Test kits and methods for lam chemiluminescent immunoassay

Abstract

The application discloses a detection kit and method for LAM chemiluminescence immunoassay, and the kit comprises: a magnetic bead reagent, wherein a LAM capture antibody is modified on the magnetic bead; a luminescent reagent, comprising a LAM detection antibody modified with a luminescent reagent; and a sample diluent, which is composed of 10-200 mM Tris-HCl buffer solution, pH 6.0-8.0, 0.1-0.5 M NaCl, 0.01-0.5 wt.% Tween-20, 0.1-5 wt.% BSA and 0.5-5 wt.% PEG. The detection kit of some examples of the application can effectively reduce the combination with non-LAM specific substances, and can improve the sensitivity and signal value of the detection system, has an optimization effect on the improvement of the reaction system, and the kit has high specificity.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of detection, and particularly relates to a detection kit and method for LAM chemiluminescence immunoassay. BACKGROUND

[0002] Lipoarabinomannan (LAM) is an important component in the cell wall of Mycobacterium tuberculosis, accounting for 15 mg / g of total bacterial weight, containing 9 monoclonal antibody binding epitopes, and is a specific antigen for binding Mycobacterium. LAM can be filtered through the kidney and can be detected in urine. Urine is sterile and easy to obtain, and LAM is a heat-stable antigen that can be detected by sensitive immunological techniques even in boiled urine. Studies have shown that urine LAM detection can improve the accuracy of the diagnosis of active tuberculosis.

[0003] The existing method for diagnosing tuberculosis by detecting LAM antibodies in the urine of HIV patients has been recognized by many scientists and WHO in succession. There are relatively mature products in the global market, including Alere Determined™ TB LAM Ag in the United States and Fujifilm SILVAMP TB LAM in Japan. The Alere Determined™ TB LAM Ag product is based on colloidal gold methodology, and in the HIV-infected group when CD4 cells are >200 / uL, the sensitivity and specificity of the detection are 4% and 99%, respectively (Alere product instructions). The Fujifilm SILVAMP TB LAM product is based on immunochromatography and colloidal gold methodology, and in the HIV-infected group when CD4 cells are >200 / uL, the sensitivity is 36% and the specificity is 99% (Li Z, Tong X, Liu S, et al. The Value of Fuji LAM in the Diagnosis of Tuberculosis: A Systematic Review and Meta-Analysis. [J]. Front Public Health, 2022: 757133.). The existing two products for detecting LAM antibodies in the urine of patients to diagnose tuberculosis are only recommended for tuberculosis screening in AIDS patients and cannot be used for tuberculosis screening in non-AIDS patients.

[0004] There is no tuberculosis screening project for non-AIDS patients based on urine LAM for non-HIV-infected populations on the market, in addition to conventional detection methods such as routine sputum smear, sputum culture, and GeneXpert.

[0005] There are registered products for tuberculosis screening based on LAM in urine, which are applied to HIV-positive patients. The content of LAM in urine is low, and the sensitivity of the reagent for detecting this index is high. At the same time, the presence of salt, urea, fat, sugar and other substances in the urine sample will affect the test results.

[0006] The existing mycobacterium tuberculosis detection method has a long cycle, complicated operation and high cost. The sample source is blood, sputum or other tissue fluid, which is obtained invasively. In addition, the urine detection products on the market are mainly applied to HIV+ patient population, and the detection method is colloidal gold. The two products are applied to auxiliary diagnosis of active tuberculosis patients, but the detection sensitivity is low, the positive detection rate in HIV+ patients is also low, and in addition, the test population is limited to HIV-positive patients, especially severe patients with concurrent HIV infection and low CD4+ T lymphocyte count (infectious disease hospital or ICU).

[0007] Chemiluminescence immunoassay is a mature detection technology. CN110988330A, CN106645729A and the like disclose a technology for detecting mycobacterium tuberculosis based on chemiluminescence immunoassay, which realizes detection by quantitatively analyzing gamma interferon. Chemiluminescence immunoassay mainly includes capturing magnetic beads, using antibodies coupled on the magnetic beads to realize enrichment of the sample, and then further performing chemiluminescence detection. Although chemiluminescence immunoassay has good sensitivity, it also has certain requirements for the sample. The urine sample is rich in salt, urea, fat and sugar, which interferes with the signal value when LAM in the sample is measured. At the same time, the content of LAM is low, which leads to considerable difficulty in detecting mycobacterium tuberculosis based on LAM. There is no related technology reported in the prior art. SUMMARY

[0008] The purpose of the present application is to overcome at least one deficiency of the prior art and provide a kit and method for LAM chemiluminescence immunoassay.

