CN117388490A - Mycobacterium tuberculosis antibody detection kit based on magnetic particle chemiluminescence method - Google Patents
Mycobacterium tuberculosis antibody detection kit based on magnetic particle chemiluminescence method Download PDFInfo
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/5695—Mycobacteria
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- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
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Abstract
The invention discloses a mycobacterium tuberculosis antibody detection kit based on a magnetic particle chemiluminescence method, and relates to the technical field of immunoassay detection, wherein the kit comprises antigen-labeled magnetic particles, acridine sulfonamide-labeled antibodies, sample diluent and quality control substances; the magnetic particle labeled mycobacterium tuberculosis specific antigen comprises 16KD,38KD and Ag85A, and the mass ratio of the protein 16KD,38KD and Ag85A in the antigen marked by the carboxyl magnetic beads is (1-3) 1 (1-3). According to the invention, the antigen is marked by carboxyl magnetic particles, and the anti-human IgG of sheep is marked by acridine sulfonamide, on one hand, the false positive factors in a blood sample are effectively reduced by adjusting the coupling and closing conditions of the magnetic particles and the composition of a system stabilizer; on the other hand, by adopting a means of mixing three antigens (16 KD,38KD and Ag 85A) of the mycobacterium tuberculosis specific protein, the specificity of detection is improved.
Description
Technical Field
The invention relates to the technical field of immunoassay detection, in particular to a mycobacterium tuberculosis antibody detection kit based on a magnetic particle chemiluminescence method.
Background
Tuberculosis (TB), the second biggest killer of humans following AIDS, is a chronic infectious disease that is mainly infected by the respiratory system, and is listed as one of the major infectious diseases in our country. The global tuberculosis death number determined by the authorities in 2020 is 130 ten thousand, which is almost twice as large as 68 ten thousand of the death number of AIDS. Among them, mycobacterium tuberculosis infection is the main cause of the infection, and many organs can be invaded, and pulmonary tuberculosis infection is the most common. Mycobacterium tuberculosis (Mycobacterium tuberculosis) is an acid-fast positive coryneform bacterium, has a size of about 1-4×0.4 μm, is flagellum-free, spore-free, incapable of producing endotoxin and endotoxin, and has the advantages of obligate aerobic, high culture requirement and slow growth. The human body is not necessarily ill after being infected by tubercle bacillus, and clinical ill is only possible to be caused when the resistance is reduced or the allergy mediated by cells is increased.
Tuberculosis is a chronic infectious disease caused by mycobacterium tuberculosis, and a bacterial remover is an important infectious source, if the tuberculosis can be diagnosed in time and reasonably treated, most of tuberculosis can be cured clinically. Tuberculosis has recently increased worldwide in incidence year by year, and particularly, it has been found that there is a tendency for the pneumonia caused by mycobacterium tuberculosis to increase, which has become a public health problem of concern. The Hillemann et al uses the Capilia TB product to detect the MPB64 antigen, and in the detection of 172 tuberculosis positive clinical specimens, the positive detection rate is 92%, and the specificity reaches 100%.
The main detection methods for the diagnosis of the mycobacterium tuberculosis at present comprise tuberculin test (PPD test), smear, tubercle bacillus gamma interferon release test, mycobacterium culture, mycobacterium tuberculosis antigen detection and the like. Clinical data show that different detection methods have certain effects on the detection rate of the mycobacterium tuberculosis, and meanwhile, the fact that the detection results with obvious differences appear when different detection methods are adopted for the same tissue of the specimen to be detected is also found. Due to the lack of effective vaccines, the lack of diagnostic reagents with high specificity and sensitivity, and the continuous emergence of drug-resistant tuberculosis, rapid and effective diagnosis is important for the control and spread of tuberculosis. Clinical studies show that the positive detection rate and the disease degree of IgG in tuberculosis patients and the immune state of the patients are closely related, and the longer the disease course of the patients is, the more serious the disease degree is, the easier immune response is induced, and the immunoglobulin such as IgG is increased.
