A method for measuring magnetic loss rate of magnetic beads

Abstract

The present invention relates to the technical field of magnetic microparticle chemiluminescence immunoassay, and specifically discloses a method for determining the magnetic loss rate of magnetic beads. The determination method comprises the following steps: constructing a standard solution with a magnetic bead concentration gradient; determining the optical density of the standard solution at each magnetic bead concentration, and drawing a magnetic bead concentration-optical density standard curve; performing magnetic separation and washing on the sample to be tested, and determining the optical density value of the sample to be tested; substituting the optical density value of the sample to be tested into the magnetic bead concentration-optical density standard curve to obtain the magnetic bead concentration of the sample to be tested, and calculating the magnetic bead amount of the sample to be tested from the magnetic bead concentration of the sample to be tested; comparing the magnetic bead amount of the sample to be tested with the total amount of magnetic beads added to the sample to be tested before magnetic separation and washing to obtain the magnetic loss rate; wherein the type of magnetic beads contained in the sample to be tested is the same as the type of magnetic beads in the magnetic bead concentration-optical density standard curve. The determination method of the present invention has the characteristics of simple testing, high efficiency and low cost.

Description

[Technical field]

[0001] The invention relates to the technical field of magnetic particle chemiluminescence immunoassay, and in particular to a method for determining magnetic bead magnetic loss rate. [Background technology]

[0002] In the field of chemiluminescence immunoassay (CLIA), magnetic particle chemiluminescence immunoassay is widely used in trace or ultra-trace non-isotope immunoassay in veterinary, medical and food analysis and scientific research due to its high sensitivity and high specificity. Magnetic particles (or magnetic beads) are the core reaction elements in magnetic particle chemiluminescence immunoassay, which are used to coat antigens or antibodies. A fixed amount of magnetic particles or magnetic beads is required in each test reaction, and the usual dosage is 5μg / test to 50μg / test.

[0003] During the magnetic particle chemiluminescence immunoassay process, at least one magnetic particle separation and cleaning is required to remove excess reactants or impurities. However, magnetic particles are often lost during the cleaning process, and the loss of magnetic particles will lead to a decrease in effective reaction, resulting in unreliable test results. The prior art uses a flow cytometer or a particle counter to detect the magnetic loss rate (the percentage of magnetic particle loss before and after the reaction) during each magnetic particle chemiluminescence immunoassay process, and the determination of the magnetic loss rate is used to assist in determining the reliability of the test results. However, both flow cytometer detection and particle counter detection have the following defects:

[0004] (1) The detection process is cumbersome and inefficient. Specifically, on the one hand, the flow cytometer or particle counter has a low throughput and low automation, and can only measure one sample at a time, and the magnetic beads need to be counted one by one; on the other hand, the magnetic particles need to be diluted to avoid aggregation of magnetic particles during direct detection on the machine, which leads to a decrease in measurement accuracy.

[0005] (2) High cost. Flow cytometers or particle counters are expensive, and the investment in equipment will make the detection cost higher. [Summary of the invention]

[0006] The purpose of the present invention is to provide a method for determining the magnetic loss rate of magnetic beads, so as to solve the problems of the existing detection methods being complicated, inefficient and costly.

[0007] In order to achieve the above technical goals, the following technical solutions are adopted:

[0008] A method for measuring magnetic bead magnetic loss rate comprises the following steps:

[0009] Constructing a standard solution with a magnetic bead concentration gradient;

[0010] Determine the optical density of the standard solution at each magnetic bead concentration;

[0011] According to the magnetic bead concentration and the optical density, a magnetic bead concentration-optical density standard curve is drawn;

[0012] Performing magnetic separation and washing on the sample to be tested, and measuring the optical density of the sample to be tested after the magnetic separation and washing;

[0013] Substituting the optical density of the sample to be tested into the magnetic bead concentration-optical density standard curve to obtain the magnetic bead concentration of the sample to be tested;

[0014] Calculating the amount of magnetic beads in the sample to be tested based on the magnetic bead concentration of the sample to be tested;

[0015] Comparing the amount of magnetic beads in the sample to be tested with the total amount of magnetic beads added to the sample to be tested before magnetic separation and washing to obtain a magnetic loss rate;

[0016] The type of magnetic beads contained in the sample to be tested is the same as the type of magnetic beads in the magnetic bead concentration-optical density standard curve.

[0017] Preferably, the magnetic separation washing comprises placing the sample to be tested in a washing solution for washing, wherein the washing solution contains phosphate buffer, sodium chloride and Tween 20;

[0018] Alternatively, the washing solution contains phosphate buffer, sodium chloride, Tween 20 and polyethylene glycol octylphenyl ether.

[0019] Preferably, when the washing solution contains phosphate buffer, sodium chloride and Tween 20, the concentration of the phosphate buffer is 10mM to 20mM, the mass fraction of the sodium chloride is 0.8% to 1.0%, the mass fraction of the Tween 20 is 0.04% to 0.08%, and the pH value of the washing solution is 7.1 to 7.3;

[0020] When the washing solution contains phosphate buffer, sodium chloride, Tween 20 and polyethylene glycol octyl phenyl ether, the concentration of the phosphate buffer is 10mM to 20mM, the mass fraction of the sodium chloride is 0.8% to 1.0%, the mass fraction of the Tween 20 is 0.04% to 0.08%, the mass fraction of the polyethylene glycol octyl phenyl ether is 0.08% to 0.12%, and the pH value of the washing solution is 7.1 to 7.3.

[0021] Preferably, the magnetic separation washing is performed 1 to 3 times;

[0022] And / or, the duration of each magnetic separation washing is 60s to 120s.

