CN114487410A - Application of H factor antibody in preparation of kit for detecting hemodialysis, peritoneal dialysis, peritonitis or glioma - Google Patents

Application of H factor antibody in preparation of kit for detecting hemodialysis, peritoneal dialysis, peritonitis or glioma Download PDF

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CN114487410A
CN114487410A CN202011268082.XA CN202011268082A CN114487410A CN 114487410 A CN114487410 A CN 114487410A CN 202011268082 A CN202011268082 A CN 202011268082A CN 114487410 A CN114487410 A CN 114487410A
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朱梅珍
刘颖冰
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Shanghai Yijue Biotechnology Co ltd
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/22—Haematology
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/70—Mechanisms involved in disease identification
    • G01N2800/7095—Inflammation

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Abstract

The invention provides an application of a factor H antibody in preparing a kit for detecting hemodialysis, peritoneal dialysis, peritonitis or glioma. The invention coats the H factor monoclonal antibody or the H factor monoclonal antibody and the polyclonal antibody, combines colloidal gold with the polyclonal antibody, can improve the sensitivity of the kit, can be used together with the polyclonal antibody, and is applied to immunoturbidimetry (immunotransmission turbidimetry, immunonephelometry, immunolatex turbidimetry), ELISA, chemiluminescence, microfluidic chip method, magnetic particle method, colloidal gold percolation method and chromatography. The invention also provides a kit, which adopts a method of H factor antigen-antibody (including monoclonal antibody and polyclonal antibody) combination reaction for detection. The kit can receive a detection report within 1 to 5 minutes, and has accurate and convenient detection result.

