Kit for detecting urine transferrin through chemiluminescence enzyme immunoassay method, and preparation method thereof

A technique of urinary transferrin and chemiluminescence enzyme is applied in the detection kit of chemiluminescence enzyme immunoassay and the field of preparation thereof, which can solve the problem that the results are easily affected by subjective factors and the sensitivity of the enzyme-linked immunosorbent assay. low specificity, poor specificity of immunoturbidimetry, etc., to achieve the effect of no radioactive contamination, wide detection range and high sensitivity

Inactive Publication Date: 2018-01-30
长春市诺克因生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Among them, the colloidal gold method has low sensitivity and is prone to false positives, and its results are easily affected by subje

Method used

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  • Kit for detecting urine transferrin through chemiluminescence enzyme immunoassay method, and preparation method thereof
  • Kit for detecting urine transferrin through chemiluminescence enzyme immunoassay method, and preparation method thereof
  • Kit for detecting urine transferrin through chemiluminescence enzyme immunoassay method, and preparation method thereof

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Experimental program
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Effect test

Embodiment 1

[0028] The enzyme labeling method of embodiment 1.UTF antibody:

[0029] Mark HRP in UTF using the simple sodium periodate method, with NaIO 4 The sugar molecules on the surface of HRP are first oxidized into aldehyde groups, and then combined with the amino groups on Ig. The yield of the obtained enzyme-labeled antibody is high. Nearly 70% of HRP is bound to Ig, and 99% of Ig is bound to the enzyme. There is no significant loss of Ig activity and is currently the most commonly used method.

[0030] The labeling principle of the simple sodium periodate method is: HRP is passed through NaIO 4 The aldolase formed after oxidation can be linked to the amino group of the antibody molecule to form Schiff's base, which can be further reduced with NaBH 4 (or ethanolamine) to generate a stable enzyme-labeled antibody.

[0031] The labeling steps of the simple sodium periodate method are as follows:

[0032] 1) Weigh 5mg of HRP and dissolve in 1ml of deionized water;

[0033] 2) Add...

Embodiment 2

[0041] Embodiment 2. Apply the detection method of kit of the present invention and the establishment of standard curve:

[0042] 1) Dilute 100 μl of enzyme-labeled antibody to 10 μg / ml with coating solution, coat it in the microwell of the antibody-coated plate, and incubate at 37°C for 10 minutes;

[0043] 2) Dilute the standard with the standard diluent to a concentration of 0, 50, 200, 600, 2000, 6000 ng / ml, add different concentrations of the standard into microwells coated with enzyme-labeled antibodies, and incubate for 45 minute;

[0044] 3) Wash 4-5 times with washing liquid and pat dry;

[0045] 4) Add 50 μl of chemiluminescent substrate solution A and 50 μl of B solution to each microwell, mix well and incubate at 37°C in the dark for 10 minutes;

[0046] 5) The antibody-coated plate is placed in a BHP-9504 microwell luminescence plate analyzer for luminescence analysis, with the antigen concentration as the abscissa and the relative light unit as the ordinate, dr...

Embodiment 3

[0047] Embodiment 3. The variation of luminescence intensity with time when the chemiluminescent substrate liquid of the kit of the present invention is used to detect UTF:

[0048] In the HRP / H2O2 / luminol / p-iodophenol enhancement system, by adding p-iodophenol as an enhancer, the luminescence intensity can be increased and the luminescence time can be prolonged. Use 10μg / ml enzyme-labeled antibody antibody coating, detect 200ng / ml concentration standard, add chemiluminescence substrate solution A solution 50μl and B solution 50μl, measure the relative light unit at different times, it can be seen that adding chemiluminescence substrate solution 10 The relative light unit reaches a peak value after 10 minutes, and then shows a plateau period of slow decline, and the plateau period can be maintained at a higher relative light unit value for more than 40 minutes (such as figure 2 shown).

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Abstract

The invention relates to a kit for detecting urine transferrin through a chemiluminescence enzyme immunoassay method, and a preparation method thereof. The kit comprises an antibody-coated plate, a urine transferrin enzyme antibody, a coating buffer solution, a blocking solution, a urine transferrin standard substance, a standard substance dilution solution, a washing solution, a sample dilution solution and a chemiluminescent substrate solution. According to the present invention, the kit can detect the UTF concentration level of human based on chemiluminescence enzyme immunoassay; the chemiluminescent substrate catalytic enzyme HRP and the anti-UTF antibody are conjugated, the obtained conjugate reacts with the sample or the antigen to form the immune complexes, the chemiluminescent substrate solution is added, the relative light unit of the chemiluminescent reaction is measured, the UTF antigen content in the standard substance/the sample is proportional to the relative light unit measured by the optical system, and through standard curve fitting, the UTF content in the urine sample is determined; and the kit of the invention has advantages of high sensitivity, strong specificity, easy operation, low cost and the like.

Description

technical field [0001] The invention relates to a kit for detecting urinary transferrin (UTF) content and a preparation method thereof, and relates to a preparation process of a kit for detecting human UTF concentration level by chemiluminescent enzyme immunoassay (CLEIA). Background technique [0002] Chronic kidney disease (CKD) and diabetic nephropathy (DN) often have the characteristics of not being detected in time, which can easily cause chronic renal failure or uremia in patients, and are the main causes of death. Especially DN, although the treatment level has been greatly improved compared with the past, it is still the main cause of end-stage renal disease, accounting for about 44% of all end-stage renal disease cases. Early diagnosis is particularly important. [0003] Traditional early diagnosis methods of kidney injury have some deficiencies in various aspects. For example, the traditional serum creatinine method for judging kidney disease has a high rate of mi...

Claims

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Application Information

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IPC IPC(8): G01N21/76G01N33/68
Inventor 周宇璠刘锋王聪张芝平王静
Owner 长春市诺克因生物科技有限公司
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