[0009] The technical solution adopted by the present application is:

[0010] In a first aspect, the present application provides:

[0011] A kit for LAM chemiluminescence immunoassay, comprising:

[0012] Magnetic bead reagent, wherein the magnetic beads are modified with LAM capture antibodies;

[0013] Luminescent reagent, including LAM detection antibodies modified with luminescent reagent;

[0014] A sample diluent, consisting of 10-200 mM Tris-HCl buffer, pH 6.0-8.0, 0.1-0.5 M NaCl, 0.01-0.5 wt.% Tween-20, 0.1-5 wt.% BSA and 0.5-5 wt.% PEG.

[0015] In some examples of the kit, the magnetic bead reagent is a magnetic bead coated with LAM capture antibody, which is obtained by covalent coupling of amino groups on the LAM capture antibody with carboxyl groups on the magnetic bead.

[0016] In some examples of the kit, the coupling ratio of the antibody to the magnetic bead is 10-50 ug antibody per mg magnetic bead.

[0017] In some examples of the kit, the working concentration of the magnetic bead reagent is 0.5-2 mg / ml.

[0018] In some examples of the kit, the luminescent reagent is a LAM detection antibody labeled with acridinium ester, which is obtained by covalent coupling of carboxyl groups on the acridinium ester with amino groups on the LAM detection antibody.

[0019] In some examples of the kit, the coupling ratio of the acridinium ester to the antibody is 20-100 ug acridinium ester per mg antibody.

[0020] In some examples of the kit, the working concentration of the luminescent reagent is 0.5-2 ug / ml.

[0021] In some examples of the kit, the PEG is PEG6000.

[0022] The second aspect of the present application provides:

[0023] A method for determining the content of LAM in a urine sample, comprising the following steps:

[0024] The magnetic bead reagent of the first aspect of the present application is added to the urine sample to be tested, and incubated under stirring for 0.5-3 hours.

[0025] The magnetic beads are separated, resuspended with the sample diluent of the first aspect of the present application, vortexed, and the vortexed solution is added to the luminescent reagent of the first aspect of the present application for machine detection to determine the content of LAM.

[0026] In some examples, the ratio of the amount of sample diluent to magnetic beads is (0.2-0.4) mL:(50 ug-300 ug).

[0027] The present application has the following beneficial effects:

[0028] The detection kit of some examples of the present application can effectively reduce the combination with non-LAM specific substances, and can improve the sensitivity and signal value of the detection system, and has an optimization effect on the improvement of the reaction system, and the kit has high specificity. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 is the detection result of the affinity of LAM capture antibody to LAM.

[0030] Figure 2 is the detection result of the affinity of LAM detection antibody to LAM.

[0031] Figure 3 is the ROC curve at the best Youden index.

[0032] Figure 4 is the comparison chart of the detection results of the LAM concentration in the urine samples of healthy people (n=106) and TB patients (n=80) by using the method of the present application. DETAILED DESCRIPTION

[0033] In a first aspect, the present application provides:

[0034] A kit for LAM chemiluminescence immunoassay, comprising:

[0035] A magnetic bead reagent, wherein the magnetic beads are modified with LAM capture antibodies;

[0036] A luminescent reagent, comprising LAM detection antibodies modified with luminescent reagents;

[0037] A sample diluent, consisting of 10-200 mM Tris-HCl buffer, pH 6.0-8.0, 0.1-0.5 M NaCl, 0.01-0.5 wt.% Tween-20, 0.1-5 wt.% BSA and 0.5-5 wt.% PEG.

[0038] In some examples of the kit, the sample diluent consists of 50 mM Tris-HCl buffer, pH 7.4, 0.15 M NaCl, 0.1 wt.% Tween-20, 1 wt.% BSA and 1 wt.% PEG.

