Protein chip and kit for detecting abnormal descarboxyprothrombin in serum, and preparation method of protein chip

Abstract

The invention relates to a protein chip and kit for detecting abnormal descarboxyprothrombin in serum, and a preparation method of the protein chip, and belongs to the protein detection technology. The protein chip is characterized in that a matrix carrying slice of the protein chip is provided with a plurality of detection sub regions, each detection sub region is used for detecting one serum sample; each detection sub region is internally provided with a detection spot region and a control spot region, each detection spot region has detection spots formed by spraying trace DCP specific antibodies, and each control spot region has control spots formed by spraying bovine serum albumin; substances on all the detection spots in the same detection spot region have the same concentration; thetotal amount of the DCP specific antibodies forming each detection spot is 3-5 nL, the concentration is 3-5 mg / ml, each detection spot is formed by a manner of spraying for 6-10 times with 300-500 pLeach time by a non-contact spotting instrument, and the diameter of the detection spots is 0.5-1 mm. The protein chip and the kit can accurately detect the abnormal decarboxylase prothrombin, and hasthe advantages of high sensitivity, time saving, convenience, economy and the like in clinical use.

Description

technical field [0001] The invention relates to protein detection technology, in particular to a protein chip for detecting abnormal decarboxylated prothrombin in serum, a kit and a preparation method thereof. Background technique [0002] Des-r-carboxy-prothrombin (Des--carboxy-prothrombin, DCP) is an abnormal prothrombin produced by hepatocellular carcinoma. Compared with normal prothrombin, the molecular structure of DCP is characterized by its alanine domain ( One or more glutamic acid (Glu) residues in the Gla domain) are not fully carboxylated into Gla, thus losing the coagulation function. Normal prothrombin is in the microsomes of liver cells, with the participation of γ-glutamyl carboxylase, coenzyme and Vitamin Kreductase, which mainly depend on vitamin K, and the 6, 7, 14, 16, 19, 20 in the structure Gla Domain , 10 Glu residues at positions 25, 26, 29 and 32 are carboxylated to Gla to become an active prothrombin. Glu residues at any one or more of the above sit...

AI Technical Summary

Problems solved by technology

[0005] Therefore, in order to ensure the accuracy, sensitivity and specificity of the test results, the above-mentioned detection methods using the immune principle have a common feature, that is, the amount of serum used for each detection reaction ranges from 50-100ul to as many as 3-5ml, correspondingly, each detection just needs a large amount of antibody to finish, for example disclosed in CN101377505A the microwell plate chemiluminescence immunoassay assay method, needs to add the coating liquid 150ul containing DCP antibody in each well, the required serum sample amount 50ul is required, and although the document discloses that the minimum detection limit can reach 4.37mAU / ml, the real clinical serum was not used in the recorded experiments to verify the specificity and accuracy of the detection, and the detection data came from normal Artificial samples obtained by spiking standards into serum

Moreover, the detection time of these methods is as long as 3-4 hours. Because according to these methods, if a high-throughput chip is made, the size of the detection spot or capture spot for each sample will be made very large, and the cost is very high. It loses the meaning of making an integrated chip, that is, it cannot achieve the purpose of high throughput and low cost, and it is not realistic to popularize it in outpatient clinics

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