[0023] Preferably, the step of determining the optical density of the standard solution comprises:

[0024] The standard solution at each magnetic bead concentration was transferred to an ELISA plate, and the optical density of the standard solution at each magnetic bead concentration was measured using an ELISA reader.

[0025] Preferably, the standard solution of each magnetic bead concentration is transferred to at least three reaction wells of the ELISA plate, and the optical density of the standard solution in each reaction well is measured using a microplate reader, and the average value is taken.

[0026] Preferably, the step of determining the optical density of the sample to be tested comprises:

[0027] The sample to be tested after the magnetic separation and washing is transferred to an ELISA plate, and the optical density of the sample to be tested is measured using an ELISA reader.

[0028] Preferably, the sample to be tested is transferred to at least three reaction wells of the ELISA plate, and the optical density of the sample to be tested at the corresponding magnetic bead concentration in each reaction well is measured using a microplate reader, and the average value is taken.

[0029] Preferably, the step of comparing the amount of magnetic beads in the sample to be tested with the total amount of magnetic beads added to the sample to be tested before magnetic separation and washing to obtain the magnetic loss rate comprises:

[0030] Calculating the amount of magnetic beads in the sample to be tested according to the magnetic bead concentration of the sample to be tested and the volume of the sample to be tested;

[0031] Calculating the difference between the amount of magnetic beads in the sample to be tested and the total amount of magnetic beads added to the sample to be tested before magnetic separation and washing;

[0032] Calculating the difference and the percentage of the total amount of magnetic beads added to the sample to be tested before magnetic separation and washing to obtain the magnetic loss rate;

[0033] And / or, after the magnetic separation and washing of the sample to be tested and before the step of determining the optical density value of the sample to be tested, the method further comprises:

[0034] The sample to be tested after magnetic separation and washing is suspended, and then the sample to be tested is transferred to an ELISA plate, and the optical density of the sample to be tested is measured using an ELISA instrument.

[0035] Preferably, the same type of magnetic beads includes the same particle size of the magnetic beads, the material constituting the magnetic beads, and the type of functional groups carried on the surface of the magnetic beads.

[0036] Compared with the prior art, the method for determining the magnetic loss rate of magnetic beads provided in the embodiment of the present invention only needs to draw a magnetic bead concentration-optical density standard curve once for the magnetic loss rate of the same type of magnetic beads, so as to quickly obtain the magnetic loss rate of the sample to be tested whose magnetic bead type is the same as the magnetic bead type of the magnetic bead concentration-optical density standard curve. The present determination method can be used to obtain batches of magnetic bead magnetic loss rate data, effectively simplifying the detection process of the magnetic bead magnetic loss rate, thereby greatly improving the detection efficiency and detection flux of the magnetic bead magnetic loss rate, and the magnetic loss rate is small. At the same time, the detection accuracy of the magnetic bead magnetic loss rate is no longer affected by the concentration of the sample to be tested, and the reliability of the magnetic bead magnetic loss rate accuracy is effectively improved, thereby effectively assisting in judging the reliability of the magnetic microparticle chemiluminescence immunoassay detection results; in addition, the equipment investment cost for the detection of the magnetic bead magnetic loss rate is also effectively reduced.

Brief Description of the Drawings

[0037] Figure 1 A simplified schematic diagram of a method for determining magnetic bead magnetic loss rate provided in an embodiment of the present invention;

[0038] Figure 2 The method for determining the magnetic loss rate of magnetic beads provided in Example 1 of the present invention is used to obtain a standard curve of magnetic bead concentration-optical density;

[0039] Figure 3 The method for determining the magnetic loss rate of magnetic beads provided in Example 7 of the present invention obtains a standard curve of magnetic bead concentration-optical density. [Specific implementation method]

[0040] The present invention is further described below in conjunction with the accompanying drawings and implementation methods. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0041] In addition, the descriptions of "first", "second", etc. in the present invention are only used for descriptive purposes and cannot be understood as indicating or implying their relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In addition, the technical solutions between the various embodiments can be combined with each other, but they must be based on the ability of ordinary technicians in the field to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be deemed that such a combination of technical solutions does not exist and is not within the scope of protection required by the present invention.

[0042] See also Figure 1 The embodiment of the present invention provides a method for determining the magnetic loss rate of a magnetic bead, which specifically includes the following steps:

[0043] (1) Construct a standard solution with a magnetic bead concentration gradient.

[0044] In step (1), multiple groups of standard solutions are constructed, and each standard solution has a magnetic bead concentration gradient. For example, in some embodiments, the standard solution is constructed according to a change rule in which the magnetic bead concentration decreases or increases by 0.01 mg / mL to 0.1 mg / mL. By setting a standard solution with a magnetic bead concentration gradient, the accuracy of the change in the magnetic bead concentration in the standard solution can be improved.

[0045] (2) Determine the optical density of the standard solution at each magnetic bead concentration.

[0046] In step (2), the standard solution under every kind of magnetic bead concentration is transferred to an ELISA plate, and an ELISA reader is used to measure the optical density of the standard solution under every kind of magnetic bead concentration. By measuring the optical density of the standard solution under every kind of magnetic bead concentration, a standard curve of magnetic bead concentration and optical density relationship can be obtained. In some embodiments, the standard solution under every kind of magnetic bead concentration is transferred to at least three reaction wells of the ELISA plate, and an ELISA reader is used to measure the optical density of the standard solution in each reaction well, and averages the values. By repeatedly testing the optical density of the standard solution under every kind of magnetic bead concentration, and calculating to obtain average values, the reliability of the standard curve of magnetic bead concentration and optical density relationship can be effectively improved.

[0047] (3) Draw a magnetic bead concentration-optical density standard curve based on the magnetic bead concentration of each standard solution and the corresponding optical density.