[0039] The magnetic bead reagent can be prepared by existing methods. In some examples of the kit, the magnetic bead reagent is a magnetic bead coated with LAM capture antibodies, which is obtained by covalent coupling of the amino group on the LAM capture antibodies with the carboxyl group on the magnetic beads.

[0040] The coupling ratio of the antibody and the magnetic beads can be adjusted according to the coupling efficiency, so as to ensure that there is enough amount of the antibody coupled on the magnetic beads to protect the capture effect. In some examples of the kit, the coupling ratio of the antibody and the magnetic beads is 10-50 ug of the antibody per mg of the magnetic beads.

[0041] The working concentration of the magnetic bead reagent can be adjusted according to the detection requirement. In some examples of the kit, the working concentration of the magnetic bead reagent is 0.5-2 mg / ml.

[0042] The luminescent reagent can be prepared by using the existing method. In some examples of the kit, the luminescent reagent is an LAM detection antibody labeled with acridan ester, which is obtained by covalent coupling of the carboxyl on the acridan ester and the amino group on the LAM detection antibody.

[0043] The coupling ratio of the acridan ester and the antibody can be adjusted according to the coupling effect, so as to ensure that there is enough amount of the luminescent reagent coupled on the antibody without affecting the specific recognition ability. In some examples of the kit, the coupling ratio of the acridan ester and the antibody is 20-100 ug of the acridan ester per mg of the antibody.

[0044] The working concentration of the luminescent reagent can be adjusted according to the detection requirement. In some examples of the kit, the working concentration of the luminescent reagent is 0.5-2 ug / ml.

[0045] In some examples of the kit, the PEG is PEG6000.

[0046] In a second aspect of the present application, there is provided:

[0047] A method for determining the content of LAM in a urine sample, comprising the following steps:

[0048] The magnetic bead reagent of the first aspect of the present application is added to the urine sample to be detected, and incubated under stirring for 0.5-3 hours.

[0049] The magnetic beads are separated, the sample diluent of the first aspect of the present application is used to resuspend the magnetic beads, vortexed, and the vortexed solution is added to the luminescent reagent of the first aspect of the present application for machine detection to determine the content of LAM.

[0050] In some examples, the use ratio of the sample diluent to the magnetic beads is (0.2-0.4) mL:(50 ug-300 ug). The specific amount can be adjusted according to the specific detection condition.

[0051] The technical solutions of the present application are further illustrated below by using examples. In the following implementation, the percentages are all mass percentages unless otherwise specified.

[0052] 1. A double antibody sandwich immunological quantitative detection method for detecting LAM antigen in urine samples using a chemiluminescence platform, and the detection process is based on a fully automatic chemiluminescence immunoassay analyzer. The method comprises the following steps:

[0053] (1) Mycobacterium tuberculosis LAM capture antibody coated microspheres are used as magnetic bead reagents;

[0054] (2) Mycobacterium tuberculosis LAM detection antibody is labeled with acridinium ester as a luminescent reagent;

[0055] (3) Mycobacterium tuberculosis LAM is gradient-diluted as a calibration curve;

[0056] (4) A certain volume of urine sample / calibrator is added to the magnetic bead reagent, incubated and then washed;

[0057] (5) A certain volume of buffer is added to resuspend the magnetic beads as a sample and loaded into the machine;

[0058] (6) A certain volume of luminescent reagent is added, incubated and then washed;

[0059] (7) A certain volume of pre-activation solution and activation solution are added;

[0060] (8) The luminescence signal value (RLU) is obtained and the data is analyzed to determine the result.

[0061] 2. The LAM capture antibody coated on the magnetic beads is incubated with the LAM in the urine sample, and then a double antibody sandwich complex is formed with the LAM detection antibody labeled with acridinium ester in the luminescent reagent. The incubation method is as follows:

[0062] (1) Take 1-10 mL of the urine sample to be tested, and add 50 ug-300 ug of magnetic bead reagent;

[0063] (2) Incubate at room temperature for 1-5 h;

[0064] (3) Use a magnetic separator to separate, discard the supernatant, and wash with 10 mM PBS (pH 7.2) 3 times;

[0065] (4) After resuspension of the magnetic beads, immediately load into the machine for detection.

[0066] 3. The LAM content in the urine is detected by a chemiluminescence detection platform.

