Raw material preparation method and detection method of vascular endothelial growth factor detection kit

Abstract

The present invention relates to the technical field of vascular endothelial growth factor detection, and discloses a raw material preparation method and a detection method of a vascular endothelial growth factor detection kit. Theraw material preparation method comprises raw material preparation, the raw material preparation comprises an antibody coated magnetic bead working solution, a VEGF labeled antibody, a VEGF calibration product, a VEGF quality control product and a sample diluent; the antibody coated magnetic bead working solution is a magnetic microsphere suspension coated with a Fab segment of a humanized VEGF antibody, and the buffer system is TBS-T (20-200 mM Tris, 0-1 M NaCl, 0.0%-1% BSA, 0.1%-0.5% Tween-20 and 0.1%-1% Proclin-300, and the pH value is 6.5-9.0). According to the present invention, the selected luminous marker is acridine salt NSP-SA-NHS; therefore, luminescence detection is carried out in homogeneous liquid, and the reaction time is shortened. Meanwhile, the bond level of C-N is larger than that of C-O, and the stability of the conjugate of the acridine salt NSP-SA-NHS and the protein is higher. Based on the use of NSP-SA-NHS, the sensitivity, the detection range and the storage time of the detection system are improved, and the reaction time is shortened.

Description

technical field [0001] The invention belongs to the technical field of vascular endothelial growth factor detection, in particular to a raw material preparation and detection method of a vascular endothelial growth factor detection kit. Background technique [0002] Vascular endothelial growth factor (VEGF) is a key regulator of physiological angiogenesis during embryogenesis, skeletal growth and reproductive function. Studies have found that VEGF is expressed in a variety of pathological and physiological conditions, so it can be used as a marker for in vitro diagnosis. At present, the in vitro detection kits for VEGF on the market mainly use quantum dot immunofluorescence method, enzyme-linked method, plate chemiluminescence method and magnetic particle chemiluminescence method. [0003] Existing test kit has the following problems at present: [0004] 1. The sensitivity and linear range need to be further improved. It has been reported that the VEGF content of healthy ...

AI Technical Summary

Problems solved by technology

Among the existing products, the product with the highest sensitivity is 0.1pg / ml, but the upper limit of its linear range is only 800pg / ml

This will result in the dilution of high VEGF concentration samples for actual detection, and the additional step will reduce the reproducibility and authenticity of the data

However, the sensitivity of products with a relatively large detection linear range is insufficient (generally greater than 1pg / ml), which may also lead to a decline in the accuracy of detection data and affect clinical judgment

[0005] 2. The process is complex

One technique is to introduce a pair of linkers between the antibody and the magnetic beads to amplify the signal ( figure 2 left); Another way is that the antibody is directly coated on the magnetic beads ( figure 2 Right), and both of these two processes need to amplify the signal, which is relatively complicated, which is not conducive to the large-scale and stable production of the kit

[0006] 3. Long detection time

Compared with the two-step method, the total incubation time required for the one-step method is shorter, generally around 25 minutes. However, the total incubation time required for the current one-step method is still relatively long, and it is impossible to provide a faster disease control plan in the clinic

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