CN111505291A - A method for eliminating the interference of giant enzyme molecules on the detection of serum enzyme concentration - Google Patents

Abstract

Translated fromChinese

本发明公开了一种排除巨酶分子对血清酶浓度检测带来干扰的方法，利用金黄色葡萄球菌表面的SpA与免疫球蛋白Fc段特异性结合的特性，可以去除“巨酶”对血清酶检测带来的干扰，对游离酶分子没有影响，同时，实验室只需要培养高表达SpA的Cowan I金葡菌，经过固定‑洗涤‑加热灭活几个步骤，可在4℃保存半年以上；与凝胶电泳、超滤、SpA琼脂糖凝胶颗粒吸附等方法比较起来，具有简便易行、经济快速、特异性强等特点，非常适合在临床上进行推广。The invention discloses a method for eliminating the interference caused by macroenzyme molecules to the detection of serum enzyme concentration. By utilizing the characteristic of the specific binding of SpA on the surface of Staphylococcus aureus to the Fc segment of immunoglobulin, the effect of "megaenzyme" on serum enzymes can be eliminated. The interference caused by the detection has no effect on free enzyme molecules. At the same time, the laboratory only needs to cultivate Cowan I Staphylococcus aureus with high SpA expression. After several steps of fixation-washing-heat inactivation, it can be stored at 4°C for more than half a year; Compared with methods such as gel electrophoresis, ultrafiltration, and SpA agarose particle adsorption, it has the characteristics of simplicity, economy, rapidity, and strong specificity, and is very suitable for clinical promotion.

Description

Translated fromChinese

一种排除巨酶分子对血清酶浓度检测带来干扰的方法A method for eliminating the interference of giant enzyme molecules on the detection of serum enzyme concentration

技术领域technical field

本发明涉及一种排除干扰的方法，特别涉及一种排除巨酶分子对血清酶浓度检测带来干扰的方法，属于免疫沉淀技术领域。The invention relates to a method for eliminating interference, in particular to a method for eliminating interference caused by macroenzyme molecules to detection of serum enzyme concentration, and belongs to the technical field of immunoprecipitation.

背景技术Background technique

巨酶是酶分子通过与血清免疫球蛋白(1型巨酶)结合或通过自身缔合 (2型巨酶)导致分子量增加而在血液中循环的酶。血清中常见的巨酶包括巨肌酸激酶(Macro-CK)、巨淀粉酶(Macro-AMY)、巨碱性磷酸酶(Macro-ALP)、巨天冬氨酸转氨酶(macro-AST)等。由于酶分子与免疫球蛋白的结合，所形成的高分子量化合物不能够通过肾脏清除，半衰期延长，可导致假阳性升高。持续增加的酶含量往往引起疾病的误诊误治，给患者带来沉重的精神负担。最近，我们报道了一例确诊的巨酶病例，该病例在6前年发现血清AST升高，虽然患者没有其它的阳性发现，但6年来一直按照“肝炎”进行治疗。我们通过免疫球蛋白沉淀技术证实是由于巨AST导致的，从而避免了不必要的治疗，消除了患者的心理负担。相关的研究成果发表在了CCLM上(Clin Chem Lab Med.2020 Mar 26；58(4):e96-e99.)。Megazymes are enzymes whose molecules circulate in the blood by binding to serum immunoglobulins (megazyme type 1) or by self-association (megazyme type 2) resulting in an increase in molecular weight. Common macroenzymes in serum include macrocreatine kinase (Macro-CK), macroamylase (Macro-AMY), macroalkaline phosphatase (Macro-ALP), macroaspartate 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 increased enzyme content often leads to misdiagnosis and mistreatment of diseases, which brings a heavy mental burden to patients. Recently, we reported a confirmed case of megaenzyme in which serum AST was found to be elevated 6 years before, and although the patient had no other positive findings, he had been treated as "hepatitis" for 6 years. We confirmed that it was caused by giant AST through immunoglobulin precipitation technology, thus avoiding unnecessary treatment and eliminating the psychological burden of patients. The related research results were published on CCLM (Clin Chem Lab Med. 2020 Mar 26; 58(4): e96-e99.).

