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Hand-foot-and-mouth disease resistant specific nanobody and titer determination method thereof

A nano-antibody and hand, foot and mouth disease technology, applied in the field of immunology, can solve the problems of side effects, poor specificity, and low purity, and achieve the effect of good water solubility, small molecular weight, and strong specificity

Inactive Publication Date: 2016-03-02
GUANGDONG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, although there are vaccines for the prevention of HFMD, clinical drugs mainly include antiviral drugs, traditional Chinese medicines and antibacterial drugs, etc., the treatment effect is not ideal, and there are side effects, which affect children's health
In addition, although there are antibodies for the treatment of hand, foot and mouth disease, due to some shortcomings of traditional antibodies, such as: low purity, poor stability and poor specificity, it has certain limitations in the treatment of hand, foot and mouth disease.

Method used

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  • Hand-foot-and-mouth disease resistant specific nanobody and titer determination method thereof
  • Hand-foot-and-mouth disease resistant specific nanobody and titer determination method thereof
  • Hand-foot-and-mouth disease resistant specific nanobody and titer determination method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Preparation of anti-hand, foot and mouth disease nanobody

[0042] 1. Antigen preparation

[0043] 1) Inoculate the EV71 virus liquid into the RD (strided muscle cell) cells grown to a monolayer, add 0.5mL to each bottle, put in CO 2 Cultivate in the incubator until about 90% of the cells appear CPE (cytopathic) and harvest, freeze and thaw 3 times at -20°C, centrifuge at 8000rpm at 4°C for 1h, collect the supernatant (virus liquid), centrifuge at 10000rpm at 4°C for 2h, discard clear, and the pellet was suspended in PBS buffer.

[0044] 2) CsCl density gradient centrifugation to purify virus antigen

[0045] Weigh 3.2g of CsCl into a 7.6mL gradient centrifuge tube, pipette gently until dissolved, centrifuge at 15000rpm at 4°C for 12h, and store at 4°C overnight. The next day, centrifuge at 20,000 rpm at 4°C for 4 hours to remove residual CsCl, discard the supernatant, add 300 μL PBS to dilute the purified virus solution and store at 4°C.

[0046] 2. Immunity

[0047...

Embodiment 2

[0053] Antibody Titer Detection of Antibody in Camel Serum Against Hand, Foot and Mouth Disease

[0054] ELISA (enzyme-linked immunosorbent assay) method is used to detect the potency of the anti-hand, foot and mouth disease nanobody of the present invention. The test results showed that the titer of the anti-hand, foot and mouth disease nanobody reached 1:20480. see results figure 2 .

[0055] Potency determination procedure

[0056] 1) Coating: Dilute the purified EV71 virus antigen with 0.01MPBS1:1000 to the optimal coating solubility and coat it on a 96-well microtiter plate (100μL / well), incubate at 37°C for 1h, and then Overnight, the next day, the liquid in the wells was shaken dry, washed once with PBST, and patted dry with absorbent paper;

[0057] 2) Blocking: block with 0.01 MPBS solution containing 3% skimmed milk powder (200 μL / well), incubate at 37°C for 1 hour, spin dry, wash with PBST 3 times, each time for 2-5 minutes, and pat dry with absorbent paper;

...

Embodiment 3

[0062] Immunobidirectional agar diffusion assay

[0063] Use a puncher to punch quincunx-like holes in the solidified agar culture dish, add EV71 virus antigen stock solution to the central hole of the quincunx hole, and add the same amount of different dilutions (1:4, 1:8, 1:16, 1:1) to the peripheral holes. 32, 1:64 and blank well PBS), the results are shown in image 3 , when the antibody was diluted at 1:4, 1:8, and 1:16, a significant white precipitation line appeared between the central antigen well and the nanobody well, but no precipitation line appeared between the blank control well PBS and the antigen well, which indicated that the prepared The nanobody has the ability to specifically bind to the EV71 antigen.

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Abstract

The invention discloses a hand-foot-and-mouth disease resistant specific nanobody and its titer determination method, and aims to provide a hand-foot-and-mouth disease resistant specific nanobody with small molecular weight, good water solubility, strong specificity and high stability and a titer determination method thereof. Technical points: the hand-foot-and-mouth disease resistant specific nanobody is prepared by the following steps successively: 1) preparing a soluble antigen by the use of inactivation purified EV71 type viral strain; 2) making the soluble antigen prepared in the step 1) into 0.8-1.2 mg / ml of an antigen solution by the use of 0.01 M of a phosphate buffer, and fully emulsifying the antigen solution by the use of a freund's complete adjuvant according to t two-humped camel he volume ratio of the antigen solution to the freund's complete adjuvant being 1:0.8-1.2 so as to prepare an immunizing antigen; 3) injecting 500-700 microliters of the immunizing antigen prepared in the step 2) at multiple sites under the skin of the back of each two-humped camel for 12-18 days of immunizing cycle, enhancing immunizing in the middle stage for three times, adopting the same primary immunization method and dosage, carrying out venous blood sampling after four immunizing cycles, collecting serum, and preserving in a refrigerator of 3-5 DEG C for standby; and 4) purifying the collected serum by a PEG three-step precipitation method and an improved water body comprehensive PEG method so as to obtain the nanobody. The invention belongs to the field of immunological technique.

Description

technical field [0001] The invention discloses an anti-hand, foot and mouth disease antibody and a method for determining its titer, in particular, an anti-hand, foot and mouth disease specific nanobody antibody and a method for determining its titer; it belongs to the technical field of immunology. Background technique [0002] Hand-foot-mouth disease (HFMD) is an acute infectious disease caused by a variety of enteroviruses. It mostly occurs in infants and young children under the age of 5, and occasionally in adults. It can cause fever and hand, foot, oral cavity Rashes and ulcers on other parts of the body are highly contagious and can be transmitted through toys, tableware, nasopharyngeal secretions, droplets, etc. Symptoms include acute onset, fever, maculopapular and herpetic rashes on the hands or soles, and rashes on the buttocks or knees. There are inflammatory flushes around the rash, and there is less fluid in the pustules; scattered herpes appear on the oral mu...

Claims

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Application Information

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IPC IPC(8): C07K16/10C07K16/06G01N33/543
CPCC07K16/1009C07K16/06G01N33/543
Inventor 赵肃清秦静崔锡平沈定吕瑞梁雨昕
Owner GUANGDONG UNIV OF TECH
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