[0048] In step (3), a linear fit is performed on the relationship between the magnetic bead concentration and the optical density to obtain a magnetic bead concentration-optical density standard curve.

[0049] (4) Performing magnetic separation and washing on the sample to be tested, and measuring the optical density of the sample to be tested after the magnetic separation and washing.

[0050] In step (4), the sample to be tested refers to a sample containing magnetic beads in its ingredients, and the type of magnetic beads contained in the sample to be tested is the same as the type of magnetic beads in the standard solution corresponding to the magnetic bead concentration-optical density standard curve in step (3). In some embodiments, the surface of the magnetic beads contained in the sample to be tested carries any functional group of amino, carboxyl, thiol, oxirane, toluenesulfonyl, chloromethyl, hydroxyl, streptavidin, protein A (Protein A), and N-succinimidyl ester. In some embodiments, an antigen or antibody is connected to the surface of the magnetic beads, and the antigen or antibody is connected to the magnetic beads through a functional group (i.e., the antigen or antibody is coated). Preferably, the antigen includes any one of cytomegalovirus (CMV) antigen, Treponema pallidum (TP) antigen, HIV (HIV) antigen, hepatitis C virus (HCV) antigen, etc. Preferably, the antibody includes any one of alpha-fetoprotein (AFP) antibody, follicle stimulating hormone (FSH) antibody, luteinizing hormone (LH) antibody, thyroid stimulating hormone (TSH) antibody, procalcitonin (PCT) antibody, carcinoembryonic antigen (CEA) antibody, etc. It is understandable that the specific category of antigen or antibody is not limited in the embodiments of the present invention. In some embodiments, the sample to be tested also contains other active substances and / or inactive substances, biological samples, and antigens, antibodies, etc. that are not connected to the surface of the magnetic beads.

[0051] It should be noted that: in the present invention, the type of magnetic beads of the sample to be tested is the same as the type of magnetic beads used to construct the standard solution in the magnetic bead concentration-optical density standard curve, which means that the particle size of the magnetic beads, the magnetic material constituting the magnetic beads, the functional groups carried by the magnetic beads, and the magnetic bead particle size, the magnetic material constituting the magnetic beads, and the functional groups carried by the magnetic beads used to construct the standard solution in the magnetic bead concentration-optical density standard curve are the same. Under the premise that the magnetic bead particle size and the magnetic bead material are the same, when the surface of the magnetic beads of the sample to be tested carries amino groups, the magnetic The surface of the magnetic beads used to construct the standard solution in the standard curve of bead concentration-optical density carries amino groups; when the surface of the magnetic beads of the sample to be tested carries hydroxyl groups, the surface of the magnetic beads used to construct the standard solution in the standard curve of magnetic bead concentration-optical density carries hydroxyl groups; when the surface of the magnetic beads of the sample to be tested carries carboxyl groups, the surface of the magnetic beads used to construct the standard solution in the standard curve of magnetic bead concentration-optical density carries carboxyl groups; when the surface of the magnetic beads of the sample to be tested carries thiol groups, the surface of the magnetic beads used to construct the standard solution in the standard curve of magnetic bead concentration-optical density carries thiol groups; etc. The same type of magnetic beads is not limited to the types of antigens and antibodies connected to the surface of the magnetic beads.

[0052] In some embodiments, the sample to be tested is placed in a washing solution for magnetic separation and washing. In some embodiments, the washing solution contains phosphate buffer (PB), sodium chloride (NaCl) and Tween-20, and the concentration of the phosphate buffer is 10mM to 20mM, the mass fraction of the sodium chloride is 0.8% to 1.0%, and the mass fraction of the Tween 20 is 0.04% to 0.08%; the pH value of the washing solution is 7.1 to 7.3. In some preferred embodiments, the washing solution contains phosphate buffer, sodium chloride and Tween 20, and the concentration of the phosphate buffer is 10mM, the mass fraction of the sodium chloride is 0.9%, and the mass fraction of the Tween 20 is 0.05%; the pH value of the washing solution is 7.2.

[0053] In other embodiments, the washing solution contains phosphate buffer, sodium chloride, Tween 20 and polyethylene glycol octyl phenyl ether (TritonX-100); and the concentration of the phosphate buffer is 10mM to 20mM, the mass fraction of the sodium chloride is 0.8% to 1.0%, the mass fraction of the Tween 20 is 0.04% to 0.08%, the mass fraction of the polyethylene glycol octyl phenyl ether is 0.08% to 0.12%, and the pH value of the washing solution is 7.1 to 7.3. In some preferred embodiments, the washing solution contains phosphate buffer, sodium chloride, Tween 20 and polyethylene glycol octyl phenyl ether; and the concentration of the phosphate buffer is 10mM, the mass fraction of the sodium chloride is 0.9%, the mass fraction of the Tween 20 is 0.05%, the mass fraction of the polyethylene glycol octyl phenyl ether is 0.1%, and the pH value of the washing solution is 7.2.

[0054] In some embodiments, based on any of the above-mentioned washing solutions, the number of magnetic separation washings is 1 to 6 times, and the time of each magnetic separation washing is 30s to 120s. By limiting the number of magnetic separation washings and the time of magnetic separation washings, the reliability of the magnetic loss rate detection results can be effectively guaranteed. If the number of magnetic separation washings is too many, such as more than 6 times, the magnetic loss amount in the magnetic separation washing process is increased, so that the reliability of the magnetic loss rate detection results decreases; similarly, if the time of each magnetic separation washing is too short, less than 30s, the reliability of the magnetic loss rate detection results also decreases. In some preferred embodiments, the number of magnetic separation washings is 1 to 3 times, and the time of each magnetic separation washing is 60s to 120s.

