CN111505291B - Method for eliminating interference of macroenzyme molecules on serum enzyme concentration detection - Google Patents
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Abstract
The invention discloses a method for eliminating interference of giant enzyme molecules on serum enzyme concentration detection, which can eliminate interference of giant enzyme on serum enzyme detection by utilizing the characteristic of specific combination of SpA on the surface of staphylococcus aureus and immunoglobulin Fc segment, 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 preserved for more than half a year at 4 ℃ through a plurality of steps of fixation, 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, practicability, economy, rapidness, 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 a giant enzyme molecule on serum enzyme concentration detection, belonging to the technical field of immunoprecipitation.
Background
Megazymes are enzymes whose molecules circulate in the blood by binding to serum immunoglobulins (type 1 megazymes) or by associating themselves (type 2 megazymes) resulting in an increase in molecular weight. Common giant enzymes in serum include giant creatine kinase (Macro-CK), giant amylase (Macro-AMY), giant alkaline phosphatase (Macro-ALP), giant aspartate aminotransferase (Macro-AST), and the like. Due to the binding of the enzyme molecule to the immunoglobulin, the resulting high molecular weight compound cannot be cleared by the kidneys, and the half-life is prolonged, which can lead to an increase in false positives. The continuously increased enzyme content often causes misdiagnosis and mistreatment of diseases, and brings heavy mental burden to patients. Recently, we reported a definite megazyme case that was found to have elevated serum AST in the last 6 years, and that was treated as "hepatitis" for 6 years, although patients had no other positive findings. We demonstrate by immunoglobulin precipitation techniques that are due to megaast, avoiding unnecessary treatment and eliminating psychological burden on patients. Related research results are published on CCLM (Clin Chem Lab Med.2020 Mar 26;58 (4): e96-e 99.).
The research reports that the giant enzyme molecules can be removed by methods such as immunoglobulin electrophoresis, PEG precipitation, ultrafiltration and the like, but the methods have a plurality of defects such as that free enzymes are precipitated by PEG at the same time, the immunoglobulin electrophoresis is time-consuming and high in cost, and the specificity of the ultrafiltration method is poor. In our reported case study, commercial protein a, protein G was used to adsorb immunoglobulins in serum, thus eliminating the interference of megaases. But also because of the high cost, this method is not likely to be popular in clinic. Therefore, a simple, easy and low-cost method is urgently needed, and the popularization in clinic is facilitated.
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 little carbohydrate, only 4 tyrosine residues, and no tryptophan. Protein a is capable of binding to the Fc portion of immunoglobulins (particularly IgG) of 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 human IgG subclasses IgM, igA and IgE and mouse IgG1 (weak), igG2a and IgG2b molecules. The staphylococcus aureus CowanI with high expression of protein A on the surface of the strain is used as a carrier, and the carrier is subjected to treatment steps such as fixation, heating inactivation and the like to interact with the giant enzyme molecules existing in serum, and separation is carried out by a centrifugal method, so that the interference of the giant enzyme molecules on serum enzyme detection is removed. The method is simple and easy to operate, has low cost, is suitable for popularization and popularizing in laboratories, and has wide application prospect.
Disclosure of Invention
The invention provides a method for eliminating interference of macro 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 a giant enzyme molecule on serum enzyme concentration detection, which comprises the following specific steps of:
step one: cowan I bacteria preparation
(A1) In order to make the SpA molecules on the surface of the Cowan I bacterial thallus more stable, firstly, fixing the bacteria by using 2% formalin solution for 2 hours at room temperature;
(A2) Placing the immobilized staphylococcus aureus in a water bath at 80 ℃ for inactivation, wherein the SpA protein is very stable to heat and does not interfere with the binding activity of the SpA, and meanwhile, the inactivated bacteria can be stored for a long time;
(A3) Washing with PBS solution containing 0.5% tween20 can remove non-covalently bound SpA protein on the surface of the bacteria, and improve the specificity of the reaction.
Step two: serum adsorption test
(B1) Extracting 5ml of fasting venous blood of a patient suspected of containing megazyme, centrifuging after serum autocongealed, and separating serum;
(B2) Uniformly mixing the Cowan I bacterial liquid in the first step with serum according to the following ratio of 1:1, and placing the mixture in a shaking table (150 rpm) at 37 ℃ for incubation for 3 hours, so that SpA protein on the surface of the thalli and an immunoglobulin Fc segment are fully combined into an immune complex;
(B3) And separating the immune complex by high-speed centrifugation, and sucking the upper layer separated serum for enzyme concentration detection.
Step three: serum enzyme concentration detection before and after adsorption and result calculation
(C1) Detecting the enzyme concentration before and after serum adsorption by a standard method;
(C2) Correction of enzyme concentration after adsorption: in the detection of enzymes and immunoglobulins, the concentration level detected after adsorption is multiplied by the dilution factor, and the ratio of the obtained value to the original concentration is the recovery rate, and the corresponding adsorption rate (%) is calculated in the following manner: 100% -recovery percent.
