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Homogenous-phase fluorescent immune reagent for rapidly and quantitatively detecting trace albumin, and preparation and detection method thereof

A technology for quantitative detection of microalbumin, which is applied in the field of medical testing, can solve the problems of large batch-to-batch differences, and achieve the effects of high cost performance, good repeatability, and improved precision and accuracy

Inactive Publication Date: 2017-05-10
SHANDONG UNIV QILU HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This method is affected by the uniformity of the latex particles, and the batch-to-batch variation is large

Method used

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  • Homogenous-phase fluorescent immune reagent for rapidly and quantitatively detecting trace albumin, and preparation and detection method thereof
  • Homogenous-phase fluorescent immune reagent for rapidly and quantitatively detecting trace albumin, and preparation and detection method thereof
  • Homogenous-phase fluorescent immune reagent for rapidly and quantitatively detecting trace albumin, and preparation and detection method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0041] The preparation method of quantitatively detecting microalbumin (MAU) homogeneous fluorescent immunological reagent comprises the steps:

[0042] 1. Preparation of anti-MAU for labeling:

[0043] Purified anti-MAU monoclonal antibody expressed by genetic engineering was selected. Eu 3+ The product code of anti-MAU monoclonal antibody for labeling is 19C7; the product code of anti-MAU monoclonal antibody for fluorescein labeling is 16A11 and 560.

[0044] 2. Preparation of rare earth element chelate labeled anti-MAU:

[0045] The mouse anti-human MAU monoclonal antibody 19C7 solution (3 mg / mL) was dialyzed twice at 4° C. with 3 L of 0.9% NaCl, 24 hr each time. Add water to adjust the concentration to 1.5mg / mL. Take 0.6mL of the antibody solution, add 1mL NaHCO 3 (0.2mol / L), and adjust the pH to 9.1 with 1mol / L NaOH. 20 μl of BHHCT methanol solution (30 μg / mL) was added dropwise to the antibody solution under stirring, and the stirring reaction was continued for 1 h...

Embodiment 2

[0051] The preparation method of the present embodiment is basically the same as that of Example 1, the difference is:

[0052] In step 2, the preparation method of rare earth element chelate-labeled anti-MAU is: dialyze the mouse anti-human MAU solution (3 mg / mL) twice at 4° C. with 3 L of 0.9% NaCl, 24 hr each time. Add water to adjust the concentration to 1.5mg / mL. Take 0.6mL of the antibody solution, add 1mL NaHCO 3 (0.2mol / L), and adjust the pH to 9.1 with 1mol / L NaOH. 20 μL of BHHBCB methanol solution (30 μg / mL) was added dropwise to the antibody solution under stirring, and the stirring reaction was continued for 1 hr. After centrifugation (10000rpm, 10min) to remove insoluble matter, put on SephadexG-25 column, use 0.05mol / L NH 4 HCO 3 (pH=8.0) to separate the labeled protein and free label. UV / visible spectrophotometer detects the A of each collection liquid 330 value, pool the solutions containing the labeled antibodies. Add final concentrations of 0.1% BSA an...

Embodiment 3

[0054] The preparation method of the present embodiment is basically the same as that of Example 1, the difference is:

[0055] In step 3, dilute anti-MAU monoclonal antibodies 16A11 and 560 with 0.1 mol / L sodium bicarbonate solution to 1 mg / mL, take 5 mL of antibody solution, add 40 mg of fluorescein DyLight-DY647 solution, and stir well. Incubate at room temperature for 1.5 hours, mixing every 15 minutes. Finally, the G25 gel column was used for column separation and purification, and the labeled fluorescein-labeled antibody was collected and diluted with 0.01mol / L phosphate buffer containing 0.05% PEG600, 3.5% BSA, 10% glycerol, and 0.05% surfactant. Seal the package with a plastic bottle and store at 4°C.

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Abstract

The invention discloses a homogenous-phase fluorescent immune reagent for rapidly and quantitatively detecting trace albumin, and a preparation and detection method thereof. The reagent consists of anti-MAU marked by rare-earth element chelate and another anti-MAU marked by a near-infrared fluorescent compound, and a series of concentration MAU calibration products. By adopting the homogenous-phase fluorescent immune rapid detection technology, and utilizing the high-sensitivity characteristic of the fluorescence, the adverse influence of the non-homogeneity of pores of a nitrocellulose membrane in a colloidal gold or fluorescent MAU dry-type immune test paper on the detection accuracy and repetition can also be avoided, and the detection sensitivity and the linear range can be greatly improved; and the reagent provided by the invention is used for detecting trace albumin in human blood, serum or plasma, has the characteristics of simplicity and rapidness in operation, sensitivity, good specificity and the like, and has good clinical application prospect.

Description

technical field [0001] The invention belongs to the field of medical testing, and in particular relates to a homogeneous fluorescent immunological reagent for rapid quantitative detection of trace albumin and a preparation and detection method. Background technique [0002] Serum albumin can pass through the glomerulus, but almost all of it is reabsorbed by the renal tubules. When the glomerulus is damaged, the amount of filtration increases, so that it exceeds the reabsorption of the renal tubules and is excreted from the urine, forming urinary microalbumin ( MAU). Urinary microalbumin refers to the excretion rate of albumin in urine exceeding 20 mg per minute or exceeding 30 mg in 24 hours, with a concentration of 30-200 mg / L, which cannot be detected by conventional methods. A large number of studies have proved that positive urinary microalbumin is an early sign of renal injury, especially glomerular injury, and its excretion is positively correlated with the degree of ...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/577G01N33/533
CPCG01N33/6893G01N33/533G01N33/577G01N2333/76G01N2800/042
Inventor 黄亚丽李新华魏芳赵珂谢爱武
Owner SHANDONG UNIV QILU HOSPITAL
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