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CN-122016427-A - Sample treatment liquid and vitamin B12Pretreatment method, detection kit, detection method and application of detection sample

CN122016427ACN 122016427 ACN122016427 ACN 122016427ACN-122016427-A

Abstract

The invention discloses a sample treatment fluid, a pretreatment method of a detection sample of vitamin B 12 , a detection kit, a detection method and application thereof, and particularly discloses a sample treatment fluid which comprises strong alkali and sarcosyl. The sample processing liquid can completely dissociate VB 12 and can maintain a relatively stable state. The invention also provides a pretreatment method of the detection sample of the vitamin B 12 , which comprises the step of mixing and reacting the sample treatment liquid according to the first aspect of the invention with a sample to be detected. The sample treatment solution provided by the invention can still enable VB 12 to dissociate from the binding protein under the condition of not adopting extremely toxic cyanide or dissociating unstable complex containing cyanide ions, and can keep a relatively stable state to participate in subsequent reactions.

Inventors

  • ZHANG CHUANGUO
  • ZHANG PING
  • LU CHUN

Assignees

  • 深圳市爱康试剂有限公司

Dates

Publication Date
20260512
Application Date
20260206

Claims (10)

  1. 1. A sample processing liquid is characterized in that, the sample processing fluid includes a strong base and sarcosyl.
  2. 2. The sample processing fluid of claim 1, wherein the sample processing fluid comprises 5-100 mg/mL strong base and 1-20 mg/mL sarcosyl.
  3. 3. A pretreatment method of a detection sample of vitamin B 12 is characterized in that the pretreatment method of the detection sample comprises the step of mixing and reacting the sample treatment liquid according to the claim 1 or 2 with a sample to be detected.
  4. 4. The pretreatment method for a test sample of vitamin B 12 as defined in claim 3, wherein the mixing volume ratio of the sample treatment solution to the sample to be tested is 1 (1-8); Preferably, the reaction time is 5-30 min; Preferably, the reaction temperature is 34-40 ℃.
  5. 5. A vitamin B 12 detection kit is characterized by comprising the sample treatment solution, streptavidin-labeled magnetic beads, inner factor-labeled biotin, a labeling enzyme and a chemiluminescent substrate solution according to claim 1 or 2.
  6. 6. The vitamin B 12 assay kit of claim 5, wherein the marker enzyme comprises vitamin B 12 derivative-labeled alkaline phosphatase; Preferably, the chemiluminescent substrate solution comprises AMPPD or APS-5.
  7. 7. The kit for detecting vitamin B 12 according to claim 5, wherein the mass ratio of streptavidin to magnetic beads in the streptavidin-labeled magnetic beads is 1 (40-60); Preferably, the molar ratio of the inner factor to the biotin in the inner factor labeled biotin is 1 (10-80).
  8. 8. A method for detecting vitamin B 12 , characterized in that the method uses the vitamin B 12 detection kit according to any one of claims 5 to 7 for detection, comprising the steps of: s1, uniformly mixing a sample to be tested and a sample treatment solution according to the volume ratio of (1-8): 1 for reaction; s2, adding the biotin marked by the inner factor into the reaction system in the step S1, and uniformly mixing and reacting; S3, adding streptavidin-labeled magnetic beads and labeling enzyme into the reaction system in the step S2, and uniformly mixing and reacting; S4, performing magnetic separation, cleaning for 2-4 times by using a cleaning solution, adding a chemiluminescent substrate solution, and then performing photoelectric signal interpretation.
  9. 9. The detection method according to claim 8, wherein the reaction time in the step S1 is 5-30 min, and the reaction temperature is 34-40 ℃; preferably, the reaction time in the steps S2 and S3 is 3-8 min, and the reaction temperature is 34-40 ℃.
  10. 10. Use of the sample processing fluid of claim 1 or 2 for detecting vitamin B 12 in a biological sample.