(C3) Comparing the serum enzyme levels before and after adsorption, and judging whether the megaenzyme interference phenomenon exists or not according to the recovery rate and the adsorption rate; if the enzyme level is in the normal reference interval after adsorption, the existence of the megazyme interference can be judged, and the result correction is carried out on the detection report.
As a preferred embodiment of the present invention, the Cowan I strain used is Staphylococcus aureus highly expressing SpA on the membrane surface.
As a preferred technical scheme of the invention, when the Cowan I staphylococcus aureus is treated, firstly, 2% formalin solution is used for fixing cells in advance; then, the mixture was washed and then inactivated 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 the interference caused by giant aspartic acid acylase (Macro-AST), giant creatine kinase (Macro-CK), giant amylase (Macro-AMY) and giant alkaline phosphatase (Macro-ALP).
The beneficial effects achieved by the invention are as follows: compared with the prior art, the method for eliminating interference of the giant enzyme molecules on the serum enzyme concentration detection has the following beneficial effects:
1. the method for eliminating interference of giant enzyme molecules on serum enzyme concentration detection of the invention utilizes the characteristic of specific combination of SpA on the surface of staphylococcus aureus and immunoglobulin Fc segment, can eliminate interference of giant enzyme on serum enzyme detection without affecting free enzyme molecules, and simultaneously, a laboratory only needs to culture Cowan I staphylococcus aureus with high SpA expression, and can be preserved for more than half a year at 4 ℃ through steps of fixation, washing and heating inactivation.
2. Compared with gel electrophoresis, ultrafiltration, spA agarose gel particle adsorption and other methods, the method for eliminating interference of the giant enzyme molecules on serum enzyme concentration detection has the characteristics of simplicity, convenience, practicability, economy, rapidness, 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 explanation only and is not intended to limit the present invention.
Example 1
The invention provides a method for eliminating interference of macro enzyme molecules on serum enzyme concentration detection, which utilizes Cowan I staphylococcus aureus to adsorb 'macro AST' in serum so as to eliminate interference of the giant AST on serum free AST detection, and comprises the following steps:
step one: cowan I bacteria preparation
(A1) Inoculating Cowan I into 200ml LB liquid medium, and shaking at 37 ℃ for overnight;
(A2) The following day the bacterial solution was centrifuged (5000 rpm,10 min), bacteria were collected and washed 2 times with PBS solution;
(A3) Bacterial immobilization: adding the washed Cowan I staphylococcus aureus into a formalin solution containing 1.5 percent, uniformly mixing, and fixing for 2 hours at room temperature;
(A4) Washing: washing with PBS solution to remove formalin in the solution, and preparing a bacterial solution with 50% concentration by using PBS;
(A5) And (3) inactivation: pouring the bacterial liquid into a conical flask (with the height of less than 1.5 cm), placing in a water bath at 80 ℃ and horizontally shaking for 5min;
(A6) Ice bath: immediately placing the conical flask on ice for cooling;
(A7) Washing: first washed once with PBS solution containing 0.5% tween20, and then with PBS without tween 20;
(A8) And (5) preserving: the washed bacterial liquid is stored in a refrigerator at the temperature of 4 ℃ and is uniformly shaken before being used. (can be preserved for half a year)
Step two: serum adsorption test
(B1) Serum preparation: extracting 5ml of fasting venous blood of a patient suspected of containing megaast, centrifuging after serum autocongealed, and separating serum;
(B2) Uniformly mixing the Cowan I bacterial liquid in the first step, sucking 500ul of the Cowan I bacterial liquid, adding the Cowan I bacterial liquid into an EP pipe, standing at room temperature for 30 minutes for rewarming, then adding 500ul of patient serum, and uniformly mixing;
(B3) Incubating in a shaker (150 rpm) at 37℃for 3 hours;
(B4) And (5) centrifuging: the above EP tube was placed in a centrifuge and centrifuged for 10 minutes at 10000rpm, the supernatant serum was carefully aspirated at 500ul and added to a fresh EP tube to determine Ig and AST concentrations.
Step three: serum AST and immunoglobulin concentration detection:
(C1) Detecting the concentration of the immunoglobulin (IgM, igG, igA) by using a Siemens BNPprospec immunonephelometer;
(C2) Detecting AST concentration before and after Cowan I adsorption by using Roche cobas c 701;
wherein, the detection reagents are all instrument matched reagents and are carried out according to standard operation rules.
Step four: result calculation
(D1) Correction of enzyme concentration after adsorption: in enzyme and immunoglobulin detection, the ratio of serum to Cowan I bacterial liquid is 1:1, so that the concentration level detected after adsorption is multiplied by a dilution factor (2 here), the ratio of the obtained value to the original concentration is the recovery rate, and the corresponding adsorption rate is calculated by: 100% -recovery percent.
(D2) And (3) results:
TABLE 1 serum immunoglobulin and AST recovery before and after Cowan I adsorption
Wherein, ig concentration unit is g/L, AST concentration unit is U/L.