[0055] By using any of the above-mentioned washing solutions for magnetic separation washing, the magnetic loss during the magnetic separation washing process can be effectively reduced, thereby improving the reliability of the magnetic loss rate detection result. It can be understood that the method for determining the magnetic loss rate of magnetic beads in the embodiment of the present invention is applicable to a variety of chemiluminescence analysis processes, including but not limited to direct chemiluminescence, enzymatic luminescence, electrochemiluminescence, etc. The method for determining the magnetic loss rate of magnetic beads in this embodiment can be applied to all reactions requiring magnetic beads.

[0056] Of course, it is understandable that the magnetic separation washing of the sample to be tested can also be set to multiple parallel groups, such as two groups, three groups, five groups, ten groups, etc., to improve the accuracy of the detection. In some embodiments, the step of the magnetic separation washing can be performed manually, or it can be implemented semi-automatically or fully automatically using some instruments, for example, using a semi-automatic or fully automatic chemiluminescence immunoassay analyzer. It is understandable that the steps of sample injection, reaction, and magnetic separation washing of the sample to be tested in a fully automatic instrument can be implemented by relying on the software of the fully automatic instrument.

[0057] In some embodiments, the sample to be tested after magnetic separation and washing is suspended before being transferred to the ELISA plate to facilitate the determination of optical density. In some embodiments, the sample to be tested after magnetic separation and washing is placed in a phosphate buffer with a concentration of 0.15mM to 0.25mM and a pH value of 7.2. By placing the sample to be tested in a phosphate buffer, the magnetic beads in the sample to be tested can be suspended in the phosphate buffer, and when the amount of magnetic beads is calculated later, the volume of the suspension formed by the sample to be tested and the phosphate buffer is directly used for calculation.

[0058] In some embodiments, the sample to be tested after magnetic separation and washing is transferred to an ELISA plate, and an ELISA reader is used to measure the optical density of the sample to be tested. In some embodiments, the sample to be tested is transferred to at least three reaction wells of the ELISA plate, and an ELISA reader is used to measure the optical density of the sample to be tested at the corresponding magnetic bead concentration in each reaction well, and the average value is taken as the optical density of the sample to be tested to calculate the amount of magnetic beads in the sample to be tested.

[0059] It should be noted that there is no order of precedence between step (4) and steps (1) to (3), and the serial numbers do not have any restrictive implications on the order of precedence. Step (4) can be performed while steps (1) to (3) are being performed, or step (4) can be performed first and then steps (1) to (3) are performed.

[0060] (5) Substituting the optical density value of the sample to be tested into the magnetic bead concentration-optical density standard curve to obtain the magnetic bead concentration of the sample to be tested.

[0061] When calculating the magnetic bead concentration of the sample to be tested, auxiliary computing equipment, such as a computer, etc., can be used to obtain the magnetic bead concentration of the sample to be tested, which is conducive to further improving the detection efficiency.

[0062] (6) Calculate the amount of magnetic beads in the sample to be tested based on the magnetic bead concentration of the sample to be tested.

[0063] In step (6), the amount of magnetic beads (m1) in the sample to be tested is calculated based on the magnetic bead concentration of the sample to be tested and the volume of the sample to be tested.

[0064] It should be noted that in step (6) of the embodiment of the present invention, the amount of magnetic beads obtained is the mass of the magnetic beads rather than the number of the magnetic beads.

[0065] (7) Compare the amount of magnetic beads in the sample to be tested with the total amount of magnetic beads added before magnetic separation and washing (M0) (i.e., the total amount of magnetic beads added for the fixation reaction of the immunobiologically active substances) to obtain the magnetic loss rate, that is, the calculation formula of the magnetic loss rate is:

[0066]

[0067] The method for determining the magnetic loss rate of magnetic beads provided in the embodiment of the present invention is different from the flow cytometer and particle counter that counts the number of magnetic beads one by one. Instead, it uses the magnetic bead concentration-optical density standard curve and the washing solution, the number of magnetic separation washings and the magnetic separation washing time to directly obtain the magnetic bead magnetic loss rate quickly and accurately based on the mass change before and after the magnetic separation washing of the magnetic beads. Such a determination method makes the determination of the magnetic loss rate of the same magnetic bead not limited by the antigen or antibody connected to the surface. More importantly, when determining the magnetic loss rate of the same magnetic bead, the same magnetic bead concentration-optical density standard curve can be used. Therefore, the method for determining the magnetic loss rate of magnetic beads provided in the embodiment of the present invention can construct a magnetic bead concentration-optical density standard curve database of various magnetic beads through a computer and other equipment. Each time the magnetic loss rate of the sample to be tested is determined, after obtaining the optical density of the sample to be tested, the corresponding magnetic bead concentration is obtained, and the magnetic bead concentration of the sample to be tested can be obtained by directly calling the magnetic bead concentration-optical density standard curve of the same magnetic bead type as the sample to be tested, and the magnetic bead amount can be obtained according to the magnetic bead concentration and the volume of the sample to be tested, thereby realizing high-throughput detection, which not only effectively improves the detection efficiency of the magnetic loss rate but also effectively reduces the magnetic loss rate of magnetic separation and washing, thereby effectively assisting in judging the reliability of the detection results of the magnetic microparticle chemiluminescence immunoassay.

[0068] In order to better illustrate the technical solution of the implementation scheme of the present invention, further explanation is given below through multiple examples.

[0069] Embodiment 1

[0070] A method for measuring magnetic bead magnetic loss rate comprises the following steps:

[0071] (11) Prepare a standard solution with a magnetic bead concentration gradient using Thermo carboxyl magnetic beads. Specifically, 2.8 μM Thermo carboxyl magnetic beads were diluted with 0.2 mM PB (pH 7.2) and then prepared into 3 mg / mL, 0.285 mg / mL, 0.27 mg / mL, 0.24 mg / mL, 0.21 mg / mL, 0.18 mg / mL and 0.15 mg / mL standard solutions.

