2 strain on pancreatic agar or other solid medium suitable for the growth of Brucella, culture at 36~38℃ for 48~72 hours, conduct pure test, and eluted culture with 0.5% phenol saline This is Brucella bacteria liquid. Inactivate the bacteria liquid at 80°C for more than 2 hours, and then perform the inactivation test. Centrifuge the bacteria liquid that passed the inactivation test at 10000g for 20 minutes, and collect the precipitate.

[0033] Take the wet...

Embodiment 2

[0147] Example 2. Sensitivity test of the kit

[0148] 1. Sensitivity comparison test

[0149] The multiple-diluted brucellosis positive serum national standard was used for the sensitivity detection of the CF-ELISA test and compared with iELISA, complement fixation test (CFT) and test tube agglutination test (SAT). The results are shown in Table 2:

[0150] Table 2. Sensitivity comparison test

[0151]

[0152]

[0153] The results show that the sensitivity of the CF-ELISA test is basically the same as that of the iELISA. According to the test results, the sensitivity of both can reach at least 0.05IU of serum antibodies (cutt off=positive serum / negative serum=S / N≥2.1), which is CFT (It can detect 50IU per ml of serum) and SAT (standard method detects 50IU per ml of serum as positive).

[0154] 2. Comparative test of sensitivity to brucellosis positive serum

[0155] 349 bovine and goat sera from the group with confirmed brucellosis infection were used for sensitivity detection of CF-...

Embodiment 3

[0160] Example 3. Kit specificity test

[0161] The 190 sera of cattle and goats from the confirmed brucella infection group were used for the specificity test of the CF-ELISA test and compared with iELISA and CFT, SAT, RBPT. Table 4: Results

[0162] Table 4. Specificity comparison test

[0163]

[0164] After 50-fold dilution of 190 Brucella positive sera, specific tests were performed on the laboratory-produced Brucella antibody CF-ELISA detection kit, and compared with iELISA, CFT, SAT, and RBPT. The results showed that The CF-ELISA kit prepared by the invention has good specificity to the serum of cattle infected with brucellosis.

[0165] Note

[0166] 1. CF-ELISA method

[0167] 1.1. Antigen coating: Dilute the purified LPS antigen to 1μg / mL with 0.05M carbonate buffer, coat a 96-well microtiter plate (Corning, 2592) at 100μL / well, and act at 2℃~8℃ for 18h the above. Add 5% gelatin (Sigma) at 100μL / well to 37°C for 2h, then 2°C to 8°C for more than 18 hours, discard the coatin...

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Abstract

The invention relates to a brucellosis complement fixation-enzyme-linked immunosorbent assay (CF-ELISA) diagnostic kit, and the kit combines a reaction system in a complement fixation test (CFT) and an enzyme marker amplification system in an enzyme-linked immunosorbent assay (ELISA). The brucellosis complement fixation-enzyme-linked immunosorbent assay (CF-ELISA) diagnostic kit has the characteristics of high sensitivity, easy and fast use, high throughput and high degree of standardization compared with a traditional complement fixation test diagnostic reagent, has the characteristics of high specificity and capability of detecting a variety of brucellosis specific antibodies compared with a traditional ELISA diagnostic kit, and is an ideal new brucellosis diagnostic tool. The kit comprises the following main components: a lipopolysaccharide LPS antigen-coated plate, strongly-positive control serum, weakly positive control serum, negative control serum, a guinea pig complement, enzyme labeled guinea pig complement C1q-B monoclonal antibody 60G4, a substrate coloring-developing solution, a stop solution and washing liquid.

Description

Technical field [0001] The invention relates to the field of biological products for detection, in particular to a brucellosis complement fixation test-enzyme-linked immunosorbent assay (CF-ELISA) antibody detection kit, which is a novel diagnostic product for brucellosis. Background technique [0002] Brucellosis is a zoonotic disease characterized by abortion and fever caused by Brucella or Brucella. Brucellosis has a long history in the world. Humans have gradually deepened their research understanding of the disease, and the diagnosis level has been continuously improved. With the continuous development of brucellosis diagnostic technology, brucellosis serological detection methods have been continuously improved and improved. Traditional diagnostic techniques such as plate agglutination test (PT) and test tube agglutination test (SAT) have gradually been adopted by Tiger Red Plate Agglutination Test (RBPT), complement fixation test (CFT), and indirect enzyme-linked immunosor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/569
CPCG01N33/56911G01N33/577G01N2333/23
Inventor 毛开荣张金亚王楠徐磊王苗苗丁家波蒋玉文朱良全程君生蒋卉蒋菲
Owner CHINA INST OF VETERINARY DRUG CONTROL
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