The results from table 1 show that: cowan I has the strongest adsorption to IgG, with giant AST and control group adsorption rates of 34.48% and 41.92%, respectively, and IgM adsorption rates lower than IgG of 17.74% and 12.7%, respectively. Giant AST serum has original AST of 109U/L, converted concentration after adsorption of 22.2%, adsorption rate of 79.63%, and AST values before and after adsorption of 161U/L and 156.8U/L, respectively, for control serum, adsorption rate of only 2.61%. From this, it can be seen that the macro AST group has a macro enzyme interference phenomenon, which results in a significant increase in the observed value of AST in serum, and considering that Cowan I bacteria cannot adsorb immunoglobulins to 100%, the actual AST concentration in serum should be less than 22.2U/L, and the result report can be made as <22.2U/L, which is in the normal reference interval.
The method for eliminating interference of giant enzyme molecules on serum enzyme concentration detection utilizes the characteristic of specific combination of SpA on the surface of staphylococcus aureus and immunoglobulin Fc segment, so that interference of giant enzyme on serum enzyme detection can be eliminated, free enzyme molecules are not affected, and meanwhile, a laboratory only needs to culture Cowan I staphylococcus aureus with high SpA expression, and the method can be stored for more than half a year at 4 ℃ through a plurality of steps of fixation, 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, practicability, economy, rapidness, strong specificity and the like, and is very suitable for clinical popularization.
Finally, it should be noted that: the foregoing description is only a preferred embodiment of the present invention, and the present invention is not limited thereto, but it is to be understood that modifications and equivalents of some of the technical features described in the foregoing embodiments may be made by those skilled in the art, although the present invention has been described in detail with reference to the foregoing embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (3)
1. The method for eliminating the interference of the giant enzyme molecules on the serum enzyme concentration detection is characterized by comprising the following specific steps of:
step one: preparing CowanI bacteria;
(A1) In order to make the SpA molecules on the surface of the CowanI bacterial thallus more stable, firstly, 2% formalin solution is used for fixation, and the fixation time and conditions are that the room temperature is fixed for 2 hours;
(A2) Placing the immobilized staphylococcus aureus in a water bath at 80 ℃ for inactivation, wherein the SpA protein is very stable to heat and does not interfere with the binding activity of the SpA, and meanwhile, the inactivated bacteria can be stored for a long time;
(A3) Washing with PBS solution containing 0.5% tween20 can remove non-covalently bound SpA protein on the surface of the bacteria, and improve the specificity of the reaction;
step two: serum adsorption test;
(B1) Extracting 5ml of fasting venous blood of a patient suspected of containing megazyme, centrifuging after serum autocongealed, and separating serum;
(B2) Uniformly mixing the cowanI bacterial liquid in the first step with serum according to the following ratio of 1:1, and placing the mixture in a shaking table at 37 ℃ for incubation for 3 hours to enable SpA protein on the surface of the thalli and an immunoglobulin Fc segment to be fully combined into an immune complex;
(B3) Separating immune complex by high-speed centrifugation, and sucking upper layer separated serum for enzyme concentration detection;
step three: detecting serum enzyme concentration before and after adsorption and calculating results;
(C1) Detecting the enzyme concentration before and after serum adsorption by a standard method;
(C2) Correction of enzyme concentration after adsorption: when detecting enzyme and immunoglobulin, multiplying the concentration level detected after adsorption by dilution times to obtain the ratio of the value to the original concentration, namely the recovery rate, wherein the corresponding adsorption rate is calculated by the following method: 100% -recovery rate;
(C3) Comparing the serum enzyme levels before and after adsorption, and judging whether the macro enzyme interference phenomenon exists in the process of judging the macro enzyme interference phenomenon, the recovery rate and the adsorption rate; if the enzyme level is in the normal reference interval after adsorption, the existence of the megazyme interference can be judged, and the result correction is carried out on the detection report;
the method is suitable for eliminating interference caused by giant aspartic acid acylases, giant creatine kinase, giant amylase and giant alkaline phosphatase.
2. The method of claim 1, wherein the CowanI strain is staphylococcus aureus with high expression of SpA on the membrane surface.
3. The method for eliminating interference of giant enzyme molecules on serum enzyme concentration detection according to claim 1, wherein when the cowan i staphylococcus aureus is treated, firstly, 2% formalin solution is used for antecedent cell fixation; then, the mixture was washed and then inactivated in a water bath at 80℃for 5 minutes.
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| US7521226B2 (en) * | 2004-06-30 | 2009-04-21 | Kimberly-Clark Worldwide, Inc. | One-step enzymatic and amine detection technique |
| US8119128B2 (en) * | 2004-10-29 | 2012-02-21 | The Regents Of The University Of Colorado, A Body Corporate | Antibodies that bind urokinase-type plasminogen activator and epitopes therefor |
| CA2646309C (en) * | 2006-04-21 | 2016-07-26 | Nanobiosym, Inc. | Single-molecule platform for drug discovery: methods and apparatuses for drug discovery, including discovery of anticancer and antiviral agents |
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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 |
| CN104792996A (en) * | 2014-01-20 | 2015-07-22 | 辽宁成大动物药业有限公司 | Rabies virus antibody (IgG) enzyme-linked immunoassay kit and detection method thereof |
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