研究报道，巨酶分子可以通过免疫球蛋白电泳、PEG沉淀、超滤等方法去除，但这些方法存在很多缺点，比如PEG同时会沉淀游离酶，免疫球蛋白电泳费时、成本高，超滤方法特异性差等。在我们报道的病例研究中，采用了商品化的蛋白A、蛋白G吸附血清中的免疫球蛋白，从而去除的巨酶的干扰。但同样由于成本较高，该方法不可能在临床中普及。因此迫切需要一种简单易行、低成本的方法，便于在临床上进行推广。Studies have reported that macroenzyme molecules can be removed by immunoglobulin electrophoresis, PEG precipitation, ultrafiltration and other methods, but these methods have many shortcomings, 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, etc. In the case study we reported, commercialized protein A and protein G were used to adsorb immunoglobulins in serum, thereby removing the interference of macroenzymes. But also due to the high cost, this method cannot be popularized in the clinic. Therefore, there is an urgent need for a simple and low-cost method, which is convenient for clinical promotion.

蛋白A(SpA)是几种金黄色葡萄球菌菌株产生的高度稳定的细胞表面受体。它由一条分子量为42kDa的多肽链组成，包含四个富含天冬氨酸和谷氨酸但不含半胱氨酸的重复域。它几乎不含碳水化合物，仅含有4个酪氨酸残基，不含色氨酸。蛋白A能够与多种物种(如人、猴子，兔子，猪，豚鼠) 的免疫球蛋白(尤其是IgG)的Fc部分结合。已显示一种蛋白质A分子同时结合至少2个IgG分子。蛋白A结合人IgG亚类IgM，IgA和IgE的Fc部分和小鼠IgG1(弱)，IgG2a和IgG2b分子。我们利用菌体表面高表达蛋白A 的金黄色葡萄球菌CowanI作为载体，经过对固定和加热灭活等处理步骤，使之与血清中存在的巨酶分子相互作用，通过离心的方法进行分离，从而去除巨酶分子对血清酶检测带来的干扰。该方法简便易行，成本低，适合实验室的推广和普及，因此具有广阔的应用前景。Protein A (SpA) is a highly stable cell surface receptor produced by several S. aureus strains. It consists of a polypeptide chain with a molecular weight of 42 kDa containing four repeating domains rich in aspartic and glutamic acid 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 two 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 cell surface, as a carrier, and through the treatment steps of fixation and heat inactivation, it interacts with the giant enzyme molecules present in the serum, and is separated by centrifugation. Remove the interference of giant enzyme molecules on serum enzyme detection. The method is simple, easy to implement, low in cost, suitable for promotion and popularization in laboratories, and therefore has broad application prospects.

发明内容SUMMARY OF THE INVENTION

本发明提出了一种排除巨酶分子对血清酶浓度检测带来干扰的方法，解决了现有技术中提出的问题。The present invention provides a method for eliminating the interference caused by macroenzyme molecules to the detection of serum enzyme concentration, and solves the problems raised in the prior art.

为了解决上述技术问题，本发明提供了如下的技术方案：In order to solve the above-mentioned technical problems, the present invention provides the following technical solutions:

本发明提供了一种排除巨酶分子对血清酶浓度检测带来干扰的方法，所述排除巨酶分子对血清酶浓度检测带来干扰的具体步骤如下：The invention provides a method for eliminating the interference of giant enzyme molecules to the detection of serum enzyme concentration, and the specific steps for eliminating the interference of giant enzyme molecules to the detection of serum enzyme concentration are as follows:

步骤一：Cowan I细菌准备Step 1: Cowan I bacterial preparation

(A1)、为使Cowan I细菌菌体表面的SpA分子更为稳定，首先利用2％福尔马林溶液进行固定，固定时间和条件为室温固定2小时；(A1), in order to make the SpA molecule on the surface of Cowan I bacteria thalline more stable, at first utilize 2% formalin solution to carry out fixation, and fixation time and condition are fixed at room temperature for 2 hours;

(A2)、将固定好的金葡菌放置在80℃水浴中进行灭活，由于SpA蛋白对热非常稳定，不会干扰SpA的结合活性，同时，灭活后的细菌可以长期保存；(A2), place the fixed Staphylococcus aureus in an 80°C water bath for inactivation, since 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)、采用含有0.5％tween 20的PBS溶液进行洗涤，可以去除菌体表面非共价结合的SpA蛋白，提高反应的特异性。(A3) Washing with a PBS solution containing 0.5% tween 20 can remove the non-covalently bound SpA protein on the bacterial surface and improve the specificity of the reaction.

