Screening kit and method for progressive muscular dystrophy of newborns

A technology for muscular dystrophy and neonates, which can be used in biomaterial analysis, instruments, measuring devices, etc., and can solve the problems of false positive results, inconsistency, and difficulty in popularization

Pending Publication Date: 2020-11-20
GUANGZHOU FENGHUA BIOENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, CK is mainly detected by biochemical methods, such as colorimetry, ultraviolet spectrophotometry and fluorescence method. The reference method for the determination of CK at home and abroad is the enzyme coupling method. The influence of released adenylate kinase (AK) and other enzymes in plasma / serum, although reagent manufacturers will add inhibitor adenosine monophosphate (AMP) or P 1 ,P 5 - Di(adenosine-5') pentalithium pentaphosphate (DAP) to inhibit the interference of AK, but the inhibition rate is only 90% to 95%. For normal specimens, an increase of 5% can be regarded as an allowable error. However, for extreme samples, its error can cause false positive results
It is extremely difficult to popularize the serum method in the screening of neonatal progressive muscular dystrophy by CK enzyme coupling method, and it is extremely difficult to achieve accuracy and stability by using the filter paper method
Moreover, the CK activity in the blood of DMD patients is usually very high in the early stage, but there are huge differences. The disease condition is inconsistent with the CK activity. In the late stage of the disease, the CK activity may not increase. There is no clear standard for how to define the relationship between the CK detection value and DMD.

Method used

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  • Screening kit and method for progressive muscular dystrophy of newborns
  • Screening kit and method for progressive muscular dystrophy of newborns
  • Screening kit and method for progressive muscular dystrophy of newborns

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] Antibody pairing detection effect of a creatine kinase isoenzyme (CK-MM) assay reagent (time-resolved fluorescent immunoassay) suitable for neonatal progressive muscular dystrophy screening:

[0087]

[0088]

[0089] From the above results, the mouse monoclonal antibody M18073101 is paired with M18121502, which has 100% reactivity with CK-MM and strong sensitivity; low reactivity with CK-MB and CK-BB, strong specificity and wide linear range.

Embodiment 2

[0091] A creatine kinase isoenzyme (CK-MM) assay reagent (time-resolved fluorescent immunoassay) suitable for neonatal progressive muscular dystrophy screening includes:

[0092] Capture antibody solid phase carrier: coated with human skeletal muscle CK-MM monoclonal antibody 5μg / mL;

[0093] Calibrator: The filter paper dry blood film calibration product contains 6 concentration points of A, B, C, D, E, F, calibrator A is zero concentration, and calibrator B, C, D, E, F contains muscle with a series of concentration gradients. Acid phosphokinase; the calibrator was traced to the standard substance GBW09167. The concentrations of A, B, C, D, E, and F were 0, 20, 100, 500, 2500, and 10 000 IU / mL, respectively.

[0094] Detection of antibody markers: Europium-labeled human skeletal muscle CK-MM monoclonal antibody.

[0095] Sample eluent: 50mmol / L, tris-hydrochloric acid buffer containing 0.02% (v / v) of pH 7.8 20ppm thimerosal, 30% (v / v) SignalBoostTM immune signal enhancer,...

Embodiment 3

[0100] The specific operations of the creatine kinase isoenzyme (CK-MM) assay reagent (time-resolved fluorescent immunoassay) of the above embodiment 2 are as follows:

[0101] 1. Reagent Preparation

[0102] 1) Capture antibody solid-phase carrier: Equilibrate the reagent and the required amount of capture antibody solid-phase carrier to room temperature (20-25°C). The rest of the capture antibody solid-phase carrier was placed in a ziplock bag in time to be sealed and stored at 2-8°C.

[0103] 2) Cleaning working solution: Add cleaning solution and purified water at a ratio of 1:2 to a clean container and mix them as a cleaning working solution.

[0104] 3) Detection antibody marker working solution: Prepare within 30 minutes before use, add the detection antibody marker and experimental buffer at a ratio of 1:1 200 to a clean disposable container and mix well; use it up for the current experiment.

[0105] 2. Detection steps

[0106] 1) Use the automatic nail punching me...

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Abstract

The invention particularly discloses a double-antibody sandwich immunoassay in-vitro diagnostic reagent suitable for screening progressive muscular dystrophy of newborns. The double-antibody sandwichimmunoassay in-vitro diagnostic reagent is used for specifically detecting creatine kinase isoenzyme CK-MM. According to the invention, a filter paper dry blood slide calibrator and a filter paper dryblood slide sample are adopted and are not influenced by AK released by red blood cells during hemolysis, so that the universality of screening the progressive muscular dystrophy of newborns is realized. The invention discloses a filter paper dry blood slide calibrator preparation method, which solves the instability of the calibrator and prevent the hydrolysis of creatine kinase isoenzyme and / orsubtype by carboxyl peptidase in blood plasma. The invention discloses a special filter paper dry blood slide sample eluent, which solves the stability problem of a to-be-detected object during sample detection and reduces the interference of external factors. After muscular dystrophy is diagnosed in the early stage and proper treatment measures are taken, the progress of the disease can be greatly delayed, the life quality of a patient is improved, and huge cost caused by blind medical treatment of the patient can also be avoided.

Description

technical field [0001] The invention relates to the technical field of screening kit preparation, in particular to a screening kit for neonatal progressive muscular dystrophy and a method thereof. Background technique [0002] Progressive muscular dystrophy is a group of inherited skeletal muscle degenerative diseases. Pathologically, it is characterized by skeletal muscle fiber degeneration and necrosis. Clinically, it is characterized by slowly progressive muscle atrophy and muscle weakness. Some types can also involve Heart, skeletal system. Traditionally divided into pseudohypertrophic muscular dystrophy (pseudohypertrophic muscular dystrophy), facioscapulohumeral muscular dystrophy (FSHD), limb girdle muscular dystrophy (LGMD), Emery-Dreifuss muscular dystrophy Dystrophy, oculopharyngeal muscular dystrophy (OPMD), ocular muscular dystrophy, distal muscular dystrophy and congenital muscular dystrophy (CMD). According to the mode of inheritance, it can be divided into s...

Claims

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Application Information

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IPC IPC(8): G01N33/573G01N33/577G01N33/543
CPCG01N33/573G01N33/577G01N33/54393G01N33/543G01N2333/9123G01N2496/05G01N2800/10G01N2800/52
Inventor 谭玉华冯建明廖三川冯淑怡邢晓敏江燚梁天铖罗海坤陈梅欣周晓姗刘远
Owner GUANGZHOU FENGHUA BIOENG
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