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Antibody screening reagent for enhancing immunosuppression turbidimetry and preparation and use method thereof

A technology for antibody screening and immunity enhancement, applied in the biological field, can solve problems such as decreased agglutination effect

Pending Publication Date: 2019-08-16
长沙文瀚生物技术有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The enhanced immunosuppression turbidimetric method of the present invention overcomes the defects of the existing enhanced immune turbidimetric method from the principle of immune response, and what it utilizes is the excess antigen curve part of immune reaction agglutination. When the antigen is detected, the antigen attached to the latex particles will produce an agglutination reaction with the antibody in the system, and the agglutination effect will reach the maximum. When the antigen exists in the measurement reaction system, because the amount of the antibody is limited, the free antigen is in excess Antigen, excess antigen will inhibit the agglutination effect of the antigen-antibody complex. Therefore, as the amount of free antigen in the reaction system increases, the agglutination effect of the antigen-antibody complex will decrease. When the antigen in the measurement system reaches a certain amount, the free antigen will Completely inhibit the combination of the antibody with the antigen on the latex. If the non-specific binding of the excess antigen and other substances in the latex and the sample can be overcome, the change in turbidity during the reaction will be zero

Method used

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  • Antibody screening reagent for enhancing immunosuppression turbidimetry and preparation and use method thereof
  • Antibody screening reagent for enhancing immunosuppression turbidimetry and preparation and use method thereof
  • Antibody screening reagent for enhancing immunosuppression turbidimetry and preparation and use method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0037] Preparation of human transferrin-latex linker (Trf-Latex) and human antibody:

[0038] Commercially available carboxyl nano latex particles (particle size 100nm, 10% concentration) for immune turbidity, take 0.2ml particles, add 0.8ml PH 4.6, 0.02M phosphate buffer (PB), -, add 20ul dicyclohexyl Carbodiimide (20mg / ml), 37°C shaker incubation reaction for 20 minutes after activation, add 0.8ml, PH7.5, 0.02M phosphate buffer (PB) and 0.2ml human transferrin (5mg / ml), Incubate on a shaker at 37°C and react for 60 minutes. After the ligation reaction is over, add pH7.0, 0.02M phosphate buffer to make it diluted to 10ml, and then add 0.1ml of 10% bovine serum albumin, 1% Tween 20 0.1ml, incubate at 37°C on a shaker, then react for 30 minutes to block and terminate the reaction, then the human transferrin-latex conjugate (Trf-Latex) can be prepared.

[0039] Human-derived transferrin (sigma) was mixed with complete Freund’s adjuvant to immunize mice and rabbits according to conve...

Embodiment 2

[0041] Antibody titer detection method: Dilute the above-mentioned tested monoclonal antibody or multi-antibody sample with water ratio, use 0.02M, PH7.0 (containing NaCl 0.15M) phosphate buffer as R1, and use the antigen-nano prepared by the above method The latex linker is R2. On the Hitachi 7170S automatic biochemical analyzer, set the measurement program according to the following parameters to determine various supernatants; measurement wavelength: 546nm; R 1 : R 2 :Sample=100:100:20, ie add R 1 After 100ul, add 20ul sample, incubate at 37℃ for 5 minutes, add R 2 100ul, read the light absorption values ​​at points 17 and 34, and calculate the difference between light absorption at points 34 and 17: △A=A34-A17. Screening of 5 different monoclonal antibodies and 6 polyclonal antibodies obtained from rabbit immunization The result is figure 1 with figure 2 As shown, the monoclonal antibody 2H5F1 is the best, and the polyclonal antibody No. 3 rabbit is the best.

[0042] Table (...

Embodiment 3

[0048] Human transferrin enhanced immunosuppressive turbidimetric analysis method and kit preparation

[0049] 3.1 Reagent R1 preparation: the human transferrin-latex conjugate (Trf-Latex) prepared above, add the preservative proclin300 to make the concentration 0.1%, where the latex concentration is 2mg / ml, and the latex to antigen mass ratio is 20 :1.

[0050] 3.2 Preparation of reagent R2: add 0.1 g bovine serum albumin, 5ml monoclonal antibody or 2.5ml polyclonal antibody, 0.1g tween20, 1ml proclin300 to every liter of 0.02M, PH7.0 (containing NaCl 0.15M) phosphate buffer, After mixing, filter through 0.22um membrane.

[0051] 3.3 Measurement procedure: implement measurement implementation according to the following measurement parameters. Measurement wavelength: 546nm; R1: R2: sample = 100: 100: 10, that is, after adding 100ul R1, add 10ul sample, keep at 37℃ for 5 minutes, add 100ul R2, read the light absorption value at 17 and 34 points, and calculate The difference in ligh...

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Abstract

The invention discloses an antibody screening reagent for enhancing immunosuppression turbidimetry and a preparation and use method thereof. The method for enhancing immunosuppression turbidimetry overcomes the defects of the method for enhancing immune turbidimetry in the prior art in the principle of immune reaction. When no antigen exists in a measurement reaction system, the antigen connectedto latex particles generates agglutination reaction with the limited amount of antibody in the system, and the agglutination effect is maximized. When an antigen exists in the measurement reaction system, since the amount of antibody is limited, free antigen is excessive antigen which can inhibit the agglutination effect of the antigen-antibody complex, therefore, as the amount of free antigen inthe reaction system increases, the agglutination effect of the antigen-antibody complex is decreased. When the amount of antigen in the measurement system reaches a certain amount, the free antigen will completely inhibit the binding of the antibody and the antigen on the latex, and if the nonspecific binding of excess antigen and other substances in the latex and sample can be overcome, the turbidity change during the reaction will be zero.

Description

Technical field [0001] This patent belongs to the field of biotechnology, and in particular relates to a screening reagent for enhancing immunosuppression turbidity antibody and its preparation and use methods. Background technique [0002] Immunoassay technology has been developed since the establishment of radioimmunoassay methods, after decades of development, combined with the principle of immunoreaction and labeling technology, established radioimmunoassay methods based on isotope labeling technology, and enzyme-linked immunoassay based on enzyme labeling technology Methods: Bioluminescence, chemiluminescence, and electrochemiluminescence luminescence immunoassay methods based on luminescent substance labeling technology, time-resolved fluorescence immunoassay methods based on metal chelate labeling technology, etc. The above analysis method relies on detecting the amount of the label in the antigen-antibody complex to determine the antigen or antibody. Its basic feature is...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N21/31
CPCG01N21/31G01N33/6854
Inventor 文建新
Owner 长沙文瀚生物技术有限责任公司
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