Chemiluminescent immunoassay (Chemilumineshcence immunoassay, CLIA) is a method of combining chemiluminescence with immunoassay. In the immune reaction system, the luminescent substance-labeled antigen reacts with the antigen to form an immune complex. In a chemiluminescent system, luminescent substances transition from a ground state to an excited state after the action of a catalyst and an oxidant, return to the ground state and emit photons, and the intensity of the photons can be detected by a luminescent signal detector and is in a certain relation with the concentration of the substances to be detected. Acridinium esters as common chemiluminescent substances for labeling can be prepared by NaOH and H 2 O 2 Is operated to emit light instantaneously. The acridinium ester compound as the marker can make the chemiluminescent immunoassay system have high marking efficiency, high speed, simple process, no need of adding catalyst and low background value. If the detected sample contains specific protein, a solid-phase complex can be formed with specific IgE antigen in serum of a patient, and the concentration of the mycobacterium tuberculosis antigen is determined by the luminous value of acridinium ester, so that the TB is diagnosed.
Disclosure of Invention
The invention aims to provide a mycobacterium tuberculosis antibody detection kit based on a magnetic particle chemiluminescence method, so as to solve the problems in the background technology.
In order to solve the technical problems, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a mycobacterium tuberculosis antibody detection kit based on a magnetic particle chemiluminescence method, comprising antigen-labeled magnetic particles, acridine sulfonamide-labeled antibodies, a sample diluent, and a quality control substance;
the magnetic particle labeled mycobacterium tuberculosis specific antigen comprises 16KD,38KD and Ag85A.
The technical scheme of the invention is further improved as follows: the mass ratio of the protein 16KD,38KD and Ag85A in the antigen marked by the carboxyl magnetic beads is (1-3): 1-3.
The technical scheme of the invention is further improved as follows: the sample diluent comprises any one or a combination of at least two of bovine serum albumin, a rheumatoid factor adsorbent, a bacteriostatic agent, urea or a blocking agent.
The technical scheme of the invention is further improved as follows: the quality control product comprises a positive quality control product and a negative quality control product, wherein the positive quality control product is a buffer solution containing humanized mycobacterium tuberculosis antibodies.
The technical scheme of the invention is further improved as follows: the mycobacterium tuberculosis antibody detection kit also comprises an excitation liquid and a washing liquid.
In a second aspect, the present invention provides a method for preparing a mycobacterium tuberculosis antibody detection kit based on a magnetic particle chemiluminescence method, the method comprising:
step one, preparing a magnetic particle marked mycobacterium tuberculosis specific protein antigen:
(1) Taking carboxyl magnetic particles, magnetically separating (1-2 min), and discarding the supernatant; then, taking a coupling buffer MES (pH=5.0-7.0) solution to redisperse the magnetic particles, oscillating for 5-10 min, magnetically separating, discarding the supernatant, and repeating for three times;
(2) Re-dispersing the magnetic particles by using a coupling buffer MES (pH=5.0-7.0), adding a coupling activating reagent EDC, and oscillating for 30-50 min at room temperature; magnetically separating, discarding supernatant;
(3) Adding antigen and diluting with MES coupling buffer solution, and vibrating at room temperature for 1.5-2.5 h; magnetically separating, discarding the supernatant, then taking a blocking solution mother solution (5% BSA) to block the system, and carrying out oscillation reaction for 1.5-2.5 h at room temperature; magnetically separating, discarding supernatant;
(4) Re-dispersing the magnetic particles by using a washing buffer TBS-T, oscillating for 5-10 min, magnetically separating, discarding the supernatant, and repeating for three times; washing the mixture once by using magnetic particle storage liquid, and redispersing antigen coupling magnetic particles in the magnetic particle storage liquid for standby at 2-8 ℃;
step two, preparing an acridine sulfonamide marked goat anti-human IgG antibody:
placing 0.2mg of goat anti-human IgG antibody into a glassware, and diluting the goat anti-human IgG antibody to 0.5-1 mg/mL with 0.1M CBS (pH 9.0-12.0) buffer solution; adding 2mg/mL NSP-SA-NHS solution into the antibody diluent, uniformly mixing, and keeping away from light at room temperature for 60-90 min;
adding 10% lysine solution, mixing uniformly, and placing at room temperature for 30-40 minutes in a dark place; dialyzing with 0.02M PBS buffer solution at 4deg.C overnight to obtain acridine sulfonamide labeled antibody mother liquor;
wherein the working concentration of the acridine sulfonamide marked goat anti-human IgG antibody is 0.5-1 mug/mL;
and thirdly, packaging the magnetic particle labeled mycobacterium tuberculosis specific protein antigen, the acridine sulfonamide labeled goat anti-human IgG, the sample diluent and the quality control product respectively to obtain the mycobacterium tuberculosis antibody detection kit.