[0072] (12) The standard solutions of step (11) were added to the ELISA plate respectively, and the optical density (i.e., OD800) of the standard solutions at each magnetic bead concentration was determined using an ELISA instrument. The standard solutions at each magnetic bead concentration were added to three wells of the ELISA plate, and the optical density of the standard solutions in the three wells was tested, and the average value was taken (i.e., the test was repeated in 3 replicate wells). The results are shown in Table 1.

[0073] Table 1

[0074] Magnetic bead concentration (mg / mL) 0 0.3 0.285 0.27 0.24 0.21 0.18 0.15 Optical density 0.005 0.2164 0.2077 0.1991 0.1839 0.1623 0.145 0.119

[0075] According to the data in Table 1, a scatter plot of magnetic bead concentration-optical density was drawn, and a linear fit was performed to obtain a standard curve of magnetic bead concentration-optical density. The results are shown in Figure 2 shown.

[0076] from Figure 2 It can be seen that the R of the fitted magnetic bead concentration-optical density standard curve is 2 =0.9947, approximately equal to 1, indicating that the relationship between magnetic bead concentration and optical density has a high linear fit, and the magnetic bead concentration and optical density show a linear correlation equation relationship, namely:

[0077] y=0.7051x+0.0107……(b);

[0078] Where x represents the concentration of magnetic beads and y represents the optical density.

[0079] (13) Carboxyl magnetic beads coated with CEA antigen were used as test samples, and the test samples were divided into 12 groups, which were magnetically separated and washed in a fully automated tube chemiluminescence instrument. The washing solution contained 10 mM phosphate buffer, 0.9% sodium chloride by mass, 0.05% Tween 20 by mass, and 0.1% polyethylene glycol octylphenyl ether by mass, and the pH value of the washing solution was 7.2.

[0080] Subsequently, each group of test samples after washing was recovered, and then the recovered test samples were resuspended in 0.2 mM PB buffer (pH = 7.2). After resuspension, they were transferred to ELISA plates and OD800 was measured using an ELISA instrument. The average value was taken and substituted into equations (b) and (a) for calculation, and the results were recorded in Table 2; flow cytometer and particle counter were used as controls, wherein the control of the flow cytometer was to directly dilute the test samples and 0.3 mg / mL magnetic bead standard by 100 times, load them respectively, read the counts, calculate the magnetic loss rate, and record the results in Table 2; the control of the particle counter was to directly dilute the test samples and 0.3 mg / mL magnetic bead standard by 100 times, load them respectively, read the counts, calculate the magnetic loss rate, and record the results in Table 2.

[0081] Table 2

[0082]

[0083] It can be seen from Table 2 that: under the same number of magnetic separations, the longer the magnetic separation time, the smaller the magnetic loss rate. If the magnetic separation time is less than 30s, a larger magnetic loss rate will appear, resulting in a reduced number of times the magnetic beads are used. At the same time, using the method for determining the magnetic loss rate of magnetic beads in this embodiment, the magnetic loss rate is close to the test results of the flow cytometer and the particle counter. It can be seen that this embodiment has the advantages of high efficiency and low cost compared to the flow cytometer or the particle counter. It can be foreseen that the determination method is close to the results tested on different detectors, indicating that the method has high detection accuracy. Moreover, under the same magnetic separation time, the number of magnetic separations increases, and the magnetic loss rate increases, that is, an increase in the number of magnetic separations will lead to an increase in the amount of magnetic bead loss.

[0084] Embodiment 2

[0085] A method for measuring magnetic bead magnetic loss rate comprises the following steps:

[0086] (21) In this embodiment, the magnetic bead concentration-optical density standard curve in Example 1 is used as the standard curve.

[0087] (22) Carboxyl magnetic beads coated with CEA antigen were used as test samples. The test samples were divided into 4 groups and magnetically separated and washed using different washing solutions in a fully automated tube chemiluminescence instrument.

[0088] Subsequently, each group of test samples after washing was recovered, and then resuspended in 0.2mM PB buffer (pH=7.2), and after resuspension, they were transferred to ELISA plates to measure OD800, and were substituted into equations (b) and (a) for calculation, and the results were recorded in Table 3; flow cytometer and particle counter were used as controls, wherein the control of flow cytometer was to directly dilute the test samples and 0.3mg / mL magnetic bead standard by 100 times, load them respectively, read the counts, calculate the magnetic loss rate, and record the results in Table 3; the control of particle counter was to directly dilute the test samples and 0.3mg / mL magnetic bead standard by 100 times, load them respectively, read the counts, calculate the magnetic loss rate, and record the results in Table 3.

[0089] Table 3

[0090]

[0091] In Table 3:

[0092] The composition of wash solution 1: 10 mM PB, pH = 7.2;

[0093] The composition of washing solution 2: 10 mM PB and 0.9% NaCl by mass, pH = 7.2;

[0094] The composition of washing solution 3-1: 10 mM PB, 0.9% by mass NaCl and 0.05% by mass Tween-20, pH = 7.2;

[0095] The composition of the washing solution 4-1: 10 mM PB, 0.9% by mass NaCl, 0.05% by mass Tween-20 and 0.1% by mass TritonX-100, pH=7.2.

[0096] It can be seen from Table 3 that when washing solutions 3-1 and 4-1 are used for magnetic separation washing, the magnetic loss rate of the sample to be tested is relatively small, that is, washing solutions 3-1 and 4-1 can effectively reduce the loss of magnetic beads in the sample to be tested during the magnetic separation washing process. Since the loss of magnetic beads is reduced each time the magnetic separation washing is performed, it is beneficial to increase the number of times the magnetic beads can be used.