[0067] Preparation of Mycobacterium tuberculosis lipoarabinomannan (LAM) reagent and detection of blank limit

[0068] 1. Main materials:

[0069] 1.1 LAM antigen, magnetic beads, purchased from a qualified supplier. LAM capture antibody, LAM detection antibody are prepared according to existing methods, of course, commercial LAM capture antibody, LAM detection antibody can also be used. The affinity of the LAM capture antibody used in the present application to LAM is shown in Figure 1 , and the affinity of the LAM detection antibody to LAM is shown in Figure 2 . Both the LAM capture antibody and the LAM detection antibody have good affinity.

[0070] 2. Method:

[0071] 2.1 LAM capture antibody coated magnetic beads

[0072] S1) Wash 140 mg of magnetic beads with Binding buffer (pH 6.0) in a magnetic separation rack for 3 times (keep the magnetic bead concentration at 20 mg / mL, and the added Binding Buffer should at least exceed the position of the magnetic magnet of the magnetic separation rack) ; the last time, place the reaction bottle on the magnetic separation rack, and when the magnetic beads are completely coagulated, carefully remove the supernatant;

[0073] S2) Add an appropriate amount of Binding Buffer to resuspend the magnetic beads, and add 5 μL of EDC and 50 uL of NHS per 10 mg of magnetic beads, and add Binding Buffer to keep the magnetic bead concentration at 20 mg / mL; rotate at room temperature for 30 min, and then wash with Binding buffer (pH 6.0) in a magnetic separation rack for 1 time; the last time, place the reaction bottle on the magnetic separation rack, and when the magnetic beads are completely coagulated, carefully remove the supernatant;

[0074] S3) Add an appropriate amount of Binding buffer (pH 6.0), and add the coating antibody according to the feeding ratio (antibody: magnetic bead = 1:40 by mass), and then add an appropriate amount of Binding buffer (pH 6.0) to keep the magnetic bead concentration at 20 mg / mL, and rotate at room temperature overnight;

[0075] S4) Add 105 μL of ethanolamine to the magnetic beads at a volume ratio of 15 μL / mL, and rotate for 30 minutes;

[0076] S5) Absorb the magnetic beads, remove the supernatant, and add magnetic bead reagent blocking solution (TBS + 1% BSA) to 20 mg / mL, and rotate for 3 hours;

[0077] S6) Wash with magnetic bead washing buffer for 3 times, and the last time, place the reaction bottle on the magnetic separation rack, and when the magnetic beads are completely coagulated, carefully remove the supernatant, and dilute the magnetic beads to 20 mg / mL with magnetic bead reagent diluent.

[0078] 2.2 LAM detection antibody labeled acridinium ester

[0079] The antibody concentration was determined by UV spectrophotometry at A280, and the antibody concentration before labeling was calculated. The labeled antibody was added to a coupling buffer sodium bicarbonate solution (0.1 M) (final antibody concentration 1 mg / mL). The acridinium ester was dissolved in DMF with a final concentration of 1 mg / mL. The DMF solution containing the acridinium ester was added to the labeled antibody dilution at a certain feed ratio, and shaken at room temperature in the dark for 40 min at a speed of 160 rpm / min. 6 uL of 10% glycine was added to terminate the reaction, and shaken at room temperature in the dark for 30 min at a speed of 160 rpm / min. Dialsing in PBS buffer at pH 6.8 for 2-3 days. 10% BSA, glycerol (glycerin) was added to the labeled product to give a final concentration of 1% BSA, 40% glycerol, and stored at -20°C for use.

[0080] 2.3 LAM calibration curve configuration

[0081] The LAM antigen was gradient diluted using the calibrator diluent at 0 pg / mL, 10 pg / mL, 100 pg / mL, 1000 pg / mL, and directly detected on the machine and detected on the machine after enrichment, respectively.

[0082] 2.4 Direct detection on the machine

[0083] (1) On the fully automatic chemiluminescent immunoassay analyzer, the instrument automatically takes 100 ul of the urine sample to be tested, and adds 10 ug-60 ug of magnetic bead reagent;

[0084] (2) Incubate at 37°C for 5-15 min;

[0085] (3) The instrument automatically washes 4-8 times;

[0086] (4) Add 50 ul of acridinium ester labeled antibody;

[0087] (5) Incubate at 37°C for 5-15 min;

[0088] (6) The instrument automatically washes 4-8 times;

[0089] (7) Add 100 ul of pre-excitation solution and excitation solution, and read the luminescence value (RLU).

