CN111505291A - Method for eliminating interference of megazyme molecules on serum enzyme concentration detection - Google Patents
Method for eliminating interference of megazyme molecules on serum enzyme concentration detection Download PDFInfo
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- CN111505291A CN111505291A CN202010289352.9A CN202010289352A CN111505291A CN 111505291 A CN111505291 A CN 111505291A CN 202010289352 A CN202010289352 A CN 202010289352A CN 111505291 A CN111505291 A CN 111505291A
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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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
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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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/305—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Micrococcaceae (F)
- G01N2333/31—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Micrococcaceae (F) from Staphylococcus (G)
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a method for eliminating interference of megazyme molecules on serum enzyme concentration detection, which can eliminate the interference of megazyme on serum enzyme detection by utilizing the characteristic of specific combination of SpA on the surface of staphylococcus aureus and Fc segment of immunoglobulin, has no influence on free enzyme molecules, and can be stored for more than half a year at 4 ℃ by only culturing Cowan I staphylococcus aureus with high expression of SpA in a laboratory and carrying out the steps of fixing, washing and heating inactivation; compared with gel electrophoresis, ultrafiltration, SpA agarose gel particle adsorption and other methods, the method has the characteristics of simplicity, convenience, quickness, economy, strong specificity and the like, and is very suitable for clinical popularization.
Description
Technical Field
The invention relates to a method for eliminating interference, in particular to a method for eliminating interference of giant enzyme molecules on serum enzyme concentration detection, and belongs to the technical field of immunoprecipitation.
Background
Megazyme is an enzyme whose enzyme molecule circulates in blood by binding to serum immunoglobulin (megazyme type 1) or causing an increase in molecular weight by self-association (megazyme type 2). megazyme commonly used in serum includes megacreatine kinase (Macro-CK), macroamylase (Macro-AMY), megaalkaline phosphatase (Macro-A L P), macroaspartate aminotransferase (Macro-AST), etc. due to the binding of the enzyme molecule to immunoglobulin, the formed high molecular weight compounds are not cleared by the kidney, the half-life is prolonged, and false positive elevation may result.continuously increased enzyme content often causes misdiagnosis of disease, placing a heavy mental burden on patients.
Studies report that megazyme molecules can be removed by immunoglobulin electrophoresis, PEG precipitation, ultrafiltration and the like, but the methods have many defects, such as that free enzymes can be precipitated by PEG at the same time, the immunoglobulin electrophoresis is time-consuming and high in cost, and the ultrafiltration method is poor in specificity. In the case study reported by us, commercial protein a and protein G were used to adsorb immunoglobulin in serum, thereby removing megaenzyme interference. However, this method cannot be clinically popularized, also because of its high cost. Therefore, a simple and easy method with low cost is urgently needed, and is convenient for clinical popularization.
Protein a (spa) is a highly stable cell surface receptor produced by several staphylococcus aureus strains. It consists of a 42kDa polypeptide chain comprising four repeat domains rich in aspartic acid and glutamic acid but free of cysteine. It contains almost no carbohydrates, only 4 tyrosine residues and no tryptophan. Protein A binds to the Fc portion of immunoglobulins (in particular IgG) from a variety of species (e.g., human, monkey, rabbit, pig, guinea pig). One protein a molecule has been shown to bind at least 2 IgG molecules simultaneously. Protein a binds to the Fc portion of the human IgG subclasses IgM, IgA and IgE and mouse IgG1 (weak), IgG2a and IgG2b molecules. Staphylococcus aureus CowanI with high protein A expression on the surface of a bacterium body is used as a carrier, and is subjected to treatment steps of fixation, heating inactivation and the like to enable the Staphylococcus aureus CowanI to interact with giant enzyme molecules existing in serum, and the giant enzyme molecules are separated by a centrifugal method, so that interference of the giant enzyme molecules on serum enzyme detection is removed. The method is simple and easy to implement, has low cost, is suitable for popularization and promotion in laboratories, and has wide application prospect.
Disclosure of Invention
The invention provides a method for eliminating interference of giant enzyme molecules on serum enzyme concentration detection, and solves the problems in the prior art.