Description

Application of H factor antibody in preparation of kit for detecting hemodialysis, peritoneal dialysis, peritonitis or glioma
Technical Field
The invention belongs to the field of bioengineering, relates to a kit, and particularly relates to an application of an H factor antibody in preparation of a kit for detecting hemodialysis, peritoneal dialysis, peritonitis or glioma.
Background
Serum factor H is a glycoprotein with the molecular weight of about 150kDa, which is synthesized and secreted mainly by liver, besides, renal mesangial cells, podocytes, renal tubular epithelial cells, monocytes and the like can also be synthesized, and the normal serum concentration is 274mg/L-501 mg/L. Factor H primarily inhibits the overactivation of the alternative complement pathway, thereby protecting host cells from damage. In addition, the H factor can be combined with fragments, DNA, histone and other components of the apoptotic cell to eliminate the apoptotic cell.
Disclosure of Invention
The invention aims to provide application of a factor H antibody in preparing a kit for detecting hemodialysis, peritoneal dialysis, peritonitis or glioma.
Furthermore, the kit comprises a detection plate, a rabbit anti-H factor antibody, a goat anti-H factor antibody conjugate marked by colloidal gold, a confining liquid, a washing liquid, a positive reference product and a negative reference product, wherein the detection plate consists of a plastic box bottom box, a water absorption layer is arranged in the plastic box bottom box, a nitrocellulose membrane is arranged on the water absorption layer, a filter screen is covered on the nitrocellulose membrane, a plastic box surface cover is arranged on the filter screen, a detection reaction hole is arranged in the center of the plastic box surface cover, the rabbit anti-H factor antibody and the goat anti-H factor antibody conjugate marked by colloidal gold are dotted on the nitrocellulose membrane corresponding to the detection reaction hole, the confining liquid is a phosphate solution containing albumin, the pH value of the phosphate solution is 7.0-7.5, and the weight percentage of bovine serum albumin in the phosphate solution is 1.0%, the washing solution is a Tris-HCl solution containing Tween-20, the pH of the Tris-HCl solution is 7.0-7.5, the weight percentage of the Tween-20 in the Tris-HCl solution is 0.05%, the mass of the goat anti-H factor antibody in each milliliter of colloidal gold conjugate is 700 mg, and the positive reference substance is the H factor antigen with the concentration of less than 274 mg/L.
Further, the detection is carried out by colloidal gold filtration, chromatography or chemiluminescence.
Furthermore, ELISA method, immunoturbidimetry, immunotransmission turbidimetry, immunoscattering turbidimetry or immunolatex enhanced turbidimetry is adopted for carrying out clinical examination and routine detection.
Furthermore, a micro-fluidic chip and a magnetic particle method are adopted for detection.
Furthermore, the rabbit anti-H factor antibody is a monoclonal antibody or a polyclonal antibody.
The invention also provides a kit for detecting hemodialysis, peritoneal dialysis, peritonitis or glioma, which adopts H factor antigen-antibody combination reaction for detection.
Furthermore, the rabbit anti-H factor antibody is a monoclonal antibody or a polyclonal antibody, so that the sensitivity and the accuracy of the reagent are improved (comprising the monoclonal antibody or the polyclonal antibody of a mouse, a rabbit, a sheep or a horse).
Use of factor H antibodies in the preparation of a marker for the detection of hemodialysis, peritoneal dialysis, peritonitis, or glioma.
The detection principle of the invention is as follows: binding reaction of H factor antibody and H factor antigen in serum.
The invention coats the H factor monoclonal antibody or the H factor monoclonal antibody and the polyclonal antibody, combines colloidal gold with the polyclonal antibody, can improve the sensitivity of the kit, can be used together with the polyclonal antibody, and is applied to immunoturbidimetry (immunotransmission turbidimetry, immunonephelometry, immunolatex turbidimetry), ELISA, chemiluminescence, microfluidic chip method, magnetic particle method, colloidal gold percolation method and chromatography.
Compared with the prior art, the invention has remarkable technical progress. The invention can be used for detecting H factor antigen in human serum samples. The kit can receive a detection report within 1 to 5 minutes, and has accurate and convenient detection result.
Detailed Description
Example 1 preparation of a kit
1.1 preparation process of colloidal gold conjugate;
1.1.1 preparing colloidal gold;
1.1.1.10.05% gold chloride solution 300 ml, heating to boil;
1.1.1.2 add 3.6 ml of 5.0% trisodium citrate;
1.1.1.3, continuously heating and boiling for 15 minutes, stopping heating, cooling to room temperature, and recovering the volume to the original volume by using distilled water;
1.1.2 labeling with colloidal gold;
1.1.2.1 colloidal gold 100 mL;
1.1.2.2 adding 2.4 ml of 1.0% anhydrous sodium carbonate solution, and adjusting the pH value to 6.4-7.2;
1.1.2.3 adding 2.5 mL goat anti-H factor antibody (1.0mg/mL), standing at room temperature for 30 min;
1.1.2.4 stirring and adding 10 XTris-HCl 20 ml to regulate pH to 7.2-7.5;
1.1.2.5 stirring and adding 2 g of BSA, 1 g of PEG20000 and 0.2 ml of ProClin-300;
1.2 detection board preparation process;
1.2.1 assembling a detection plate, taking a bottom cover of a plastic box, placing a water absorption layer, placing a nitrocellulose membrane on the water absorption layer, covering a surface cover of the plastic box, and pressing;
1.2.2 detection of Point antibodies: rabbit anti-H factor antibody (or alternatively, goat, horse, mouse anti-GPI antibody or monoclonal antibody) (1.0mg/mL)1.0 mL was added 1.0M NaHCO30.1 ml, and mixing evenly;
1.2.3 taking 2.0uL of detection point antibody, and adding the detection point antibody on a nitrocellulose membrane at the center of a reaction hole of the detection plate to prepare a detection plate;
1.3 preparing sealing liquid and washing liquid;
1.3.1 preparing a sealing liquid, 1.0 percent of bovine serum albumin and 0.02M phosphate solution with the pH value of 7.0-7.5;
1.3.2 preparing a washing solution, 0.05 percent of Tween-20 and 0.05M of Tris-HCl solution with the pH7.0-7.5;
1.4 determining a normal reference range, and selecting a sample with the H factor antigen content of about 274mg/L as sensitivity detection because the method is qualitative detection and the detection point of the sample H factor antigen content in the normal reference range does not develop color;
1.4.1 methods of experiment:
1. before use, the reagent and the sample are taken out, and are placed for 15-30 minutes at room temperature to be recovered to the room temperature;
2. taking out the detection plate;
3. adding 2 drops of sealing liquid into the center of a reaction hole of the detection plate until the sealing liquid completely permeates;
4. adding 40 microliters of the sample to be detected into the center of the reaction hole of the detection plate by using a pipettor until the sample completely permeates into the reaction hole;
5. adding 4 drops of washing liquid into the center of the reaction hole of the detection plate until the washing liquid completely permeates into the reaction hole;