步骤二：血清吸附试验Step 2: Serum adsorption test

(B1)、抽取怀疑含有巨酶的患者空腹静脉血5ml，待血清自凝后离心，分离血清；(B1), extract 5ml of fasting venous blood from patients suspected of containing macroenzyme, and centrifuge after serum self-coagulation to separate serum;

(B2)、将步骤一中的Cowan I菌液混匀，与血清按照1：1的比例进行混匀，置于37℃摇床(150rpm)孵育3小时，使菌体表面的SpA蛋白与免疫球蛋白Fc段充分结合成免疫复合物；(B2), mixing the Cowan I bacterial liquid in step 1, mixing with serum according to a ratio of 1:1, and placing it on a 37° C. shaker (150 rpm) to incubate for 3 hours, so that the SpA protein on the surface of the bacterium and the immune system The globulin Fc segment is fully combined into an immune complex;

(B3)、通过高速离心的方式对免疫复合物进行分离，吸取上层分离血清进行酶浓度检测。(B3), the immune complexes are separated by high-speed centrifugation, and the upper layer separated serum is drawn to detect the enzyme concentration.

步骤三：吸附前后血清酶浓度检测和结果计算Step 3: Serum enzyme concentration detection and result calculation before and after adsorption

(C1)、通过标准方法检测血清吸附前后的酶浓度；(C1), detect the enzyme concentration before and after serum adsorption by standard method;

(C2)、吸附后酶浓度校正：在酶和免疫球蛋白检测时，将吸附后检测到的浓度水平乘以稀释倍数，得到的数值与原始浓度的比值，即为回收率，相应的吸附率(％)计算方式为：100％-回收率％。(C2) Correction of enzyme concentration after adsorption: When detecting enzyme and immunoglobulin, 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 (%) The calculation method is: 100%-recovery %.

(C3)、比较吸附前后血清酶的水平，以及回收率和吸附率，判断是否存在巨酶干扰现象；如吸附后酶水平处于正常参考区间，可以判断存在巨酶干扰，对检测报告进行结果更正。(C3), compare the levels of serum enzymes before and after adsorption, as well as the recovery rate and adsorption rate, to determine whether there is giant enzyme interference; if the enzyme level after adsorption is in the normal reference range, it can be judged that there is giant enzyme interference, and the test report is corrected. .

作为本发明的一种优选技术方案，所利用的Cowan I菌株是高表达膜表面SpA的金黄色葡萄球菌。As a preferred technical solution of the present invention, the Cowan I strain used is Staphylococcus aureus highly expressing SpA on the membrane surface.

作为本发明的一种优选技术方案，对Cowan I金黄色葡萄球菌进行处理时，首先利用2％福尔马林溶液先行细胞固定；然后经洗涤后用80℃的水浴5 分钟进行灭活。As a preferred technical solution of the present invention, when dealing with Cowan I Staphylococcus aureus, firstly use 2% formalin solution for cell fixation; then wash and inactivate in 80°C water bath for 5 minutes.

作为本发明的一种优选技术方案，本发明的方法适用于排除巨天冬氨酸酰基转移酶(Macro-AST)、巨肌酸激酶(Macro-CK)、巨淀粉酶(Macro-AMY) 和巨碱性磷酸酶(Macro-ALP)造成的干扰。As a preferred technical solution of the present invention, the method of the present invention is suitable for eliminating macroaspartate acyltransferase (Macro-AST), macrocreatine kinase (Macro-CK), macroamylase (Macro-AMY) and Interference caused by macroalkaline phosphatase (Macro-ALP).

本发明所达到的有益效果是：本发明的一种排除巨酶分子对血清酶浓度检测带来干扰的方法与现有技术相比，具有以下的有益效果：The beneficial effects achieved by the present invention are: a method for eliminating the interference of giant enzyme molecules to the detection of serum enzyme concentration of the present invention has the following beneficial effects compared with the prior art:

1、本发明的排除巨酶分子对血清酶浓度检测带来干扰的方法利用金黄色葡萄球菌表面的SpA与免疫球蛋白Fc段特异性结合的特性，可以去除“巨酶”对血清酶检测带来的干扰，对游离酶分子没有影响，同时，实验室只需要培养高表达SpA的Cowan I金葡菌，经过固定-洗涤-加热灭活几个步骤，可在4℃保存半年以上。1. The method for eliminating the interference of giant enzyme molecules to the detection of serum enzyme concentration of the present invention utilizes the characteristic of the specific binding of SpA on the surface of Staphylococcus aureus to the immunoglobulin Fc segment, and can remove the "giant enzyme" to the serum enzyme detection band. At the same time, the laboratory only needs to cultivate Cowan I Staphylococcus aureus with high SpA expression. After several steps of fixation-washing-heat inactivation, it can be stored at 4°C for more than half a year.