In a third aspect, the present invention also provides a method of using a mycobacterium tuberculosis antibody detection kit based on a magnetic particle chemiluminescence method, the method of using the kit comprising the steps of: adding a magnetic particle marked mycobacterium tuberculosis specific protein antigen into a sample to be detected, performing first incubation, washing, adding acridine sulfonamide marked goat anti-human IgG, performing second incubation, washing, and detecting to obtain a detection result.
According to the invention, a two-step two-washing method is adopted to detect a sample to be detected, namely, the sample and the magnetic bead labeled antigen are mixed, incubated and washed, then the acridine sulfonamide labeled antibody is added, incubated and washed, and a two-step one-washing method (directly mixing the sample, the carboxyl magnetic bead labeled antigen and the acridine sulfonamide labeled antibody), incubated and washed, is adopted, and the detection effect of the two-step two-washing method is better.
Preferably, the sample to be tested is serum or plasma; the absolute value of the relative deviation of the detection luminescence values of different sample types (plasma/serum) of the same sample is smaller than 10%, and the detection results of the plasma and the serum are considered to be the same.
Diluting a sample to be tested by a sample diluent; diluting the sample by 25 times, 50 times, 100 times, 200 times and 400 times respectively by using a sample diluent; when the diluted sample was detected by chemiluminescence, the degree of discrimination was maximized when the dilution factor of the sample was 100 times.
In the invention, the optimal reaction conditions are tested by respectively adding 50, 75 and 100 mu L of sample, 25, 50 and 75 mu L of magnetic particle labeled antigen, 30, 40 and 50 mu L of magnetic particle adding amount, 4, 6, 8 and 10min of first-step reaction time, 1, 0.5, 0.25 and 0.125 mu g/mL of optimal acridine sulfonamide labeled goat anti-human IgG concentration, 4, 6, 8 and 10min of second-step reaction time, and 1, 2, 3 and 4 times of washing times.
The dilution factor of the sample to be measured is 1 (90-150);
the antigen-labeled carboxyl magnetic beads have the quantity of 40 mug/mL and the antigen-labeled carboxyl magnetic beads have the quantity of 40-60 mug/mL;
preferably, the working concentration of the acridine sulfonamide marked sheep anti-human IgG is 0.5-1 mug/mL; for example, it may be 0.5. Mu.g/mL, 0.75. Mu.g/mL, 0.8. Mu.g/mL, 0.9. Mu.g/mL, or 1. Mu.g/mL, and preferably 1. Mu.g/mL.
Preferably, the first incubation time is 5-15 min; for example, it may be 5min, 6min, 7min, 8min, 9min, 10min, 12min, 14min or 15min, preferably 10min.
Preferably, the second incubation time is 5 to 15min, for example, 5min, 6min, 7min, 8min, 9min, 10min, 12min, 14min or 15min, and preferably 10min.
Preferably, the index I in the detection result is more than or equal to 1.0, and the sample to be detected is positive;
and if the index I in the detection result is smaller than 1.0, the sample to be detected is negative.