[0090] 2.5 Detection on the machine after enrichment

[0091] (1) Take 1-10 mL of the urine sample to be tested, and add 50 ug-300 ug of magnetic bead reagent;

[0092] (2) Incubate at room temperature for 1-5 h;

[0093] (3) Use a magnetic separator to separate, and discard the supernatant;

[0094] (4) Resuspend the magnetic beads with 0.3 ml diluent (50 mM TBS + 0.15 M NaCl + 0.1% Tween 20 + 1% BSA, pH 7.4), vortex well, and immediately run on the machine.

[0095] 3. Results

[0096] The test results are shown in Table 1.

[0097] Table 1

[0098]

[0099] From the data in Table 1, it can be seen that after using magnetic bead enrichment, the sensitivity of the detection system is significantly improved, and the detection limit can reach 2.6 pg / mL.

[0100] Effect of different sample diluents on test results

[0101] 1. Main materials:

[0102] 1.1 Magnetic bead diluent: Control group: 50 mM TBS + 0.15 M NaCl + 0.1% Tween 20 + 1% BSA, pH 7.4; Experimental group: 50 mM TBS + 0.15 M NaCl + 0.1% Tween 20 + 1% BSA + 1% PEG6000, pH 7.4.

[0103] Comparative Example 1: 50 mM TBS + 0.15 M NaCl + 0.1% Tween 20 + 1% BSA, pH 7.4

[0104] Comparative Example 2: 50 mM TBS + 0.15 M NaCl + 1% BSA + 1% PEG6000, pH 7.4

[0105] Comparative Example 3: 50 mM TBS + 0.15 M NaCl + 0.1% Tween 20 + 1% PEG6000, pH 7.4

[0106] Example 1: 50 mM TBS + 0.15 M NaCl + 0.1% Tween 20 + 1% BSA + 1% PEG6000, pH 7.4

[0107] 1.2 Calibration curve, luminescent reagent, magnetic bead reagent.

[0108] 2. Method:

[0109] (1) Take 1-10 mL of the urine sample to be tested, add 50 ug-300 ug of magnetic bead reagent;

[0110] (2) Incubate at room temperature for 1-5 h;

[0111] (3) Use a magnetic separator to separate, discard the supernatant;

[0112] (4) Resuspend the magnetic beads with the control and experimental magnetic bead diluents, vortex well, and immediately detect.

[0113] 3. Results

[0114] The detection results are shown in Table 2.

[0115] Table 2

[0116]

[0117] From the data in Table 2, it can be seen that the diluent resuspended magnetic beads of Comparative Example 2 have improved sensitivity compared to Comparative Example 1; the diluent of Comparative Example 3 has improved sensitivity compared to Comparative Example 1, but the detection background value is high; by using Example 1, the detection sensitivity is improved compared to Comparative Example 1, and the background signal value does not change significantly. It shows that BSA, Tween and PEG have unpredictable effects on the detection of LAM. The sample diluent of Example 1 has the best effect.

[0118] Specificity experiment

[0119] 1. Main materials:

[0120] 1.1 Calibration curve, luminescent reagent, magnetic bead reagent

[0121] 1.2 Clinical samples: healthy human urine samples

[0122] 2. Method: LAM enrichment

[0123] (1) Take 1-10 mL of the urine sample to be tested, add 50 ug-300 ug of magnetic bead reagent;

[0124] (2) Incubate at room temperature for 1-5 h;

[0125] (3) Use a magnetic separator to separate, discard the supernatant;

[0126] (4) Resuspend the magnetic beads with the sample diluent of Example 1, and immediately detect.

[0127] 3. Results

[0128] The detection results are shown in Table 3.

[0129] Table 3

[0130]

[0131] From table 3, 97.5% of 60 healthy human urine samples were used as the cut-off value, only 1 sample showed false positive, the specificity was 98.33%.