2、本发明的排除巨酶分子对血清酶浓度检测带来干扰的方法与凝胶电泳、超滤、SpA琼脂糖凝胶颗粒吸附等方法比较起来，具有简便易行、经济快速、特异性强等特点，非常适合在临床上进行推广。2. Compared with methods such as gel electrophoresis, ultrafiltration, SpA agarose gel particle adsorption, etc., the method for eliminating the interference caused by giant enzyme molecules to the detection of serum enzyme concentration has the advantages of simplicity, economy, rapidity and strong specificity. and other characteristics, it is very suitable for clinical promotion.

具体实施方式Detailed ways

以下对本发明的优选实施例进行说明，应当理解，此处所描述的优选实施例仅用于说明和解释本发明，并不用于限定本发明。The preferred embodiments of the present invention will be described below, and it should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, but not to limit the present invention.

实施例1Example 1

本发明提供了一种排除巨酶分子对血清酶浓度检测带来干扰的方法，利用CowanI金黄色葡萄球菌吸附血清中的“巨AST”，以排除其对血清游离AST检测的干扰，更为具体的步骤为：The invention provides a method for eliminating the interference caused by giant enzyme molecules to the detection of serum enzyme concentration, using CowanI Staphylococcus aureus to absorb "giant AST" in serum to eliminate its interference to the detection of serum free AST, more specifically The steps are:

步骤一：Cowan I细菌准备Step 1: Cowan I bacterial preparation

(A1)、将Cowan I接种于200ml LB液体培养基中，置于37℃摇床培养过夜；(A1), inoculate Cowan I in 200ml LB liquid culture medium, place 37 ℃ of shakers to cultivate overnight;

(A2)、第二天将菌液离心(5000rpm，10分钟)，收集细菌，PBS溶液洗涤2次；(A2), the next day the bacterial liquid was centrifuged (5000rpm, 10 minutes), the bacteria were collected, and the PBS solution was washed twice;

(A3)细菌固定：将洗涤后的Cowan I金葡菌加入到含有1.5％福尔马林溶液，混匀，室温下固定2小时；(A3) Bacterial fixation: The washed Cowan I Staphylococcus aureus was added to a solution containing 1.5% formalin, mixed, and fixed at room temperature for 2 hours;

(A4)洗涤：PBS溶液洗涤，以去除溶液中的福尔马林，然后利用PBS 配制成50％浓度的菌液；(A4) Washing: washing with PBS solution to remove formalin in the solution, and then using PBS to prepare a bacterial solution with a concentration of 50%;

(A5)灭活：将菌液倒入锥形瓶内(少于1.5cm高度)，置于80℃水浴，水平摇动5min；(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)、冰浴：将锥形瓶立即放置在冰上进行降温；(A6), ice bath: place the Erlenmeyer flask on ice immediately to cool down;

(A7)、洗涤：首先用含有0.5％tween 20的PBS溶液洗涤一次，再用不含tween 20的PBS洗涤；(A7), washing: first wash once with PBS solution containing 0.5% tween 20, and then wash with PBS without tween 20;

(A8)、保存：洗涤后的菌液保存于4℃冰箱，用前需摇匀。(可保存半年)(A8) Storage: The washed bacterial solution is stored in a refrigerator at 4°C, and needs to be shaken well before use. (can be stored for half a year)

步骤二：血清吸附试验Step 2: Serum adsorption test

(B1)、血清准备：抽取怀疑含有巨AST的患者空腹静脉血5ml，待血清自凝后离心，分离血清；(B1), Serum preparation: extract 5ml of fasting venous blood from patients suspected of containing giant AST, centrifuge the serum after self-coagulation, and separate the serum;

(B2)、将步骤一中的Cowan I菌液混匀，吸取500ul加入到EP管内，室温静置30分钟复温，然后加入患者血清500ul，混匀；(B2), the Cowan I bacterial liquid in the step 1 is mixed, draw 500ul and join in the EP tube, leave standstill at room temperature for 30 minutes to rewarm, then add patient serum 500ul, mix;

(B3)、置于37℃摇床(150rpm)孵育3小时；(B3), incubate at 37°C on a shaker (150rpm) for 3 hours;

(B4)、离心：将上述EP管置于离心机中离心10分钟，转速为10000rpm，小心吸取上层血清500ul，加入到新的EP管，待测Ig和AST浓度。(B4), centrifugation: the above-mentioned EP tube was placed in a centrifuge for 10 minutes, the rotating speed was 10000rpm, and 500ul of the upper serum was carefully drawn, and added to a new EP tube to measure the Ig and AST concentrations.