The numerical ranges of the present invention include not only the recited point values, but also any point values between the recited numerical ranges that are not recited, and for the sake of brevity, the present invention is not intended to be exhaustive of the specific point values that are included in the recited ranges.
By adopting the technical scheme, compared with the prior art, the invention has the following technical progress:
1. the invention provides a mycobacterium tuberculosis antibody detection kit based on a magnetic particle chemiluminescence method, which selects carboxyl magnetic particles to mark antigen and acridine sulfonamide to mark sheep anti-human IgG, on one hand, false positive factors in a blood sample are effectively reduced by adjusting magnetic particle coupling, closing conditions and system stabilizer composition; on the other hand, by adopting a means of mixing three antigens (16 KD,38KD and Ag 85A) of the mycobacterium tuberculosis specific protein, the specificity of detection is improved.
2. The invention provides a mycobacterium tuberculosis antibody detection kit based on a magnetic particle chemiluminescence method, which adopts 16KD,38KD and Ag85A as antigens of mycobacterium tuberculosis specific proteins, recombinant proteins have dominant epitopes of original protein sequences, have higher hydrophilic index, antigen index and surface possibility, and can obviously improve detection sensitivity.
3. The invention provides a mycobacterium tuberculosis antibody detection kit based on a magnetic particle chemiluminescence method, and the detection kit has high sensitivity and precision. Compared with manual operation, the automatic device has the advantages of high accuracy, more accurate sample adding time and reaction time, greatly improved detection accuracy and repeatability, simple system and low background value. Compared with luminol and its derivative and other luminescent material, the main system has small acridinium ester molecular weight, easy combination with protein, high labeling stability, great luminous intensity, fast release, no need of catalyst and reinforcing agent, and capacity of emitting light in H2O 2-containing alkaline environment, low cost, simple operation and high speed. The system is combined with the full-automatic chemiluminescence determinator, an operator only needs to complete the loading and setting procedures, and the rest is automatically completed by the determinator; on the other hand, the system detection speed is faster, and the result can be obtained after 40 min.
Drawings
FIG. 1 is a schematic diagram of a preparation flow of a kit of the present invention;
FIG. 2 is a schematic diagram of the use flow of the kit of the present invention.
Detailed Description
The invention is further illustrated by the following examples:
the present invention provides three aspects:
1-2, the invention provides a mycobacterium tuberculosis antibody detection kit based on a magnetic particle chemiluminescence method, which comprises antigen-labeled magnetic particles, acridine sulfonamide-labeled antibodies, a sample diluent and a quality control substance;
magnetic particle labeled mycobacterium tuberculosis specific antigens include 16kd,38kd and Ag85A.
Wherein the mass ratio of the protein 16KD,38KD and Ag85A in the antigen marked by the carboxyl magnetic beads is (1-3): 1-3, wherein the sample diluent comprises any one or a combination of at least two of bovine serum albumin, a rheumatoid factor adsorbent, a bacteriostat, urea or a blocking agent, the quality control products comprise positive quality control products and negative quality control products, the positive quality control products are buffer solutions containing humanized mycobacterium tuberculosis antibodies, and the mycobacterium tuberculosis antibody detection kit also comprises excitation solution and washing solution.