Description

Sample treatment fluid, pretreatment method of detection sample of vitamin B 12, detection kit, detection method and application of detection kit Technical Field The invention belongs to the technical field of immunodiagnosis, and particularly relates to a sample treatment fluid, a pretreatment method of a detection sample of vitamin B 12, a detection kit, a detection method and application thereof. Background Vitamin B 12（VB12) is a water-soluble vitamin, since it contains cobalt element, also known as cobalamin. The presence of cobalt causes the crystals of VB 12 to appear red, and therefore VB 12 is also known as a red vitamin. VB 12 is the latest vitamin of the B group found by humans so far, and is also the only vitamin containing metal elements, and the structural body is a corrinoid ring, and a Co atom is arranged in the center of the ring. VB 12 has a molecular formula of CoC 63H88N14 P and a relative molecular weight of 1355.38, and is the vitamin with the largest molecular weight and the most complex structure in the B vitamins. VB 12 is readily soluble in water and ethanol and does not exist stably in strong acid or alkali solutions. Vitamin B 12 is present in plasma in the main form of methylcobalamin (MeCbl), usually in combination with transcobalamin. About 90% of vitamin B 12 in plasma is stored as transcobalamin I, the remaining 10% being responsible for physiological transport by transcobalamin II. This binding form aids in the stable presence and transport of vitamin B 12 in plasma. The current immunological methods for detecting vitamin B 12 mainly comprise a radioimmunoassay, an enzyme-linked immunoassay, a chemiluminescent immunoassay and the like. In all the detection methods, the sample needs to be pretreated, VB 12 is dissociated from the binding protein and is converted into a stable cyanocobalamine form, so that the subsequent concentration measurement is facilitated. The dissociation solution for sample pretreatment of the imported commercial detection kit mainly comprises strong alkali, dithiothreitol (DTT) serving as a reducing agent and cyanide, and VB 12 can be released by damaging a binding protein structure through the strong alkali and the DTT and form stable cyanocobalamin with the cyanide. However, sodium cyanide and potassium cyanide are extremely toxic compounds, are strictly regulated in use, are unfavorable for experimental operation, and have the possibility of environmental pollution. The existing proposal uses cyanide ion complex to replace cyanide, the cyanide ion complex is difficult to dissociate to form highly toxic cyanide ions, so the toxicity is very low, and the cyanide ion complex is not a controlled compound, but has the defects of low dissociation degree and unstable dissociation degree, thereby adversely affecting the detection result. Disclosure of Invention The present invention aims to solve at least one of the technical problems in the prior art described above. Therefore, the invention provides a sample treatment liquid which can completely dissociate VB 12 and can keep a relatively stable state. The invention also provides a pretreatment method of the detection sample of vitamin B 12. The invention also provides a vitamin B 12 detection kit. The invention also provides a detection method of the vitamin B 12. The invention also provides application of the sample treatment fluid. According to a first aspect of the present invention, there is provided a sample processing liquid, characterized in that the sample processing liquid comprises a strong base and sarcosyl. In some embodiments of the invention, the sample treatment fluid comprises 5-100 mg/mL strong base and 1-20 mg/mL sarcosyl. In some embodiments of the invention, the sample treatment fluid comprises 5-50 mg/mL strong base and 1-5 mg/mL sarcosyl. In some embodiments of the invention, the strong base comprises any one of lithium hydroxide, sodium hydroxide, potassium hydroxide. According to a second aspect of the present invention, there is provided a pretreatment method for a detection sample of vitamin B 12, which comprises mixing and reacting the sample treatment solution according to the first aspect of the present invention with a sample to be tested. In some embodiments of the present invention, the mixing volume ratio of the sample processing liquid to the sample to be tested is 1 (1-8). In some embodiments of the present invention, the mixing volume ratio of the sample processing liquid to the sample to be tested is 1 (1-3). In some embodiments of the invention, the reaction time is 5 to 30 minutes. In some embodiments of the invention, the reaction time is 5 to 15 minutes. In some embodiments of the invention, the reaction temperature is 34 ℃ to 40 ℃. According to a third aspect of the invention, a vitamin B 12 detection kit is provided, wherein the vitamin B 12 detection kit comprises the sample treatment solution, streptavidin-labeled magnetic beads, inner factor-labeled biotin,