CN111505291B - A method for excluding the interference of giant enzyme molecules on the detection of serum enzyme concentration - Google Patents

Abstract

The invention discloses a method for eliminating the interference of giant enzyme molecules on the detection of serum enzyme concentration. The specific binding characteristics of SpA on the surface of Staphylococcus aureus and the Fc segment of immunoglobulin can be used to remove the influence of "giant enzyme" on serum enzyme concentration. The interference caused by the detection has no effect on the free enzyme molecules. At the same time, the laboratory only needs to cultivate Cowan I S. aureus with high expression of SpA. After several steps of fixing-washing-heating inactivation, it can be stored at 4°C for more than half a year; Compared with gel electrophoresis, ultrafiltration, SpA agarose gel particle adsorption and other methods, it has the characteristics of simplicity, convenience, economy, speed, and strong specificity, and is very suitable for clinical promotion.

Description

A method for excluding the interference of giant enzyme molecules on the detection of serum enzyme concentration

technical field

The invention relates to a method for eliminating interference, in particular to a method for eliminating interference caused by macrozyme molecules to detection of serum enzyme concentration, and belongs to the technical field of immunoprecipitation.

Background technique

Megazymes are enzyme molecules that circulate in the blood either by binding to serum immunoglobulins (type 1 megazyme) or by self-association (type 2 megazyme) 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 so on. Due to the combination of enzyme molecules and immunoglobulins, the formed high-molecular-weight compounds cannot be cleared by the kidneys, and the half-life is prolonged, which can lead to increased false positives. The continuously increasing enzyme content often leads to misdiagnosis and mistreatment of diseases, which brings heavy mental burden to patients. Recently, we reported a confirmed case of macrozyme in which elevated serum AST was found 6 years ago, although the patient had no other positive findings, but had been treated as "hepatitis" for 6 years. We confirmed by immunoglobulin precipitation technique that it was caused by giant AST, thus avoiding unnecessary treatment and eliminating the psychological burden on patients. Related research results were published on CCLM (Clin Chem Lab Med. 2020 Mar 26; 58(4):e96-e99.).

Studies have reported that giant enzyme molecules can be removed by immunoglobulin electrophoresis, PEG precipitation, ultrafiltration and other methods, but these methods have many disadvantages, such as PEG will precipitate free enzymes at the same time, immunoglobulin electrophoresis is time-consuming and costly, and ultrafiltration methods are specific Poor sex and so on. In the case study we reported, commercialized protein A and protein G were used to adsorb immunoglobulin in serum, thereby removing the interference of macrozyme. However, due to the high cost, this method cannot be popularized in clinic. Therefore, there is an urgent need for a simple, low-cost method that is easy to promote clinically.

Protein A (SpA) is a highly stable cell surface receptor produced by several strains of S. aureus. It consists of a polypeptide chain with a molecular weight of 42 kDa containing four repeat domains rich in aspartate and glutamate but not cysteine. It contains almost no carbohydrates, only 4 tyrosine residues, and no tryptophan. Protein A is capable of binding to the Fc portion of immunoglobulins (especially IgG) of various species (eg, human, monkey, rabbit, pig, guinea pig). One protein A molecule has been shown to bind at least 2 IgG molecules simultaneously. Protein A binds the Fc portion of human IgG subclasses IgM, IgA and IgE and mouse IgG1 (weak), IgG2a and IgG2b molecules. We use Staphylococcus aureus CowanI, which highly expresses protein A on the surface of the bacteria, as a carrier, and through the treatment steps of fixation and heat inactivation, make it interact with the giant enzyme molecule in the serum, and separate it by centrifugation, so that Remove the interference of giant enzyme molecules on serum enzyme detection. The method is simple and easy to implement, low in cost, suitable for promotion and popularization in laboratories, and thus has broad application prospects.

Contents of the invention

The invention proposes a method for eliminating the interference of giant enzyme molecules on the detection of serum enzyme concentration, and solves the problems raised in the prior art.