In order to solve the technical problems, the invention provides the following technical scheme:
the invention provides a method for eliminating interference of megazyme molecules on serum enzyme concentration detection, which comprises the following specific steps of:
the method comprises the following steps: cowan I bacterial preparation
(A1) In order to stabilize the SpA molecules on the surface of the Cowan I bacteria, firstly, fixing the Cowan I bacteria by using 2% formalin solution for 2 hours at room temperature;
(A2) the fixed staphylococcus aureus is placed in a water bath at the temperature of 80 ℃ for inactivation, the SpA protein is very stable to heat, the binding activity of SpA cannot be interfered, and meanwhile, the inactivated staphylococcus aureus can be stored for a long time;
(A3) and washing with PBS (phosphate buffer solution) containing 0.5% tween 20 can remove SpA protein non-covalently bound on the surface of the thallus and improve the specificity of the reaction.
Step two: serum adsorption test
(B1) 5ml of fasting venous blood of a patient suspected of containing megazyme is extracted, and after the serum is subjected to self-coagulation, the serum is centrifuged to separate the serum;
(B2) uniformly mixing the Cowan I bacterial liquid in the step I, and mixing the Cowan I bacterial liquid with serum according to the ratio of 1: 1, placing the mixture in a shaking table (150rpm) at 37 ℃ for incubation for 3 hours to ensure that SpA protein on the surface of the thallus and an Fc segment of immunoglobulin are fully combined into an immune complex;
(B3) and separating the immune complex by a high-speed centrifugation mode, and sucking upper layer separated serum for enzyme concentration detection.
Step three: detection of serum enzyme concentration before and after adsorption and result calculation
(C1) Detecting the enzyme concentration before and after the adsorption of the serum by a standard method;
(C2) and correcting enzyme concentration after adsorption: when detecting enzyme and immunoglobulin, the concentration level detected after adsorption is multiplied by dilution times to obtain the ratio of the value to the original concentration, namely the recovery rate, and the corresponding adsorption rate (%) is calculated in the following way: 100% -recovery rate.
(C3) Comparing the levels of serum enzymes before and after adsorption, the recovery rate and the adsorption rate, and judging whether a giant enzyme interference phenomenon exists; if the enzyme level after adsorption is in a normal reference interval, judging that giant enzyme interference exists, and correcting the result of the detection report.
As a preferred technical scheme of the invention, the Cowan I strain utilized is staphylococcus aureus highly expressing SpA on the surface of a membrane.
As a preferred technical scheme of the invention, when treating Cowan I staphylococcus aureus, firstly, 2% formalin solution is used for firstly fixing cells; then, after washing, inactivation was carried out in a water bath at 80 ℃ for 5 minutes.
As a preferred embodiment of the present invention, the method of the present invention is suitable for eliminating interference caused by megaaspartate acyltransferase (Macro-AST), megacreatine kinase (Macro-CK), macroamylase (Macro-AMY) and megaalkaline phosphatase (Macro-A L P).
The invention has the following beneficial effects: compared with the prior art, the method for eliminating the interference of the megazyme molecules on the detection of the serum enzyme concentration has the following beneficial effects:
1. the method for eliminating the interference of megazyme molecules on the serum enzyme concentration detection utilizes the specific combination characteristic of SpA on the surface of staphylococcus aureus and Fc segment of immunoglobulin, can eliminate the interference of megazyme on the serum enzyme detection, has no influence on free enzyme molecules, and can be stored at 4 ℃ for more than half a year only by culturing Cowan I staphylococcus aureus with high SpA expression in a laboratory and carrying out the steps of fixing, washing and heating inactivation.
2. Compared with methods such as gel electrophoresis, ultrafiltration, SpA agarose gel particle adsorption and the like, the method for eliminating the interference of giant enzyme molecules on the detection of the serum enzyme concentration has the characteristics of simplicity, convenience, quickness, economy, strong specificity and the like, and is very suitable for clinical popularization.
Detailed Description
The following description of the preferred embodiments of the present invention is provided for the purpose of illustration and description, and is in no way intended to limit the invention.
