Neutrophile granulocyte gelatinase related lipid transport protein detection kit and preparation method thereof

Abstract

The invention discloses a neutrophile granulocyte gelatinase related lipid transport protein detection kit and a preparation method thereof. The detection kit is composed of a reagent I and a reagent II independent from each other; the reagent I comprises the components of biobuffer, surfactant, coagulant, preservative, stabilizer, blocker, chelating agent and water; and the reagent II comprises the components of rubber latex particle of enveloping neutrophile granulocyte gelatinase related lipid transport protein antibody, biobuffer, chelating agent, surfactant, preservative, suspending agent, sealing agent, stabilizer and water. According to the invention, the neutrophile granulocyte gelatinase related lipid transport protein antibody of the detection kit can not only be combined with the neutrophile granulocyte gelatinase related lipid transport protein antigen, but also be combined with the MMP-9 / NGAL (matrix metallopeptidase-9 / (neutrophil gelatinase associated lipocalin) compound, thereby avoiding the influence of MMP-9 / NGAL compound to the detecting result, and having the advantages of good specificity, high sensitivity, wide linear range, favorable stability and the like.

Description

technical field

[0001] The present invention relates to a kit for detecting lipocalin content, in particular to a kit for detecting the content of neutrophil gelatinase-associated lipocalin (NGAL) in a human body and a preparation method thereof, and the present invention further relates to the detection kit The method for detecting NGAL content in a human body belongs to the field of detection of NGAL content in a human body. Background technique

[0002] Recent studies have found that a new member of the lipocalin family, neutrophil gelatinase-associated lipocalin (NGAL), is associated with acute kidney injury (acute kidney injury, AKI) and Chronic kidney disease (CKD) caused by various causes is closely related, and it is a new marker for predicting AKI, as well as a biological indicator for monitoring the progress of CKD and renal function damage.

[0003] NGAL is a member of the lipocalin family. It was discovered and isolated in human neutrophils by Kjeldsen et al. in...

Examples

Embodiment 1

[0047] Preliminary Example 1 Screening of NGAL Antibody

[0048] 1. Preparation of MMP-9 affinity chromatography column: Mix protein MMP-9 (purchased from R&D Company) and packed CNBr-Sepharose4B (Pharmacia product) at a ratio of 5-7 mg / mL, shake at 4°C for 20 hours, make it cross-linked. Then wash with 0.1 mol / L pH 8.0 carbonate buffer solution for 3 times, then add an equal amount of 0.1 mol / L ethanolamine-HCl and shake at 4°C for 1 hour to block the remaining activated groups. Finally, wash with 0.01mol / L Tris-HCl (TB), pack the column, and equilibrate. The volume of the column is selected as 10mL.

[0049] 2. Preparation of MMP-9 / NGAL complex affinity chromatography column: protein NGAL (purchased from R&D Company) was prepared into a 2 mg / mL solution, and the NGAL protein solution was passed through the MMP-9 affinity column at a flow rate of 1 mL / min. Chromatographic column, the amount of protein solution is 60mL; then excess NGAL protein is eluted with 0.1mol / L pH8.0 ...

Embodiment 2

[0051] Preparation of latex particles coated with NGAL antibody in preliminary example 2

[0052] The preparation process of latex particles coated with NGAL antibody is as follows:

[0053] ①Preparation of carboxylated polystyrene latex solution: Weigh 1.0g of dry polystyrene latex particles with a particle size of 105nm, add 100mL of 0.12M phosphate buffer (pH7.5) to disperse, add 40mg of 1-ethyl -3-(3-Dimethylaminopropyl)carbodiimide (EDC), then add 20mg of thio-NHS, stir the reaction at room temperature for 0.5 hours, centrifuge to discard the supernatant, and then use 100mL of 0.12M phosphate buffer The carboxylated modified polystyrene latex obtained by liquid dilution, dispersion and precipitation latex;

[0054] ② Preparation of NGAL antibody solution: Dissolve 250 μg of the NGAL antibody screened in Preliminary Example 1 with 0.5 mL of deionized water to obtain an NGAL antibody solution;

[0055] ③ Preparation of latex particles coated with NGAL antibody: Take 100 μ...

