Search

CN-122017228-A - Colloidal gold method for rapidly detecting ACE inhibition activity and test paper preparation

CN122017228ACN 122017228 ACN122017228 ACN 122017228ACN-122017228-A

Abstract

The invention relates to the technical field of activity detection, and particularly discloses a colloidal gold method for rapidly detecting ACE inhibition activity and test paper preparation. The test paper comprises a sample pad, a gold-labeled pad, a nitrocellulose membrane and a water absorption pad, wherein the gold-labeled pad is loaded with colloidal gold-labeled angiotensin converting enzyme, and the nitrocellulose membrane is provided with a detection line coated with an angiotensin II antibody and a quality control line coated with an anti-ACE antibody. During detection, the ACE inhibitor in the sample can inhibit enzyme activity and influence aggregation of colloidal gold on a detection line, so that quick judgment is realized through color comparison of the detection line and a quality control line. The invention also provides a preparation method and a detection method of the test paper, which have the advantages of simple and rapid operation, no need of large-scale instruments, low cost, suitability for field detection and the like, and can be widely applied to drug screening, food detection and clinical sample primary screening.

Inventors

  • DU PENG
  • ZHANG JIANING
  • WANG YUHAN
  • Yu Ruxue
  • ZHANG GUOFANG
  • LIU LIBO
  • LI CHUN

Assignees

  • 东北农业大学

Dates

Publication Date
20260512
Application Date
20260128

Claims (8)

  1. 1. Test paper for rapidly detecting ACE inhibitory activity by a colloidal gold method, which is characterized by comprising the following components: The sample pad, the gold mark pad, the nitrocellulose membrane and the water absorbing pad are sequentially stuck on the bottom plate; The gold-labeled pad is loaded with angiotensin converting enzyme labeled by colloidal gold; The nitrocellulose membrane is provided with a detection line and a quality control line, the detection line is coated with an angiotensin II antibody, and the quality control line is coated with an anti-ACE antibody.
  2. 2. The test strip of claim 1, wherein the method for preparing the colloidal gold-labeled angiotensin converting enzyme comprises: Adjusting pH of the colloidal gold solution to 7.4-7.8, adding angiotensin converting enzyme, reacting at room temperature for 10-30 min, adding blocking agent for blocking, centrifuging, and re-suspending in buffer solution containing stabilizer.
  3. 3. The test paper according to claim 2, wherein the colloidal gold solution is prepared by a method comprising: adding chloroauric acid solution into ultrapure water, heating to boil, rapidly adding sodium citrate solution, continuously heating until the solution turns into wine red or mauve, cooling, and fixing volume.
  4. 4. The test strip of claim 3, wherein the sodium citrate solution is added in an amount of 1.5 to 2.5 times the volume of the chloroauric acid solution.
  5. 5. The test strip of claim 1, wherein the coating concentration of the angiotensin II antibody on the test line is 0.4-0.8 mg/ml and the coating concentration of the anti-ACE antibody on the quality control line is 1.0 mg/ml.
  6. 6. The test paper according to claim 1, wherein the sample pad is pretreated with a buffer solution containing a surfactant and a stabilizer and then dried, and the gold-labeled pad is pretreated with a buffer solution containing a surfactant and a saccharide and then dried.
  7. 7. A method of preparing a test strip according to any one of claims 1 to 6, comprising the steps of: (1) Preparing a colloidal gold solution; (2) Preparing colloidal gold labeled angiotensin converting enzyme; (3) Respectively spraying an angiotensin II antibody and an anti-ACE antibody on a nitrocellulose membrane to form a detection line and a quality control line; (4) And sequentially adhering the pretreated sample pad, the gold-labeled pad loaded with colloidal gold-labeled enzyme, the nitrocellulose membrane and the water absorption pad to a bottom plate.
  8. 8. A method of detecting ACE inhibitory activity using the test strip of any one of claims 1 to 6, comprising: Dripping a sample to be detected to a sample pad, and observing the color development conditions of the detection line and the quality control line after reacting for 10-20 minutes; if the quality control line develops color and the detection line develops color deeply, judging that the color is negative; If the quality control line develops color and the detection line develops light color or does not develop color, judging that the color is positive; If the quality control line does not develop, the control line is judged to be invalid.