Example 1
The invention provides a method for eliminating interference of megazyme molecules on serum enzyme concentration detection, which utilizes CowanI staphylococcus aureus to adsorb 'megaast' in serum so as to eliminate interference on serum free AST detection, and comprises the following specific steps:
the method comprises the following steps: cowan I bacterial preparation
(A1) Inoculating Cowan I into 200ml of L B liquid culture medium, and performing shake culture at 37 ℃ overnight;
(A2) centrifuging the bacteria solution the next day (5000rpm, 10 minutes), collecting bacteria, and washing with PBS solution for 2 times;
(A3) and (3) bacteria fixation: adding the washed Cowan I staphylococcus aureus into a 1.5% formalin solution, uniformly mixing, and fixing for 2 hours at room temperature;
(A4) washing: washing with a PBS (phosphate buffer solution) solution to remove formalin in the solution, and preparing a bacterial solution with a concentration of 50% by using the PBS;
(A5) inactivation: pouring the bacterial liquid into a conical flask (less than 1.5cm in height), placing in 80 deg.C water bath, and horizontally shaking for 5 min;
(A6) and ice bath: immediately placing the conical flask on ice for cooling;
(A7) and washing: first washed once with PBS solution containing 0.5% tween 20 and then washed with PBS without tween 20;
(A8) and (4) preservation: the washed bacterial liquid is stored in a refrigerator at 4 ℃ and is shaken up before use. (can be stored for half a year)
Step two: serum adsorption test
(B1) And serum preparation: 5ml of fasting venous blood of a patient suspected of containing giant AST is extracted, centrifugation is carried out after serum is self-coagulated, and serum is separated;
(B2) uniformly mixing the Cowan I bacterial liquid obtained in the step one, sucking 500ul of the mixture, adding the mixture into an EP tube, standing at room temperature for 30 minutes for rewarming, then adding 500ul of serum of a patient, and uniformly mixing;
(B3) incubating the cells in a shaker (150rpm) at 37 ℃ for 3 hours;
(B4) and centrifuging: placing the EP tube in a centrifuge for 10 minutes, centrifuging at 10000rpm, carefully sucking 500ul of upper serum, adding into a new EP tube, and measuring the Ig and AST concentrations.
Step three: serum AST and immunoglobulin concentration detection:
(C1) detecting the concentration of immunoglobulin (IgM, IgG and IgA) by using a Siemens BNProspec immune turbidimeter;
(C2) detecting AST concentrations before and after Cowan I adsorption by using Roche cobas c 701;
wherein, the detection reagents are all instrument matching reagents and are carried out according to standard operation procedures.
Step four: calculation of results
(D1) And correcting enzyme concentration after adsorption: in the detection of enzyme and immunoglobulin, the ratio of serum to Cowan I bacterial liquid is 1: 1, so that the concentration level detected after adsorption is multiplied by the dilution factor (here, 2), and the ratio of the obtained value to the original concentration is the recovery rate, and the corresponding adsorption rate is calculated by the following method: 100% -recovery rate.
(D2) And the result is as follows:
TABLE 1 recovery of serum immunoglobulins and AST before and after Cowan I adsorption
Wherein the Ig concentration unit is g/L concentration unit is U/L.
The results in Table 1 show that Cowan I has the strongest adsorption effect on IgG, the adsorption rates of giant AST and a control group are 34.48% and 41.92% respectively, the adsorption rate of IgM is lower than that of IgG, and is 17.74% and 12.7% respectively, the original AST of giant AST serum is 109U/L, the converted concentration after adsorption is 22.2%, and the adsorption rate is 79.63%, while for the control serum, the AST values before and after adsorption are 161U/L and 156.8U/L respectively, and the adsorption rate is only 2.61%.