[0097] Embodiment 3

[0098] A method for measuring magnetic bead magnetic loss rate comprises the following steps:

[0099] (31) In this embodiment, the magnetic bead concentration-optical density standard curve in Example 1 is used as the standard curve.

[0100] (32) Carboxyl magnetic beads coated with CEA antigen were used as test samples. The test samples were divided into 4 groups and magnetically separated and washed using different washing solutions in a fully automated tube chemiluminescence instrument.

[0101] Subsequently, each group of test samples after washing was recovered, and then resuspended in 0.2mM PB buffer (pH=7.2), and after resuspension, they were transferred to ELISA plates to measure OD800, and were substituted into equations (b) and (a) for calculation, and the results were recorded in Table 4; flow cytometer and particle counter were used as controls, wherein the control of flow cytometer was to directly dilute the test samples and 0.3mg / mL magnetic bead standard by 100 times, load them respectively, read the counts, calculate the magnetic loss rate, and record the results in Table 4; the control of particle counter was to directly dilute the test samples and 0.3mg / mL magnetic bead standard by 100 times, load them respectively, read the counts, calculate the magnetic loss rate, and record the results in Table 4.

[0102] Table 4

[0103]

[0104] In Table 4:

[0105] The composition of washing solution 3-2: 20 mM PB, 0.8% by mass NaCl and 0.08% by mass Tween-20, pH = 7.2;

[0106] The composition of washing solution 4-2: 15 mM PB, 1.0% by mass NaCl, 0.04% by mass Tween-20 and 0.12% by mass TritonX-100, pH=7.1.

[0107] It can be seen from Table 4 that when washing solutions 3-2 and 4-2 are used for magnetic separation washing, the magnetic loss rate of the sample to be tested is relatively small, that is, washing solutions 3-2 and 4-2 can effectively reduce the loss of magnetic beads in the sample to be tested during the magnetic separation washing process.

[0108] Embodiment 4

[0109] A method for measuring magnetic bead magnetic loss rate comprises the following steps:

[0110] (41) In this embodiment, the magnetic bead concentration-optical density standard curve in Example 1 is used as the standard curve.

[0111] (42) Carboxyl magnetic beads coated with CEA antigen were used as test samples. The test samples were divided into 4 groups and magnetically separated and washed using different washing solutions in a fully automated tube chemiluminescence instrument.

[0112] Subsequently, each group of test samples after washing was recovered, and then resuspended in 0.2mM PB buffer (pH=7.2). After resuspension, they were transferred to ELISA plates to measure OD800, and were substituted into equations (b) and (a) for calculation, and the results were recorded in Table 5; flow cytometer and particle counter were used as controls, wherein the control of flow cytometer was to directly dilute the test samples and 0.3mg / mL magnetic bead standard by 100 times, load them respectively, read the counts, calculate the magnetic loss rate, and record the results in Table 5; the control of particle counter was to directly dilute the test samples and 0.3mg / mL magnetic bead standard by 100 times, load them respectively, read the counts, calculate the magnetic loss rate, and record the results in Table 5.

[0113] Table 5

[0114]

[0115] In Table 5:

[0116] The composition of washing solution 3-3: 15 mM PB, 1.0% by mass NaCl and 0.06% by mass Tween-20, pH = 7.2;

[0117] The composition of washing solution 4-3: 20 mM PB, 0.8% by mass NaCl, 0.08% by mass Tween-20 and 0.08% by mass TritonX-100, pH=7.3.

[0118] It can be seen from Table 5 that when washing solutions 3-3 and 4-3 are used for magnetic separation and washing, the magnetic loss rate of the sample to be tested is relatively small, and when washing solutions 3-3 and 4-3 are used for magnetic loss rate tests of flow cytometer and particle counter respectively, the results are close to the magnetic loss rate obtained by the measurement method of this embodiment, that is, washing solutions 3-3 and 4-3 can also effectively reduce the loss of magnetic beads in the sample to be tested during the magnetic separation and washing process.

[0119] Embodiment 5

[0120] A method for measuring magnetic bead magnetic loss rate comprises the following steps:

[0121] (51) In this embodiment, the magnetic bead concentration-optical density standard curve in Example 1 is used as the annotation curve.

[0122] (52) Prepare the following six samples to be tested:

[0123] Among them, the components of the sample 1 to be tested: AFP antibody coated carboxyl magnetic beads (Thermo, magnetic bead particle size is 2.8 μM);

[0124] Components of the test sample 2: FSH antibody-coated carboxyl magnetic beads (Thermo, magnetic bead particle size 2.8 μM);

[0125] Components of the test sample 3: LH antibody-coated carboxyl magnetic beads (Thermo, magnetic bead particle size 2.8 μM);

[0126] Components of the test sample 4: TSH antibody-coated carboxyl magnetic beads (Thermo, magnetic bead particle size 2.8 μM);

[0127] Composition of the test sample 5: CMV virus antigen coated carboxyl magnetic beads (Thermo, magnetic bead particle size 2.8 μM);

[0128] Components of the test sample 6: TP antigen coated carboxyl magnetic beads (Thermo, magnetic bead particle size is 2.8 μM).

[0129] The washing solution 4-1 in Example 2 was used as the washing solution, and magnetic separation and washing were performed in a fully automated tubular chemiluminescence instrument.