步骤三：血清AST和免疫球蛋白浓度检测：Step 3: Serum AST and immunoglobulin concentration detection:

(C1)、利用西门子BNProspec免疫比浊仪检测免疫球蛋白(IgM、IgG、 IgA)浓度；(C1), use Siemens BNProspec immunoturbidimeter to detect the concentration of immunoglobulin (IgM, IgG, IgA);

(C2)利用Roche cobas c701检测Cowan I吸附前后的AST浓度；(C2) utilize Roche cobas c701 to detect the AST concentration before and after Cowan I adsorption;

其中，检测试剂均为仪器配套试剂，按照标准操作规程进行。Among them, the detection reagents are all instrument supporting reagents, which are carried out in accordance with the standard operating procedures.

步骤四：结果计算Step 4: Result calculation

(D1)、吸附后酶浓度校正：在酶和免疫球蛋白检测时，因血清与Cowan I菌液按1：1进行混合，故将吸附后检测到的浓度水平乘以稀释倍数(此处为2)，得到的数值与原始浓度的比值，即为回收率，相应的吸附率计算方式为：100％-回收率％。(D1) Correction of enzyme concentration after adsorption: in the detection of enzyme and immunoglobulin, since 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)、结果：(D2), result:

表1.Cowan I吸附前后血清免疫球蛋白和AST回收率Table 1. Recovery of serum immunoglobulin and AST before and after Cowan I adsorption

其中，Ig浓度单位为g/L，AST浓度单位为U/L。Among them, the unit of Ig concentration is g/L, and the unit of AST concentration is U/L.

由表1结果显示：Cowan I对IgG吸附作用最强，巨AST和对照组吸附率分别为34.48％和41.92％，IgM吸附率低于IgG，分别为17.74％和12.7％。巨AST血清原始AST为109U/L，吸附后的换算浓度为22.2％，吸附率为79.63％，而对于对照血清，吸附前后的AST值分别为161U/L和156.8U/L，吸附率仅为2.61％。由此可见，巨AST组存在巨酶干扰现象，导致血清中AST的实测值明显增高，考虑到Cowan I细菌吸附免疫球蛋白并不能达到100％，实际血清中的AST浓度应小于22.2U/L，可按<22.2U/L进行结果报告，介于正常参考区间。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 rate of IgM is lower than that of IgG, which are 17.74% and 12.7%, respectively. The original AST of giant AST serum was 109 U/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 161 U/L and 156.8 U/L, respectively, and the adsorption rate was only 2.61%. It can be seen that in the giant AST group, there is giant enzyme interference, which leads to a significant increase in the measured value of AST in serum. Considering that Cowan I bacteria cannot adsorb immunoglobulin to 100%, the actual concentration of AST in serum should be less than 22.2U/L , the results can be reported at <22.2U/L, which is within the normal reference range.

本发明的排除巨酶分子对血清酶浓度检测带来干扰的方法利用金黄色葡萄球菌表面的SpA与免疫球蛋白Fc段特异性结合的特性，可以去除“巨酶”对血清酶检测带来的干扰，对游离酶分子没有影响，同时，实验室只需要培养高表达SpA的Cowan I金葡菌，经过固定-洗涤-加热灭活几个步骤，可在 4℃保存半年以上；与凝胶电泳、超滤、SpA琼脂糖凝胶颗粒吸附等方法比较起来，具有简便易行、经济快速、特异性强等特点，非常适合在临床上进行推广。The method for eliminating the interference caused by giant enzyme molecules to the detection of serum enzyme concentration of the present invention utilizes the characteristic of the specific binding of SpA on the surface of Staphylococcus aureus to the Fc segment of immunoglobulin, and can remove the interference caused by "macroenzyme" to serum enzyme detection. It 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-heat inactivation, it can be stored at 4°C for more than half a year; with gel electrophoresis Compared with other methods such as , ultrafiltration, SpA agarose particle adsorption, etc., it has the characteristics of simplicity, economy, rapidity, and strong specificity, and is very suitable for clinical promotion.

最后应说明的是：以上所述仅为本发明的优选实施例而已，并不用于限制本发明，尽管参照前述实施例对本发明进行了详细的说明，对于本领域的技术人员来说，其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。Finally, it should be noted that the above descriptions are only preferred embodiments of the present invention, and are 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, the The technical solutions described in the foregoing embodiments may be modified, or some technical features thereof may be equivalently replaced. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within 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).