6. adding 3 drops of colloidal gold conjugate in the center of the reaction hole of the detection plate until the colloidal gold conjugate completely permeates;
7. adding 4 drops of washing liquid into the center of the reaction hole of the detection plate, and observing the result visually after the washing liquid completely permeates;
[ interpretation of test results ]
When a user performs sample detection, the following situations may occur:
a. a red spot is arranged at the center detection point of the reaction hole of the detection plate, and a positive result is obtained;
b. the detection point at the center of the reaction hole of the detection plate has no red spot, and the result is negative.
[ REFERENCE VALUES (REFERENCE RANGE) ]
Based on the normal reference range of the H factor antigen determination kit, the H factor antigen content is 274 mg/L-508 mg/L, no red spot exists at the center detection point of the reaction hole of the detection plate in the normal reference range of the sample H factor antigen content, and negative results are displayed
The result is;
normal reference range: negative results were shown (indicating that the antigen content of factor H was between 274mg/L and 508 mg/L).
[ interpretation of test results ]
1. Negative result, indicating that the H factor antigen content of the sample is 274 mg/L-508 mg/L which is a normal range;
2. a positive result indicates that the H factor antigen content > 508mg/L in the sample is in an abnormal range.
[ limitations of assay methods ]
1. The method can only qualitatively determine the H factor in the human serum sample and is only used as auxiliary diagnosis;
2. stability of reaction spots: within 30 minutes the positive spots did not fade and the negative results remained unchanged.
Results of 20 hemodialysis tests:
positive for Positive for Positive for Positive for Positive for
Positive for Positive for Positive for Positive for Positive for
Positive for Positive for Positive for Positive for Positive for
Positive for Positive for Positive for Positive for Positive for
20 healthy persons (all negative in liver function, kidney function, HBsAg, HIV, hepatitis C, etc.) were negative between 274mg/L and 508 mg/L.
Negative of Negative of Negative of Negative of Negative of
Negative of Negative of Negative of Negative of Negative of
Negative of Negative of Negative of Negative of Negative of
Negative of Negative of Negative of Negative of Negative of
Results of 20 patients and 20 healthy people are compared and completely accord with clinical diagnosis.
Results of 60 peritoneal dialysis:
positive for Positive for Positive for Positive for Positive for Positive for Positive for Positive for Positive for Positive for
Positive for Positive for Positive for Positive for Positive for Positive for Positive for Positive for Positive for Positive for
Positive for Positive for Positive for Positive for Positive for Positive for Positive for Positive for Positive for Positive for
Positive for Positive for Positive for Positive for Positive for Positive for Positive for Positive for Positive for Positive for
Positive for Positive for Positive for Positive for Positive for Positive for Positive for Positive for Positive for Positive for
Positive for Positive for Positive for Positive for Positive for Positive for Positive for Positive for Positive for Positive for
Example 2: immunolatex turbidimetry kit (peritoneal dialysis)
The instrument comprises the following steps: 7100 Biochemical instrument
The operation method comprises the following steps: the samples were assayed using Hitachi 7100 Biochemical Analyzer. In the step of measuring the sample by using a biochemical analyzer, after adding the reagent I R1 into the sample, incubating the sample at 37 ℃ for 5min, reading the sample A1 at the 1 st point, adding the reagent II R2, incubating the sample at 37 ℃ for 5min, reading the sample A2 at the 2 nd point, wherein the sample A is sample A2-sample A1; the method also comprises a step of measuring the calibrator by using a Hitachi 7100 biochemical analyzer, wherein in the step of measuring the calibrator by using the biochemical analyzer, the calibrator is added into a reagent I R1, then the mixture is incubated at 37 ℃ for 5min, the calibrator A1 at the 1 st point is read, the reagent II R2 is added, the mixture is incubated at 37 ℃ for 5min, and the calibrator A2 at the 2 nd point is read, wherein the calibrator A is calibrator A2-calibrator A1;
Figure BDA0002776824000000041
the parameters measured above were: temperature 37 ℃, wavelength 600nm or 340nm, R1: 160uL R2: 40 uL. Sample or calibrator 3 μ l, R1: 240 μ l, R2: 60 μ l, reaction time 10 min.
20 normal persons (negative for liver function, kidney function, HBsAg, HIV, hepatitis C, etc.)
301mg/L 362mg/L 465mg/L 496mg/L 299mg/L
289mg/L 418mg/L 378mg/L 483mg/L 455mg/L
449mg/L 450mg/L 321mg/L 340mg/L 436mg/L
345mg/L 372mg/L 335mg/L 367mg/L 492mg/L
Concentrations between 274mg/L and 508mg/L are normal.
The clinical diagnosis results of 20 healthy people are between 274mg/L and 508mg/L, are negative and meet the clinical diagnosis standard.
Test results for 60 peritoneal dialysis patients
Figure BDA0002776824000000051
Concentrations between 274mg/L and 508mg/L are normal.
The results of 60 cases of clinical diagnosis are below 274mg/L or above 508mg/L, are positive and accord with the clinical diagnosis standard.
Example 3: 20 specimens from hemodialysis patients (immunoturbidimetry)
The instrument comprises the following steps: 7100 Biochemical instrument
The operation method is the same as that of example 2
Figure BDA0002776824000000052
Kit parameters: wavelength 600nm or 340nm, R1: 160uL R2: 40 uL.
20 examples of detection results:
137mg/L 245mg/L 746mg/L 257mg/L 239mg/L
218mg/L 220mg/L 635mg/L 674mg/L 513mg/L
506mg/L 130mg/L 146mg/L 208mg/L 115mg/L
155mg/L 783mg/L 233mg/L 264mg/L 160mg/L
concentrations between 274mg/L and 508mg/L are normal.
The clinical diagnosis result of 20 patients is more than 508mg/L, and the patients are positive and accord with the clinical diagnosis standard.
Example 4: 20 examples chemiluminescence method
The chemiluminescence detection method comprises the following steps:
1. the instrument comprises the following steps: MPC-1 chemiluminescence determinator
2. Fixing the anti-human H factor antibody on the surface of a solid phase, adding a sample to be detected and a marked anti-human H factor antibody, incubating, and then washing with a washing solution; meanwhile, setting a reference of the H factor standard substance;
3. adding a chemiluminescent substrate working solution, and standing for a period of time;
4. and measuring the luminous value to obtain the H factor concentration value of the sample to be measured.
50 samples of peritonitis patients (chemiluminescence method)
Figure BDA0002776824000000061
Results of 50 cases of detection: concentrations between 274mg/L and 508mg/L are normal.
The clinical diagnosis result of 50 patients is more than 508mg/L, is positive and accords with the clinical diagnosis standard.
The above examples of the present invention are merely examples for clearly illustrating the present invention and are not intended to limit the embodiments of the present invention. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.

