Brucellosis cf-elisa antibody detection kit

A brucellosis and antibody detection technology, applied in measurement devices, instruments, scientific instruments, etc., can solve problems such as missed detection of brucellosis antibodies, improve detection sensitivity, easily standardize reagents, and achieve high-throughput detection. Effect

Active Publication Date: 2018-07-20
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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1, preparation and assembly of the main components of the kit

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

[0030] The main components of this kit: antigen-coated 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 chromogenic 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°C for 48-72 hours, conduct a pure test, and elute the culture with 0.5% phenol saline This is the Brucella bacteria liquid. The bacteria liquid was inactivated at 80°C for more than 2 hours, and then the inactivation test was carried out. Centrifuge the bacteria solution that passed the inactivation test at 10,000 g for 20 min to...

Embodiment 2

[0147] Embodiment 2, kit sensitivity test

[0148] 1. Sensitivity comparison test

[0149] National standards for brucellosis-positive sera in multiple dilutions were used to test the sensitivity of CF-ELISA and compared with iELISA, complement fixation test (CFT) and tube agglutination test (SAT). The results are shown in Table 2:

[0150] Table 2. Sensitivity comparison tests

[0151]

[0152]

[0153] The results show that the sensitivity of CF-ELISA test is basically the same as that of iELISA. According to the test results, the sensitivity of both can reach at least 0.05IU serum antibody detection (cutt off=positive serum / negative serum=S / N≥2.1), which is CFT (50 IU per milliliter of serum can be detected) and 100 times of SAT (50 IU per milliliter of serum can be detected by the standard method is positive).

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

[0155] 349 bovine and sheep sera from confirmed brucellosis infection groups wer...

Embodiment 3

[0160] Embodiment 3, kit specificity test

[0161] 190 bovine and sheep sera confirmed without Brucella infection were used for the specificity test of CF-ELISA test, and compared with iELISA and CFT, SAT, RBPT. The results are in Table 4 below:

[0162] Table 4. Specificity comparison tests

[0163]

[0164] After diluting 190 Brucella positive sera by 50 times, the specificity test was carried out on the Brucella antibody CF-ELISA detection kit trial-produced in the laboratory, and compared with iELISA, CFT, SAT, RBPT, the results showed that the The CF-ELISA kit prepared by the invention has good specificity to the serum of cattle and sheep infected with brucellosis herds.

[0165] notes

[0166] 1. CF-ELISA method

[0167] 1.1. Antigen coating: Dilute the purified LPS antigen to 1 μg / mL with 0.05M carbonate buffer, coat 96-well microtiter plate (Corning, 2592) at 100 μL / well, and react at 2°C to 8°C for 18 hours above. Add 5% gelatin (Sigma) at 100 μL / well to seal...

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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 brucellosis diagnostic product. Background technique [0002] Brucellosis is a zoonotic infectious disease characterized by abortion and fever caused by Brucella or Brucella. The existence of brucellosis in the world has a long history. Human beings have gradually deepened their research understanding of the disease, and the level of diagnosis has been continuously improved. With the continuous development of brucellosis diagnostic technology, the serological detection methods of brucellosis have been continuously improved and improved. Traditional diagnostic techniques such as plate agglutination test (PT) and test tube agglutination test (SAT) are gradually replaced by highly sensitive and specific tiger red plate agglutination test (RBPT)...

Claims

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Application Information

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Patent Type & Authority Patents(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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