Embodiment 3

[0056] Preliminary Example 3 Preparation of latex particles coated with NGAL antibody

[0057] The preparation process of latex particles coated with NGAL antibody is as follows:

[0058] ①Preparation of carboxylated modified polystyrene latex solution: Weigh 1.0g of dry polystyrene latex particles with a particle size of 130nm, add 100mL of 0.12M phosphate buffer (pH7.5) to disperse, add 40mg of 1-ethyl -3-(3-Dimethylaminopropyl)carbodiimide (EDC), then add 20mg of thio-NHS, stir the reaction at room temperature for 0.5 hours, centrifuge to discard the supernatant, and then use 100mL of 0.12M phosphate buffer The carboxylated modified polystyrene latex obtained by liquid dilution, dispersion and precipitation latex;

[0059] ② Preparation of NGAL antibody solution: Dissolve 250 μg of the NGAL antibody screened in Preliminary Example 1 with 0.5 mL of deionized water to obtain an NGAL antibody solution;

[0060]③ Preparation of latex particles coated with NGAL antibody: Take ...

technical field

[0001] The present invention relates to a kit for detecting lipocalin content, in particular to a kit for detecting the content of neutrophil gelatinase-associated lipocalin (NGAL) in a human body and a preparation method thereof, and the present invention further relates to the detection kit The method for detecting NGAL content in a human body belongs to the field of detection of NGAL content in a human body. Background technique

[0002] Recent studies have found that a new member of the lipocalin family, neutrophil gelatinase-associated lipocalin (NGAL), is associated with acute kidney injury (acute kidney injury, AKI) and Chronic kidney disease (CKD) caused by various causes is closely related, and it is a new marker for predicting AKI, as well as a biological indicator for monitoring the progress of CKD and renal function damage.

[0003] NGAL is a member of the lipocalin family. It was discovered and isolated in human neutrophils by Kjeldsen et al. in...

Examples

Embodiment 1

[0047] Preliminary Example 1 Screening of NGAL Antibody

[0048] 1. Preparation of MMP-9 affinity chromatography column: Mix protein MMP-9 (purchased from R&D Company) and packed CNBr-Sepharose4B (Pharmacia product) at a ratio of 5-7 mg / mL, shake at 4°C for 20 hours, make it cross-linked. Then wash with 0.1 mol / L pH 8.0 carbonate buffer solution for 3 times, then add an equal amount of 0.1 mol / L ethanolamine-HCl and shake at 4°C for 1 hour to block the remaining activated groups. Finally, wash with 0.01mol / L Tris-HCl (TB), pack the column, and equilibrate. The volume of the column is selected as 10mL.

[0049] 2. Preparation of MMP-9 / NGAL complex affinity chromatography column: protein NGAL (purchased from R&D Company) was prepared into a 2 mg / mL solution, and the NGAL protein solution was passed through the MMP-9 affinity column at a flow rate of 1 mL / min. Chromatographic column, the amount of protein solution is 60mL; then excess NGAL protein is eluted with 0.1mol / L pH8.0 ...

Embodiment 2

[0051] Preparation of latex particles coated with NGAL antibody in preliminary example 2

[0052] The preparation process of latex particles coated with NGAL antibody is as follows:

[0053] ①Preparation of carboxylated polystyrene latex solution: Weigh 1.0g of dry polystyrene latex particles with a particle size of 105nm, add 100mL of 0.12M phosphate buffer (pH7.5) to disperse, add 40mg of 1-ethyl -3-(3-Dimethylaminopropyl)carbodiimide (EDC), then add 20mg of thio-NHS, stir the reaction at room temperature for 0.5 hours, centrifuge to discard the supernatant, and then use 100mL of 0.12M phosphate buffer The carboxylated modified polystyrene latex obtained by liquid dilution, dispersion and precipitation latex;

[0054] ② Preparation of NGAL antibody solution: Dissolve 250 μg of the NGAL antibody screened in Preliminary Example 1 with 0.5 mL of deionized water to obtain an NGAL antibody solution;

[0055] ③ Preparation of latex particles coated with NGAL antibody: Take 100 μ...

Embodiment 3

[0056] Preliminary Example 3 Preparation of latex particles coated with NGAL antibody

[0057] The preparation process of latex particles coated with NGAL antibody is as follows:

[0058] ①Preparation of carboxylated modified polystyrene latex solution: Weigh 1.0g of dry polystyrene latex particles with a particle size of 130nm, add 100mL of 0.12M phosphate buffer (pH7.5) to disperse, add 40mg of 1-ethyl -3-(3-Dimethylaminopropyl)carbodiimide (EDC), then add 20mg of thio-NHS, stir the reaction at room temperature for 0.5 hours, centrifuge to discard the supernatant, and then use 100mL of 0.12M phosphate buffer The carboxylated modified polystyrene latex obtained by liquid dilution, dispersion and precipitation latex;

[0059] ② Preparation of NGAL antibody solution: Dissolve 250 μg of the NGAL antibody screened in Preliminary Example 1 with 0.5 mL of deionized water to obtain an NGAL antibody solution;

[0060]③ Preparation of latex particles coated with NGAL antibody: Take ...

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