Claims (9)

  1. Use of a factor H antibody in the manufacture of a kit for the detection of hemodialysis, peritoneal dialysis, peritonitis or glioma.
  2. 2. Use according to claim 1, characterized in that: the kit comprises a detection plate, a rabbit anti-H factor antibody, a goat anti-H factor antibody conjugate marked by colloidal gold, a sealing liquid, a washing liquid, a positive reference product and a negative reference product, wherein the detection plate consists of a plastic box bottom box, a water absorption layer is arranged in the plastic box bottom box, a nitrocellulose membrane is arranged on the water absorption layer, a filter screen is covered on the nitrocellulose membrane, a plastic box surface cover is arranged on the filter screen, a detection reaction hole is arranged in the center of the plastic box surface cover, the rabbit anti-H factor antibody and the goat anti-H factor antibody conjugate marked by the colloidal gold are dotted on the nitrocellulose membrane corresponding to the detection reaction hole, the sealing liquid is a phosphate solution containing bovine serum albumin, the pH value of the phosphate solution is 7.0-7.5, and the weight percentage of the bovine serum albumin in the phosphate solution is 1.0%, the washing solution is a Tris-HCl solution containing Tween-20, the pH of the Tris-HCl solution is 7.0-7.5, the weight percentage of the Tween-20 in the Tris-HCl solution is 0.05%, the mass of the goat anti-H factor antibody in each milliliter of colloidal gold conjugate is 700 mg, and the positive reference substance is the H factor antigen with the concentration of less than 274 mg/L.
  3. 3. Use according to claim 1, characterized in that: and (3) adopting a colloidal gold percolation method and a chromatography method or a chemiluminescence method for detection.
  4. 4. Use according to claim 1, characterized in that: and performing clinical examination by ELISA, immunoturbidimetry, immunotransmission turbidimetry, immunoscattering turbidimetry or immunolatex-enhanced turbidimetry.
  5. 5. Use according to claim 1, characterized in that: and detecting by adopting a micro-fluidic chip or a magnetic particle method.
  6. 6. Use according to claim 1, characterized in that: the rabbit anti-H factor antibody is a monoclonal antibody or a polyclonal antibody.
  7. 7. Use according to claim 1, characterized in that: the H factor antibody comprises monoclonal antibody or polyclonal antibody of mouse, rabbit, sheep or horse.
  8. 8. A kit for detecting hemodialysis, peritoneal dialysis, peritonitis or glioma, comprising: detection was performed using a factor H antigen-antibody binding reaction.
  9. Use of factor H antibodies in the preparation of a marker for the detection of hemodialysis, peritoneal dialysis, peritonitis, or glioma.
CN202011268082.XA 2020-11-13 2020-11-13 Application of H factor antibody in preparation of kit for detecting hemodialysis, peritoneal dialysis, peritonitis or glioma Pending CN114487410A (en)

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