[0130] After washing, each sample to be tested was recovered, and then resuspended in 0.2 mM PB buffer (pH = 7.2), and then transferred to an ELISA plate to measure OD800, and then substituted into equations (b) and (a) for calculation, and the results were recorded in Table 6. The flow cytometer and particle counter were used as controls, wherein the control of the flow cytometer was to directly dilute the sample to be tested and the 0.3 mg / mL magnetic bead standard by 100 times, load the sample, read the count, calculate the magnetic loss rate, and record the results in Table 6; the control of the particle counter was to directly dilute the sample to be tested and the 0.3 mg / mL magnetic bead standard by 100 times, load the sample, read the count, calculate the magnetic loss rate, and record the results in Table 6.

[0131] Table 6

[0132]

[0133] It can be seen from Table 6 that, under the same magnetic separation washing time and the same number of magnetic separation washings, different antibodies or antigens coated on the magnetic beads have little effect on the results of the magnetic loss rate determination; it can also be seen that the magnetic loss rate increases as the number of magnetic separation washings increases from 3 to 6 times.

[0134] Embodiment 6

[0135] A method for measuring magnetic bead magnetic loss rate comprises the following steps:

[0136] (61) In this embodiment, the magnetic bead concentration-optical density standard curve in Example 1 is used as the annotation curve.

[0137] (62) AFP antibody-coated carboxyl magnetic beads (Thermo, magnetic bead particle size of 2.8 μM) were used as test samples and divided into six groups. Washing solution 4-1 in Example 2 was used as washing solution, and each group of test samples was subjected to magnetic separation and washing in a fully automated tube chemiluminescence instrument.

[0138] After washing, each group of samples to be tested were recovered, and then resuspended in 0.2 mM PB buffer (pH = 7.2). After resuspending, they were transferred to enzyme-immunoassay plates to measure OD800, and were substituted into equations (b) and (a) for calculation, and the results were recorded in Table 7. Flow cytometer and particle counter were used as controls, wherein the control of flow cytometer was to directly dilute the sample to be tested and 0.3 mg / mL magnetic bead standard by 100 times, load the sample, read the count, calculate the magnetic loss rate, and record the results in Table 7; the control of particle counter was to directly dilute the sample to be tested and 0.3 mg / mL magnetic bead standard by 100 times, load the sample, read the count, calculate the magnetic loss rate, and record the results in Table 7.

[0139] Table 7

[0140]

[0141] It can be seen from Table 7 that when the number of magnetic separation washings is between 1 and 6 times, the magnetic loss rate is small; when the number of magnetic separation washings exceeds 6 times, the magnetic loss rate increases, which will increase the loss of magnetic beads.

[0142] Embodiment 7

[0143] A method for measuring magnetic bead magnetic loss rate comprises the following steps:

[0144] (71) Thermo amino magnetic beads were used to prepare a standard solution with a magnetic bead concentration gradient. Specifically, 2.8 μM Thermo amino magnetic beads were diluted with 0.2 mM PB (pH 7.2) and then prepared into 3 mg / mL, 0.285 mg / mL, 0.27 mg / mL, 0.24 mg / mL, 0.21 mg / mL, 0.18 mg / mL and 0.15 mg / mL standard solutions.

[0145] (72) The standard solution of step (71) was added to the ELISA plate respectively, and the optical density (i.e., OD800) of the standard solution at each magnetic bead concentration was measured in three replicate wells, and the average value was taken. The results are shown in Table 8.

[0146] Table 8

[0147] Magnetic bead concentration (mg / mL) 0 0.3 0.285 0.27 0.24 0.21 0.18 0.15 Optical density 0.003 0.3678 0.3461 0.3283 0.2964 0.2654 0.2257 0.1994

[0148] According to the data in Table 8, a scatter plot of magnetic bead concentration-optical density was drawn, and a linear fit was performed to obtain a standard curve of magnetic bead concentration-optical density. The results are shown in Figure 3 shown.

[0149] from Figure 3 It can be seen that the R of the fitted magnetic bead concentration-optical density standard curve is 2 =0.9978, approximately equal to 1, indicating that the magnetic bead concentration-optical density has a high linear fit, and the magnetic bead concentration and optical density show a linearly related equation relationship, namely:

[0150] y=1.1974x+0.0093……(c);

[0151] Where x represents the concentration of magnetic beads and y represents the optical density.

[0152] (73) Prepare the following two samples to be tested and two washing solutions:

[0153] Among them, the components of the sample 1 to be tested: AFP antibody-coated amino magnetic beads (Thermo, magnetic bead concentration 2.8 μM);

[0154] Components of the test sample 2: FSH antibody-coated amino magnetic beads (Thermo, magnetic bead concentration 2.8 μM);

[0155] The composition of wash solution 1: 10 mM PB, pH = 7.2;

[0156] The composition of the washing solution 4-1: 10 mM PB, 0.9% by mass NaCl, 0.05% by mass Tween-20 and 0.1% by mass TritonX-100, pH=7.2.

[0157] According to the magnetic separation time and washing conditions in Table 9, magnetic separation and washing were performed in a fully automated tubular chemiluminescence instrument.

[0158] Subsequently, the samples to be tested were recovered after separation and washing, and then resuspended in 0.2 mM PB buffer (pH = 7.2). After resuspension, they were transferred to ELISA plates to measure OD800, which was then entered into equations (c) and (a) for calculation, and the results were recorded in Table 9.

[0159] The flow cytometer and the particle counter were used as controls. The control for the flow cytometer was to directly dilute the sample to be tested and the 0.3 mg / mL magnetic bead standard by 100 times, load the sample, read the count, calculate the magnetic loss rate, and record the results in Table 9; the control for the particle counter was to directly dilute the sample to be tested and the 0.3 mg / mL magnetic bead standard by 100 times, load the sample, read the count, calculate the magnetic loss rate, and record the results in Table 9.