In order to solve the problems of the technologies described above, the present invention provides the following technical solutions:

The present invention provides a method for eliminating the interference caused by macrozyme molecules to the detection of serum enzyme concentration. The specific steps for eliminating the interference caused by macrozyme molecules to the detection of serum enzyme concentration are as follows:

Step 1: Cowan I bacteria preparation

(A1), in order to make the SpA molecule on the surface of the Cowan I bacterial thalline more stable, at first utilize 2% formalin solution to fix, and the fixation time and condition are fixed at room temperature for 2 hours;

(A2), placing the fixed Staphylococcus aureus in an 80°C water bath for inactivation, because the SpA protein is very stable to heat, it will not interfere with the binding activity of SpA, and at the same time, the inactivated bacteria can be stored for a long time;

(A3), using the PBS solution containing 0.5% tween 20 to wash, can remove the non-covalently bound SpA protein on the surface of the thalline, and improve the specificity of the reaction.

Step 2: Serum Adsorption Test

(B1), extract 5ml of fasting venous blood from patients suspected of containing macrozyme, centrifuge after the serum autocoagulates, and separate the serum;

(B2), mix the Cowan I bacteria solution in step 1, mix it with the serum at a ratio of 1:1, and incubate it on a shaker (150rpm) at 37°C for 3 hours, so that the SpA protein on the surface of the bacteria and the immune The Fc portion of the globulin is fully combined into an immune complex;

(B3). The immune complex is separated by high-speed centrifugation, and the serum separated from the upper layer is drawn to detect the enzyme concentration.

Step 3: Serum enzyme concentration detection and result calculation before and after adsorption

(C1), detect the enzyme concentration before and after serum adsorption by standard method;

(C2) Enzyme concentration correction after adsorption: When detecting enzymes and immunoglobulins, multiply the concentration level detected after adsorption by the dilution factor, and the ratio of the obtained value to the original concentration is the recovery rate, and the corresponding adsorption rate (%) calculation method is: 100%-recovery rate%.

(C3), compare the serum enzyme levels before and after adsorption, as well as the recovery rate and adsorption rate, to determine whether there is macrozyme interference; if the enzyme level after adsorption is within the normal reference range, it can be judged that there is macrozyme interference, and the test report is corrected. .

As a preferred technical solution of the present invention, the Cowan I bacterial strain utilized is Staphylococcus aureus highly expressing SpA on the membrane surface.

As a preferred technical scheme of the present invention, when Cowan I Staphylococcus aureus is processed, at first utilize 2% formalin solution to fix the cells first; then inactivate them in a water bath at 80° C. for 5 minutes after washing.

As a preferred technical scheme of the present invention, the method of the present invention is suitable for eliminating giant aspartate acyltransferase (Macro-AST), giant creatine kinase (Macro-CK), giant amylase (Macro-AMY) and Interference caused by giant alkaline phosphatase (Macro-ALP).

The beneficial effects achieved by the present invention are: compared with the prior art, a method for eliminating the interference of giant enzyme molecules on the detection of serum enzyme concentration has the following beneficial effects:

1. The method of the present invention that eliminates the interference of giant enzyme molecules on the detection of serum enzyme concentration utilizes the specific binding characteristics of SpA on the surface of Staphylococcus aureus and the Fc segment of immunoglobulin, and can remove the "giant enzyme" on the serum enzyme detection band. Interference from the outside has no effect on free enzyme molecules. At the same time, the laboratory only needs to cultivate Cowan I S. aureus with high expression of SpA. After several steps of fixation-washing-heating inactivation, it can be stored at 4°C for more than half a year.

2. Compared with methods such as gel electrophoresis, ultrafiltration, and SpA agarose gel particle adsorption, the method of eliminating the interference of giant enzyme molecules to the detection of serum enzyme concentration is simple, easy to implement, economical, fast, and specific. And other characteristics, very suitable for clinical promotion.

Detailed ways

Preferred embodiments of the present invention are described below, and it should be understood that the preferred embodiments described here are only used to illustrate and explain the present invention, and are not intended to limit the present invention.