[0132] Accuracy determination experiment

[0133] 1、Main materials:

[0134] 1.1 Calibration curve, luminescent reagent, magnetic bead reagent

[0135] 1.2 Clinical samples: 17 urine samples of patients with positive bacteria (using bacteria culture as the gold standard)

[0136] 2、Method: LAM enrichment

[0137] (1) Take 1-10 mL of the urine sample to be tested, add 50 ug-300 ug of magnetic bead reagent;

[0138] (2) Incubate at room temperature for 1-5 h;

[0139] (3) Use magnetic separator for separation, discard the supernatant;

[0140] (4) After resuspension of the sample diluent magnetic beads of Example 1, immediately detect by machine.

[0141] 3、Results

[0142] 15 out of 17 samples were detected positive, the detection rate was 88.24%, and the accuracy was high.

[0143] Application of the qualitative product in clinic

[0144] 1、Main materials:

[0145] 1.1 Calibration curve, luminescent reagent, magnetic bead reagent

[0146] 1.2 Cut-off value establishment Clinical samples: 50 tuberculosis samples, 60 healthy human samples were used to establish the cut-off level of the kit detection system.

[0147] 1.3 Clinical verification samples: The cut-off level is the judgment standard, 80 urine samples of patients diagnosed with tuberculosis in clinic and 106 urine samples of healthy people were used for clinical verification of specificity and accuracy.

[0148] 2、Method: LAM enrichment

[0149] (1) Take 1-10 mL of the urine sample to be tested, add 50 ug-300 ug of magnetic bead reagent;

[0150] (2) Incubate at room temperature for 1-5 hours;

[0151] (3) Separate using a magnetic separator, discard the supernatant;

[0152] (4) After resuspension of the sample dilution magnetic beads of Example 1, immediately perform machine detection.

[0153] 3. Results

[0154] 3.1 At the optimal Youden index, the ROC curve AUC area is 0.799, P < 0.0001, the sensitivity is 72.0% ( Figure 3 ), and the specificity is 93.8% in order to ensure product specificity. The selected qualitative product cutoff is 0.97, and the specificity is 100%, and large clinical sample verification is performed.

[0155] 3.2 The selected clinical verification samples: 80 urine samples of patients diagnosed with pulmonary tuberculosis, and 106 urine samples of healthy people, the verification results are as follows: the positive detection rate of LAM in 80 pulmonary tuberculosis samples is 55%, and the positive detection rate in 106 healthy samples is 0% ( Figure 4 ).

[0156] The above is a further detailed description of the present application, which cannot be considered as a limitation of the specific implementation of the present application. For ordinary skilled persons in the technical field to which the present application belongs, simple deductions or replacements without departing from the concept of the present application are within the protection scope of the present application.

Claims

1. A kit for LAM chemiluminescent immunoassay, comprising: a magnetic bead reagent, wherein the magnetic beads are modified with LAM capture antibody, and the coupling ratio of the antibody to the magnetic beads is 10-50 ug antibody per mg magnetic beads; a luminescent reagent, which is LAM detection antibody labeled with acridan ester, and is obtained by covalent coupling of the carboxyl group on the acridan ester with the amino group on the LAM detection antibody, and the coupling ratio of the acridan ester to the antibody is 20-100 ug acridan ester per mg antibody; a sample diluent, which is composed of 50 mM Tris-HCl buffer, pH 7.4, 0.15 M NaCl, 0.1 wt.% Tween-20, 1 wt.% BSA and 1 wt.% PEG6000.

2. The kit of claim 1, wherein The magnetic bead reagent is magnetic beads coated with LAM capture antibody, and is obtained by covalent coupling of the amino group on the LAM capture antibody with the carboxyl group on the magnetic beads.

3. The kit according to claim 1 or 2, characterized in that, The working concentration of the magnetic bead reagent is 0.5-2 mg / ml.

4. The kit according to claim 1 or 2, characterized in that, The working concentration of the luminescent reagent is 0.5-2 ug / ml.

5. A method for determining the content of LAM in a urine sample, comprising the following steps: adding the magnetic bead reagent of claim 1 or 2 to the urine sample to be tested, and incubating under stirring for 0.5-3 hours; separating the magnetic beads, resuspending the magnetic beads with the sample diluent of claim 1, vortexing, taking the vortexed solution, adding the luminescent reagent of claim 1, and detecting on a machine to determine the content of LAM.

6. The method of claim 5, wherein, The ratio of the amount of the sample diluent to the magnetic beads is (0.2-0.4) mL:(50 ug-300 ug).

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