The method for eliminating the interference of megazyme molecules on the detection of the concentration of the serum enzyme utilizes the characteristic of specific combination of the SpA on the surface of the staphylococcus aureus and the Fc segment of the immunoglobulin, can eliminate the interference of megazyme on the detection of the serum enzyme, has no influence on free enzyme molecules, and meanwhile, a laboratory only needs to culture Cowan I staphylococcus aureus with high SpA expression, and can be stored at 4 ℃ for more than half a year through the steps of fixing, washing and heating inactivation; compared with gel electrophoresis, ultrafiltration, SpA agarose gel particle adsorption and other methods, the method has the characteristics of simplicity, convenience, quickness, economy, strong specificity and the like, and is very suitable for clinical popularization.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (4)
1. A method for eliminating interference of megazyme molecules on serum enzyme concentration detection is characterized by comprising the following specific steps of:
the method comprises the following steps: cowan I bacterial preparation
(A1) In order to stabilize the SpA molecules on the surface of the Cowan I bacteria, firstly, fixing the Cowan I bacteria by using 2% formalin solution for 2 hours at room temperature;
(A2) the fixed staphylococcus aureus is placed in a water bath at the temperature of 80 ℃ for inactivation, the SpA protein is very stable to heat, the binding activity of SpA cannot be interfered, and meanwhile, the inactivated staphylococcus aureus can be stored for a long time;
(A3) and washing with PBS (phosphate buffer solution) containing 0.5% tween 20 can remove SpA protein non-covalently bound on the surface of the thallus and improve the specificity of the reaction.
Step two: serum adsorption test
(B1) 5ml of fasting venous blood of a patient suspected of containing megazyme is extracted, and after the serum is subjected to self-coagulation, the serum is centrifuged to separate the serum;
(B2) uniformly mixing the Cowan I bacterial liquid in the step I, and mixing the Cowan I bacterial liquid with serum according to the ratio of 1: 1, placing the mixture in a shaking table (150rpm) at 37 ℃ for incubation for 3 hours to ensure that SpA protein on the surface of the thallus and an Fc segment of immunoglobulin are fully combined into an immune complex;
(B3) and separating the immune complex by a high-speed centrifugation mode, and sucking upper layer separated serum for enzyme concentration detection.
Step three: detection of serum enzyme concentration before and after adsorption and result calculation
(C1) Detecting the enzyme concentration before and after the adsorption of the serum by a standard method;
(C2) and correcting enzyme concentration after adsorption: when detecting enzyme and immunoglobulin, the concentration level detected after adsorption is multiplied by dilution times to obtain the ratio of the value to the original concentration, namely the recovery rate, and the corresponding adsorption rate (%) is calculated in the following way: 100% -recovery rate.
(C3) Comparing the levels of serum enzymes before and after adsorption, the recovery rate and the adsorption rate, and judging whether a giant enzyme interference phenomenon exists; if the enzyme level after adsorption is in a normal reference interval, judging that giant enzyme interference exists, and correcting the result of the detection report.
2. The method of claim 1, wherein the Cowan I strain is Staphylococcus aureus highly expressing SpA on the membrane surface.
3. The method of claim 1, wherein the treatment of Cowan I S.aureus is performed by first fixing the cells with 2% formalin solution; then, after washing, inactivation was carried out in a water bath at 80 ℃ for 5 minutes.
4. The method of claim 1, wherein the method is suitable for eliminating interference caused by megaaspartate acyltransferase (Macro-AST), megacreatine kinase (Macro-CK), giant amylase (Macro-AMY) and megaalkaline phosphatase (Macro-A L P).
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| CN1723220A (en) * | 2003-11-13 | 2006-01-18 | 韩美药品工业株式会社 | A pharmaceutical composition comprising an immunoglobulin FC region as a carrier |
| US20050281803A1 (en) * | 2004-03-16 | 2005-12-22 | The Regents Of The University Of Michigan | Methods and compositions for using aleveolar macrophage phospholipase A2 |
| US20060003336A1 (en) * | 2004-06-30 | 2006-01-05 | Kimberly-Clark Worldwide, Inc. | One-step enzymatic and amine detection technique |
| US20090232799A1 (en) * | 2004-10-29 | 2009-09-17 | The Regents Of The University Of Colorado | Antibodies that bind urokinase-type plasminogen activator and epitopes therefor |
| US20070254279A1 (en) * | 2006-04-21 | 2007-11-01 | Anita Goel | Single molecule drug discovery |
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