Dot-ELISA (enzyme-linked immunosorbent essay) quick detection reagent kit for toxoplasmosis serum antibodies of cats

A serum antibody and Toxoplasma gondii technology, which is applied in the direction of measuring devices, instruments, and biological material analysis, can solve the problem of weak antigen-antibody binding and achieve economic benefits, high sensitivity and specificity, and detection efficiency high effect

Inactive Publication Date: 2017-01-25
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Dot-ELISA not only retains the advantages of conventional ELISA, but also overco

Method used

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  • Dot-ELISA (enzyme-linked immunosorbent essay) quick detection reagent kit for toxoplasmosis serum antibodies of cats
  • Dot-ELISA (enzyme-linked immunosorbent essay) quick detection reagent kit for toxoplasmosis serum antibodies of cats
  • Dot-ELISA (enzyme-linked immunosorbent essay) quick detection reagent kit for toxoplasmosis serum antibodies of cats

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Preparation of early stage samples of indirect Dot-ELISA

[0033] (1) The NC membrane was soaked in distilled water for 20 minutes. Make a circular membrane of appropriate size, put it into a 96-well plate after drying, coat it with protein, add 5% skimmed milk powder; the blocking time is 1h; wash 3 times for 5min each time.

[0034] (2) Antigen preparation: Toxoplasma gondii surface antigen SAG3 gene was expressed and identified by genetic engineering technology, and the Toxoplasma gondii SAG3 protein was finally obtained.

[0035] (3) Use a micropipette to spot the antigen mixture in the middle of the NC membrane. After drying, 5% skim milk powder is used to seal; after sealing, the NC membrane is washed in PBST to obtain a recombinant antigen-coated NC membrane. , also known as rapid diagnostic diaphragm. Store in a cool and dry place for later use;

[0036] The primary antiserum was collected by vein and then centrifuged to remove the supernatant;

[0037] (4) ...

Embodiment 2

[0045] Establishment of indirect Dot-ELISA method

[0046] 1. The optimal dilution of the serum to be tested

[0047] Take 2 μg of Toxoplasma gondii SAG3 protein-coated membrane, and use PBST to dilute the positive reference serum 1:100 backwards to 1:25600, carry out the test according to Example 1, and finally determine that the optimal dilution of the serum to be tested is 1 : 1600. (See figure 1 )

[0048] 2. The optimal working concentration of HRP-labeled goat anti-cat IgG

[0049] The HRP-labeled goat anti-cat IgG was diluted to 1:7000 according to the ratio of 1:1000, and the test was carried out according to Example 1, and finally the optimal working concentration was determined to be 1:5000. (See figure 2 )

[0050] 3. Optimal working concentration of coating antigen

[0051] Judgment criteria are based on the ratio of positive and negative reference serum A450 and the degree of color development of the membrane. The optimal antigen coating amount is 0.125 μ...

Embodiment 3

[0062] Assembling the components of an indirect ELISA kit for detecting serum antibodies to Toxoplasma gondii in cats

[0063] The test kit includes the following components:

[0064] 1. Toxoplasma gondii surface antigen SAG3 protein (stored at -20°C);

[0065] 2. Coating solution (0.05M carbonate buffer) pH (9.6): weigh NaCO 3 1.59g; NaHCO 3 2.93g; distilled water 80mL, after it dissolves, adjust the pH value to 9.6, and dilute to 100 mL;

[0066] 3. Store in a vacuum bag after nitrocellulose membrane (NC) is perforated;

[0067] 4. Blocking solution: Weigh 5g skimmed milk powder and add PBST to dissolve it, then dilute to 100mL;

[0068] 5. Washing solution and sample diluent (PBST): Weigh 8g of NaCl; 0.2g of KCl; NaHPO 4 .12H 2 O 2.9g; KH 2 PO 4 0.2g; Tween-20 0.5Ml;

[0069] 6. TMB substrate chromogenic solution: Substrate A solution (weigh TMB 200mg, add 100mL absolute ethanol, adjust pH value to 5.0, add double distilled water to make up to 1000mL), and substra...

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Abstract

The invention particularly discloses a Dot-ELISA (enzyme-linked immunosorbent essay) quick detection reagent kit for toxoplasmosis serum antibodies of cats. Nitrocellulose (NC) membranes are perforated and then are fixed into 96 orifice plates during experiments, toxoplasmosis SAG3 proteins are enveloped in the NC membranes, to-be-detected antibodies are added into the toxoplasmosis SAG3 proteins and react to the toxoplasmosis SAG3 proteins, and enzyme-labeled secondary antibodies are added into the toxoplasmosis SAG3 proteins to obtain antigen-antibody compositions; color development solution is added into the antigen-antibody compositions, and results are judged after reaction is terminated; the results are positive if dots on the NC membranes are brown and are negative if colors are not changed. The Dot-ELISA quick detection reagent kit has the advantages that the NC membranes are enveloped by the aid of recombinant antigens, and accordingly the storage time can be prolonged; diaphragms can be detached to be independently stored, and accordingly the NC membranes can be prevented from being contaminated; the Dot-ELISA quick detection reagent kit is high in sensitivity and specificity, special instruments can be omitted, serum samples can be screened on a large screen, and accordingly the Dot-ELISA quick detection reagent kit has high practical application value.

Description

technical field [0001] The invention specifically discloses a Dot-ELISA rapid detection kit for cat toxoplasma gondii serum antibody, and further provides a preparation method of the Dot-ELISA rapid detection kit for feline toxoplasma gondii serum antibody, belonging to the technical field of parasite detection methods. Background technique [0002] Toxoplasma gondii ( Toxoplasmosis ) is an obligate intracellular parasite with a wide range of hosts, among which cats and felines are the ultimate hosts of the disease. Cats are the most common animal infected with the disease in nature, and they are also an important source of infection for humans. People with abnormal immune function in the population usually have secondary diseases and even cause death of patients. After pregnant women are infected with Toxoplasma gondii, there will be miscarriage, premature delivery, stillbirth and so on. [0003] At present, the main methods used to detect Toxoplasma gondii antibodies ar...

Claims

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Application Information

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IPC IPC(8): G01N33/569G01N33/544G01N33/535
CPCG01N33/56905G01N33/535G01N33/544G01N2333/45
Inventor 宫鹏涛刘伟建杨正涛张西臣寇金华李建华杨举李赫李棕松
Owner JILIN UNIV
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