[0160] Table 9

[0161]

[0162] It can be seen from Table 9 that when the magnetic separation washing time is 90 s, the washing liquid in the magnetic separation washing is washing liquid 4-1, and the washing is performed 3 times and 6 times, the magnetic loss rate is low.

[0163] In summary, it can be seen that the method for determining the magnetic loss rate of magnetic beads provided in the embodiment of the present invention, when the sample to be tested is magnetically separated and washed 1 to 3 times with washing solution 3-1, washing solution 3-2, washing solution 3-3, washing solution 4-1, washing solution 4-2 and washing solution 4-3 respectively, and the time of each magnetic separation washing is 30s to 120s, the magnetic loss rate of the obtained magnetic beads is small, so that the magnetic beads can be used more times, which is beneficial to improve the effectiveness of the magnetic beads. Due to the determination method of the present invention, the magnetic loss rate is small, which can effectively assist in judging the reliability of the detection results of the magnetic particle chemiluminescence immunoassay; in addition, the method for determining the magnetic loss rate of magnetic beads provided in the embodiment of the present invention has the characteristics of simple detection process, high efficiency and low detection cost compared with existing determination methods such as flow cytometers and particle counters.

[0164] The above description is only an implementation mode of the present invention. It should be pointed out that, for ordinary technicians in this field, improvements can be made without departing from the creative concept of the present invention, but these all belong to the protection scope of the present invention.

Claims

1. A method for measuring magnetic bead magnetic loss rate, characterized in that: The following steps are involved: Constructing a standard solution with a magnetic bead concentration gradient; Determine the optical density of the standard solution at each magnetic bead concentration; According to the magnetic bead concentration and the optical density, a magnetic bead concentration-optical density standard curve is drawn; Performing magnetic separation and washing on the sample to be tested, and measuring the optical density of the sample to be tested after the magnetic separation and washing; Substituting the optical density of the sample to be tested into the magnetic bead concentration-optical density standard curve to obtain the magnetic bead concentration of the sample to be tested; Calculating the amount of magnetic beads in the sample to be tested based on the magnetic bead concentration of the sample to be tested; Comparing the amount of magnetic beads in the sample to be tested with the total amount of magnetic beads added to the sample to be tested before magnetic separation and washing to obtain a magnetic loss rate; Wherein, the type of magnetic beads contained in the sample to be tested is the same as the type of magnetic beads in the magnetic bead concentration-optical density standard curve; The magnetic separation washing comprises placing the sample to be tested in a washing solution for washing, wherein the washing solution contains phosphate buffer, sodium chloride and Tween 20; the concentration of the phosphate buffer is 10mM to 20mM, the mass fraction of the sodium chloride is 0.8% to 1.0%, the mass fraction of the Tween 20 is 0.04% to 0.08%, and the pH value of the washing solution is 7.1 to 7.3; Alternatively, the washing solution contains phosphate buffer, sodium chloride, Tween 20 and polyethylene glycol octyl phenyl ether; the concentration of the phosphate buffer is 10mM to 20mM, the mass fraction of the sodium chloride is 0.8% to 1.0%, the mass fraction of the Tween 20 is 0.04% to 0.08%, the mass fraction of the polyethylene glycol octyl phenyl ether is 0.08% to 0.12%, and the pH value of the washing solution is 7.1 to 7.3; The magnetic separation washing times are 1 to 6 times; The duration of each magnetic separation and washing is 30 s to 120 s.

2. The method for measuring magnetic bead magnetic loss rate according to claim 1, characterized in that: The step of measuring the optical density of the standard solution comprises: The standard solution at each magnetic bead concentration was transferred to an ELISA plate, and the optical density of the standard solution at each magnetic bead concentration was measured using an ELISA reader.

3. The method for measuring magnetic bead magnetic loss rate according to claim 2, characterized in that: The standard solution of each magnetic bead concentration is transferred to at least three reaction wells of the ELISA plate, and the optical density of the standard solution in each reaction well is measured using a microplate reader, and the average value is taken.

4. The method for measuring magnetic bead magnetic loss rate according to claim 1, characterized in that: The step of measuring the optical density of the sample to be tested comprises: The sample to be tested after the magnetic separation and washing is transferred to an ELISA plate, and the optical density of the sample to be tested is measured using an ELISA reader.

5. The method for measuring magnetic bead magnetic loss rate according to claim 4, characterized in that: The sample to be tested is transferred to at least three reaction wells of the ELISA plate, and the optical density of the sample to be tested at the corresponding magnetic bead concentration in each reaction well is measured using an ELISA instrument, and the average value is taken.

6. The method for measuring magnetic bead magnetic loss rate according to claim 1, characterized in that: The step of comparing the amount of magnetic beads in the sample to be tested with the total amount of magnetic beads added to the sample to be tested before magnetic separation and washing to obtain the magnetic loss rate comprises: Calculating the amount of magnetic beads in the sample to be tested according to the magnetic bead concentration of the sample to be tested and the volume of the sample to be tested; Calculating the difference between the amount of magnetic beads in the sample to be tested and the total amount of magnetic beads added to the sample to be tested before magnetic separation and washing; Calculating the difference and the percentage of the total amount of magnetic beads added to the sample to be tested before magnetic separation and washing to obtain the magnetic loss rate; And / or, after the magnetic separation and washing of the sample to be tested and before the step of determining the optical density value of the sample to be tested, the method further comprises: The sample to be tested after magnetic separation and washing is suspended, and then the sample to be tested is transferred to an ELISA plate, and the optical density of the sample to be tested is measured using an ELISA instrument.

7. The method for measuring magnetic bead magnetic loss rate according to claim 1, characterized in that: The same type of magnetic beads includes the same particle size of the magnetic beads, the material constituting the magnetic beads, and the type of functional groups carried on the surface of the magnetic beads.

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