Description

Colloidal gold method for rapidly detecting ACE inhibition activity and test paper preparation Technical Field The invention relates to the technical field of activity detection, in particular to a colloidal gold method for rapidly detecting ACE inhibition activity and test paper preparation. Background Angiotensin converting enzyme inhibitors (angiotenin-ConvertingEnzymeInhibitors, ACEIs) are first-line core drugs for the clinical treatment of cardiovascular diseases such as hypertension and heart failure. Its action target-Angiotensin Converting Enzyme (ACE) -is one of the key enzymes for regulating blood pressure of human body. Angiotensin converting enzyme is a zinc-containing dipeptide carboxypeptidase that is widely found in human endothelial cells, epithelial cells and plasma. It is a core catalytic enzyme of renin-angiotensin system (Renin-AngiotensinSystem, RAS), and mainly performs two key physiological functions of catalyzing the conversion of angiotensin I (AngiotensinI, angI) into angiotensin II (AngiotensinII, angII), wherein AngII is a powerful vasoconstrictor and can promote secretion of aldosterone and increase retention of sodium and water, and the two key physiological functions together lead to elevation of blood pressure. Degrading bradykinin (Bradykinin) which is a peptide substance having vasodilatory action. Thus, ACE is responsible for the dual mechanisms of "producing a boosting substance (AngII)" and "eliminating a lowering substance (bradykinin)". A substance that inhibits ACE activity means that it reduces AngII production, thereby directly dilating blood vessels and reducing peripheral resistance. The degradation of bradykinin is retarded, and the vasodilation effect is indirectly enhanced. The synergy of the two effects makes ACE inhibitor become high-efficiency and reliable antihypertensive drugs (such as captopril, enalapril and the like). Currently, the mainstream methods for detecting ACE inhibitory activity in the laboratory mainly include: (1) High Performance Liquid Chromatography (HPLC), directly and quantitatively measuring the generation amount of an ACE catalytic reaction product AngII, incubating ACE and a substrate (Ang I) with a sample to be detected, separating Ang I and Ang II by using the HPLC after the reaction is ended, quantifying the peak area of Ang II by a detector (such as ultraviolet or mass spectrum), and calculating the inhibition rate. (2) Spectrophotometry/fluorescence spectrophotometry, using synthetic chromogenic or fluorogenic substrates (most commonly, hippophae-histidyl-leucine, HHL). ACE hydrolyzes HHL to release hippuric acid and histidyl-leucine (HL), which can react with derivatizing agents (e.g., phthalaldehyde) to produce a strong fluorescent product, and after enzymatic reaction, derivatizing agents are added and fluorescence intensity is measured using a fluorescence spectrophotometer to indirectly calculate enzyme activity. The method can also be used for directly detecting the absorbance of the hippuric acid by using an ultraviolet-visible spectrophotometer. However, both high performance liquid chromatography and spectrophotometry are required to include incubation, reaction, sample injection analysis, and the like, and involve multi-step pipetting, pH adjustment, reaction control, and special equipment, so a new technique for detecting ACE inhibition activity, which can overcome the above drawbacks, is fast, simple, low in cost, does not need large-scale instruments, and is suitable for field detection, is urgently needed in the art. Disclosure of Invention The embodiment of the application solves the problem that complex processes and equipment are needed in the prior art by providing a colloidal gold method for rapidly detecting ACE inhibitory activity and preparing test paper. The embodiment of the application provides a test paper for rapidly detecting ACE inhibition activity by a colloidal gold method, which comprises the following components: The sample pad, the gold mark pad, the nitrocellulose membrane and the water absorbing pad are sequentially stuck on the bottom plate; The gold-labeled pad is loaded with angiotensin converting enzyme labeled by colloidal gold; The nitrocellulose membrane is provided with a detection line and a quality control line, the detection line is coated with an angiotensin II antibody, and the quality control line is coated with an anti-ACE antibody. Further, the preparation method of the colloidal gold-labeled angiotensin converting enzyme comprises the following steps: Adjusting pH of the colloidal gold solution to 7.4-7.8, adding angiotensin converting enzyme, reacting at room temperature for 10-30 min, adding blocking agent for blocking, centrifuging, and re-suspending in buffer solution containing stabilizer. Further, the preparation method of the colloidal gold solution comprises the following steps: adding chloroauric acid solution into ultrapure water, heating to boil, rapidly adding sodi