Example 1

The present invention provides a method for eliminating the interference of giant enzyme molecules on the detection of serum enzyme concentration, using CowanI Staphylococcus aureus to absorb the "giant AST" in serum to eliminate its interference to the detection of serum free AST, more specifically The steps are:

Step 1: Cowan I bacteria preparation

(A1), inoculate Cowan I in 200ml LB liquid medium, and place it in a shaker at 37°C for overnight culture;

(A2), the next day, the bacteria solution was centrifuged (5000rpm, 10 minutes), the bacteria were collected, and washed twice with PBS solution;

(A3) Bacterial fixation: add the washed Cowan I Staphylococcus aureus to a solution containing 1.5% formalin, mix well, and fix at room temperature for 2 hours;

(A4) Washing: washing with PBS solution to remove formalin in the solution, and then using PBS to prepare a 50% bacterial solution;

(A5) Inactivation: Pour the bacterial solution into a conical flask (less than 1.5cm in height), place it in a water bath at 80°C, and shake it horizontally for 5 minutes;

(A6), ice bath: place the Erlenmeyer flask on ice immediately for cooling;

(A7), washing: first wash once with PBS solution containing 0.5% tween 20, then wash with PBS without tween 20;

(A8) Storage: Store the washed bacterial solution in a refrigerator at 4°C and shake well before use. (can be stored for half a year)

Step 2: Serum Adsorption Test

(B1) Serum preparation: Take 5ml of fasting venous blood from patients suspected of containing giant AST, centrifuge after the serum autocoagulates, and separate the serum;

(B2), mix the Cowan I bacterial solution in step 1, draw 500ul and add it in the EP tube, let stand at room temperature for 30 minutes to rewarm, then add 500ul of patient serum, and mix;

(B3), incubate on a shaker (150 rpm) at 37°C for 3 hours;

(B4), centrifugation: place the above EP tube in a centrifuge for 10 minutes at a speed of 10,000 rpm, carefully absorb 500ul of the upper serum, add it to a new EP tube, and test the Ig and AST concentrations.

Step 3: Detection of serum AST and immunoglobulin concentration:

(C1), using the Siemens BNProspec immunoturbidimeter to detect the concentration of immunoglobulins (IgM, IgG, IgA);

(C2) Use Roche cobas c701 to detect the concentration of AST before and after Cowan I adsorption;

Among them, the detection reagents are all supporting reagents for the instrument, and are carried out in accordance with the standard operating procedures.

Step 4: Calculation of Results

(D1), correction of enzyme concentration after adsorption: when detecting enzymes and immunoglobulins, since the serum and Cowan I bacterial solution are mixed at a ratio of 1:1, the concentration level detected after adsorption is multiplied by the dilution factor (here is 2) The ratio of the obtained value to the original concentration is the recovery rate, and the corresponding adsorption rate calculation method is: 100%-recovery rate%.

(D2), the result:

Table 1. Recovery rate of serum immunoglobulin and AST before and after Cowan I adsorption

Wherein, the unit of Ig concentration is g/L, and the unit of AST concentration 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 the control group are 34.48% and 41.92%, respectively, and the adsorption rates of IgM are lower than IgG, which are 17.74% and 12.7%, respectively. The original AST of giant AST serum was 109U/L, the converted concentration after adsorption was 22.2%, and the adsorption rate was 79.63%, while for the control serum, the AST values before and after adsorption were 161U/L and 156.8U/L, and the adsorption rate was only 2.61%. It can be seen that there is a phenomenon of giant enzyme interference in the giant AST group, which leads to a significant increase in the measured value of AST in the serum. Considering that the adsorption of immunoglobulin by Cowan I bacteria cannot reach 100%, the actual concentration of AST in the serum should be less than 22.2U/L , the results can be reported according to <22.2U/L, which is within the normal reference interval.

The method for eliminating the interference of giant enzyme molecules on the detection of serum enzyme concentration utilizes the specific binding characteristics of SpA on the surface of Staphylococcus aureus and the Fc segment of immunoglobulins, and can remove the interference caused by "giant enzymes" on serum enzyme detection. Interference has no effect on free enzyme molecules. At the same time, the laboratory only needs to cultivate Cowan I Staphylococcus aureus with high expression of SpA. After several steps of fixation-washing-heating inactivation, it can be stored at 4°C for more than half a year; and gel electrophoresis Compared with other methods, such as ultrafiltration, SpA agarose gel particle adsorption, etc., they are simple, easy, economical, fast, and specific, and are very suitable for clinical promotion.

Finally, it should be noted that: the above is only a preferred embodiment of the present invention, and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, for those skilled in the art, it still The technical solutions recorded in the foregoing embodiments may be modified, or some technical features thereof may be equivalently replaced. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within 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.