Double-label immunodetection method containing internal reference and application of double-label immunodetection method

A detection method and immunoassay technology, applied in the field of double-labeled immunoassay with internal reference

Pending Publication Date: 2022-01-28
GUANGZHOU WONDFO BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, limited by other interference factors, including light source stability, indoor temperature, instrument temperature, sampling accuracy, reagent accuracy, etc., the total precision of the kit is usually only 10%.

Method used

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  • Double-label immunodetection method containing internal reference and application of double-label immunodetection method
  • Double-label immunodetection method containing internal reference and application of double-label immunodetection method
  • Double-label immunodetection method containing internal reference and application of double-label immunodetection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] The preparation of the double-labeled kit of embodiment 1cTnI and the establishment of detection method

[0067] Taking the detection of troponin I (cTnI) as an example, the preparation method of the double-labeled kit for detecting cTnI is as follows:

[0068] Step 1: Preparation of cTnI antibody and chicken IgY coated plates. Add 4 μg / mL cTnI antibody (Fepone Bio cTnI-MCAB-29, epitope 24-40aa) and 1 μg / mL chicken IgY (Biolab F050315) to the coating solution at the same time, mix for half an hour, take Add 100 μl to the coated plate and let it stand at 4°C for 22 hours. After washing the coated plate, add blocking solution, block at 37°C for 3 hours, and dry it for later use.

[0069] Step 2: Preparation of europium-labeled cTnI antibody. The β-diketone chelating agent europium and cTnI antibody (Fepeng biological cTnI-MCAB-22, epitope 86-90aa) were routinely labeled. After purification, the europium-labeled cTnI antibody was obtained with a concentration of 0.2 mg...

Embodiment 2

[0090] Example 2 Comparison of detection precision of cTnI double-labeled kit

[0091] Use the double-labeled kit for detecting cTnI of the present invention and the single-labeled detection kit for using self-made europium-labeled cTnI antibody (compared with the cTnI double-labeled kit of the present invention) to detect two high and low quality controls (self-made cTnI quality control) Products, the concentration is 2500pg / mL and 100pg / mL, antigen: sea peptide biological recombinant human cardiac troponin I8RTI7; quality control dilution: 100mM Tris-HCl, 5% BSA, 0.1% P300 preservative, PH7.8 ), wherein the double-labeled kit adopts the detection method described in Example 1, and the single-labeled detection kit is operated according to the detection method of the conventional single-labeled kit, and the detection is repeated 10 times, and the precision is calculated, and the results are as follows:

[0092] table 2-1

[0093]

[0094] Table 2-2

[0095]

[0096] Re...

Embodiment 3

[0098] Embodiment 3 internal reference marker concentration selection

[0099] 1) Selection of quality control concentration: Use ratios of samarium-labeled goat anti-chicken IgY (0.2mg / mL) to 1:2500, 1:5000, 1:10000, 1:20000 respectively with europium-labeled cTnI antibody (0.2mg / mL)1 : 1000 At the same time, add labeling buffer and mix well, as cTnI double labeling (reagent).

[0100] 2) Detection method:

[0101] Using these four double-labeled reagents, refer to the preparation method and detection method of the double-labeled kit described in Example 1, and simultaneously detect the serum samples from the same source.

[0102] Eu will be detected separately 3+ Signal and Sm 3+ The signal is calculated as the relative signal value of the serum sample by the formula: (T-B1) / (C-B2). Substitute the relative signal value into the fitting curve, and inversely calculate the cTnI concentration value of the sample.

[0103] 3) Test results

[0104] Using the above 4 groups o...

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Abstract

The invention relates to a double-labeled immunodetection method containing an internal reference, which is used for detecting a target protein in a sample. The method comprises the following steps of labeling a second binding protein of a target protein and an internal reference quality controller by adopting different markers, and substituting a marker signal value into a correction formula established by an inventor for calculation; substituting the calculation result into a fitting curve established based on the standard substance, and reversely solving the target protein concentration of the actual sample. The method can effectively reduce a problem of poor precision caused by the influence of interference factors on the detection result of the immunodetection reagent so that detection precision can be improved to CV less than 5%; based on the method, the inventor also designs a double-labeling kit for detecting troponin I, the influence of interference factors on a detection result can be effectively reduced, so detection precision is improved, and a powerful guarantee is provided for improving detection accuracy of existing immune quantitative analysis.

Description

technical field [0001] The invention relates to the field of in vitro diagnosis, in particular to a double-labeled immunoassay method containing an internal reference and its application. Background technique [0002] Antibody-based immune response is the most basic and common method for specific object detection, and has been widely used in life science research and clinical detection. It is difficult to realize the simultaneous analysis of multiple components. Even the fluorescent immunoassay labeled with fluorescent quantum dots is difficult to realize the simultaneous analysis of many components. More importantly, these conventional immunoassay methods will eventually go through a reaction process, such as The color reaction or luminescence reaction can be detected by the detector, and the stability and consistency of this reaction will directly affect the accuracy of the quantitative results. Therefore, it is necessary to develop a precise immunological quantitative ana...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/577G01N33/58G01N33/543
CPCG01N33/68G01N33/6854G01N33/577G01N33/582G01N33/54306G01N33/54326G01N2333/4712
Inventor 李奕辉钟日晟黄赞力黄岭芳杜嘉铭杨雄
Owner GUANGZHOU WONDFO BIOTECH
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