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Latex-enhanced two-antibody competitive immunoturbidimetric assay kit as well as preparation and application methods thereof

An immunoturbidimetric and latex-enhanced technology, which is applied in the biological field and can solve problems such as differences, impact thresholds, and incomplete quality of antigens and antibodies

Active Publication Date: 2018-10-16
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

In order to avoid false negative results due to excess antigen, some people use the method of pre-reaction threshold judgment for the determination of excess antigen. Through short-term pre-reaction, the threshold value of turbidity change is given to screen out the samples with excess antigen, and dilute This method can effectively avoid "false negative" results, but for different analytes, due to different antibodies, the given thresholds are different. In addition, the quality of antigens and antibodies in different batches is not complete. The same also affects the setting of the threshold value. In addition, this method is different from the conventional immunoassay process. It needs to design a special measurement program or special equipment, and this analysis process cannot be realized on a general automatic biochemical analyzer.

Method used

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  • Latex-enhanced two-antibody competitive immunoturbidimetric assay kit as well as preparation and application methods thereof
  • Latex-enhanced two-antibody competitive immunoturbidimetric assay kit as well as preparation and application methods thereof
  • Latex-enhanced two-antibody competitive immunoturbidimetric assay kit as well as preparation and application methods thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] 1.1 Preparation of rabbit anti-mouse IgG secondary antibody-latex conjugate (YSab-Latex)

[0052] Commercially available carboxyl nano-latex particles (particle size 100nm, 10% concentration) for immunoturbidity, take 0.2ml particles, add PH 4.6, 0.02M phosphate buffer (PB) 0.8ml, dilute to 1% particle concentration, Add 20ul of dicyclohexylcarbodiimide (20mg / ml), incubate at 37°C for 20 minutes to activate, then add 0.5ml, pH7.5, 0.02M phosphate buffer (PB) and 0.02ml purified rabbit anti-mouse Antibody (10mg / ml), keep warm on a shaking table at 37°C and react for 60 minutes. After the conjugation reaction is completed, add pH 7.5, 0.02M phosphate buffer to dilute to 10ml, and then add 10% bovine serum albumin 0.1 ml, 1% tween0.1ml, incubated on a shaker at 37°C for 30 minutes, blocked and terminated the reaction, the rabbit anti-mouse secondary antibody-latex conjugate (YSab-Latex) can be prepared.

[0053] 1.2 Preparation of goat anti-rabbit IgG secondary antibody-l...

Embodiment 2

[0056] Titer determination of primary antibody:

[0057] 2.1 Determination of mouse monoclonal antibody titer: Dilute the detected mouse anti-human transferrin, D-dimer (DD2) monoclonal antibody (Tfr) with water ratio to 0.02M, pH7.0 (including NaCl 0.15M) phosphate buffer solution is R1, the rabbit anti-mouse IgG secondary antibody-latex conjugate (YSab-Latex) prepared by the above method is R2, on the Hitachi 7170S automatic biochemical analyzer, according to the following parameters Set the measurement program to measure the primary antibody solution with different dilutions; measurement wavelength: 546nm; R 1 :R 2 : Sample=100:100:20, that is, after adding 100ul R1, add 20ul sample, keep warm at 37°C for 5 minutes, then add 100ul R 2 , read the light absorption value at point 17 and point 34, and calculate the light absorption difference between point 34 and point 17: △A=A34-A17. The dilution with the largest difference is the working concentration (titer) of the primar...

Embodiment 3

[0063] 3.1 Preparation of rabbit anti-mouse IgG secondary antibody kit:

[0064] 3.1.1 Rabbit anti-mouse IgG secondary antibody D-D kit:

[0065] Reagent R1 preparation: the rabbit anti-mouse IgG secondary antibody-latex conjugate (YSab-Latex) prepared above was added with preservative proclin300 to make the concentration 0.1%, wherein the latex concentration was 2mg / ml.

[0066]Reagent R2 preparation: Add 1 gram of bovine serum albumin, 0.1 gram of tween20, 1ml of proclin300 to each liter of 0.02M, PH7.0 (containing NaCl 0.15M) phosphate buffer solution, mix well, and filter through a 0.22um membrane , Dilute the mouse anti-human D-D monoclonal antibody 1:100 with the above liquid.

[0067] 3.1.2 Rabbit anti-mouse IgG secondary antibody Tf kit:

[0068] Preparation of reagent R1: (same as D-D kit R1 above) add preservative proclin300 to the rabbit anti-mouse IgG secondary antibody-latex conjugate (YSab-Latex) prepared above to make the concentration 0.1%, and the latex conc...

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Abstract

The invention discloses a latex-enhanced two-antibody competitive immunoturbidimetric assay kit as well as preparation and application methods thereof. According to the precipitation or agglutinationphenomenon of an immune complex formed by an antigen-antibody reaction and the principle of latex-enhanced immunological turbidimetry, on the basis of precipitation or agglutination formed by secondary antibody and primary antibody reaction, a secondary antibody-latex connector is prepared, antibody titer of primary antibodies is determined by the secondary antibody-latex connector, the quantity of the primary antibodies is limited, and during measuring of a reaction system, when antigens exist, the antigens compete with secondary antibodies for binding sites of the primary antibodies, so thatthe primary antibodies and the secondary antibodies are inhibited from forming the immune complex and agglutination or precipitation effect is reduced. A latex particle preparation process can be standardized by universal secondary antibody-latex, the product quality difference of the kits prepared from different kinds of latex and different antibodies with different methods is reduced, and the method is particularly suitable for clinical kit preparation; when the antigens are excessive, a false-negative result caused by hook effect can be eliminated.

Description

technical field [0001] This patent belongs to the field of biotechnology, and in particular relates to a latex-enhanced secondary antibody competitive immunoturbidimetric assay kit and its production and use methods. Background technique [0002] Immunoturbidimetric analysis methods have been widely used clinically, and dozens of kits have been used clinically. At the same time, the discovery of new biomarkers will continue to promote the production of this new kit. At present, there are mainly two types. One is to directly add the antibody into the reaction system. Under certain conditions, the antigen and antibody react to form an immune complex, which causes agglutination or precipitation to change the turbidity of the system. This turbidity change is related to the system. There is a positive correlation between the antigen concentration in the sample, and the antigen concentration in the sample can be determined accordingly. This method is usually called turbidimetry or...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
CPCG01N33/68
Inventor 文建新刘株漆乐新
Owner 长沙文瀚生物技术有限责任公司
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