In a second aspect, as shown in fig. 1-2, the present invention provides a method for preparing a mycobacterium tuberculosis antibody detection kit based on a magnetic particle chemiluminescence method, the method comprising:
step one, preparing a magnetic particle marked mycobacterium tuberculosis specific protein antigen:
(1) Taking carboxyl magnetic particles, magnetically separating (1-2 min), and discarding the supernatant; then, taking a coupling buffer MES (pH=5.0-7.0) solution to redisperse the magnetic particles, oscillating for 5-10 min, magnetically separating, discarding the supernatant, and repeating for three times;
(2) Re-dispersing the magnetic particles by using a coupling buffer MES (pH=5.0-7.0), adding a coupling activating reagent EDC, and oscillating for 30-50 min at room temperature; magnetically separating, discarding supernatant;
(3) Adding antigen and diluting with MES coupling buffer solution, and vibrating at room temperature for 1.5-2.5 h; magnetically separating, discarding the supernatant, then taking a blocking solution mother solution (5% BSA) to block the system, and carrying out oscillation reaction for 1.5-2.5 h at room temperature; magnetically separating, discarding supernatant;
(4) Re-dispersing the magnetic particles by using a washing buffer TBS-T, oscillating for 5-10 min, magnetically separating, discarding the supernatant, and repeating for three times; washing the mixture once by using magnetic particle storage liquid, and redispersing antigen coupling magnetic particles in the magnetic particle storage liquid for standby at 2-8 ℃;
step two, preparing an acridine sulfonamide marked goat anti-human IgG antibody:
placing 0.2mg of goat anti-human IgG antibody into a glassware, and diluting the goat anti-human IgG antibody to 0.5-1 mg/mL with 0.1M CBS (pH 9.0-12.0) buffer solution; adding 2mg/mL NSP-SA-NHS solution into the antibody diluent, uniformly mixing, and keeping away from light at room temperature for 60-90 min;
adding 10% lysine solution, mixing uniformly, and placing at room temperature for 30-40 minutes in a dark place; dialyzing with 0.02M PBS buffer solution at 4deg.C overnight to obtain acridine sulfonamide labeled antibody mother liquor;
wherein the working concentration of the acridine sulfonamide marked goat anti-human IgG antibody is 0.5-1 mug/mL;
and thirdly, respectively packaging the magnetic particle marked mycobacterium tuberculosis specific protein antigen, the acridine sulfonamide marked sheep anti-human IgG, the sample diluent and the quality control product to obtain the mycobacterium tuberculosis antibody detection kit.
In a third aspect, as shown in fig. 1-2, the present invention provides a method for using a mycobacterium tuberculosis antibody detection kit based on a magnetic particle chemiluminescence method, the method for using the kit comprising the steps of: adding the sample to be detected and the magnetic particles marked by the antigen into a reaction cup, incubating and magnetically washing, adding the acridine sulfonamide marked antibody, incubating and magnetically washing for the second time, and detecting to obtain a detection result.
Wherein the sample to be tested is serum or plasma;
diluting a sample to be tested by a sample diluent;
the dilution factor of the sample to be measured is 1 (90-150);
the antigen labeling carboxyl magnetic beads have the quantity of 40 mug/mL and the antigen labeling quantity of 40-60 mug/mL;
the working concentration of the acridine sulfonamide marked sheep anti-human IgG is 0.5-1 mug/mL;
the first incubation time is 5-15 min;
the second incubation time is 5-15 min, and the index I in the detection result is more than or equal to 1.0, so that the sample to be detected is positive; and if the index I in the detection result is less than 1.0, the sample to be detected is negative.
Example 1
The implementation provides a mycobacterium tuberculosis antibody detection kit based on magnetic particle chemiluminescence, which specifically comprises the following components:
the kit comprises a carboxyl magnetic bead labeled mycobacterium tuberculosis specific protein antigen, an acridine sulfonamide labeled goat anti-human IgG antibody, a sample diluent, a positive quality control product, a negative quality control product, a sample diluent and a washing liquid.
The preparation method of the mycobacterium tuberculosis antibody detection kit based on magnetic particle chemiluminescence comprises the following steps:
(1) Preparing a magnetic particle marked mycobacterium tuberculosis specific protein antigen:
1) Taking carboxyl magnetic particles, magnetically separating (1-2 min), and discarding the supernatant; then, taking a coupling buffer MES (pH=5.0-7.0) solution to redisperse the magnetic particles, oscillating for 5-10 min, magnetically separating, discarding the supernatant, and repeating for three times;
2) Re-dispersing the magnetic particles by using a coupling buffer MES (pH=5.0-7.0), adding a coupling activating reagent EDC, and oscillating for 30-50 min at room temperature; magnetically separating, discarding supernatant;
3) Adding antigen and diluting with MES coupling buffer solution, and vibrating at room temperature for 1.5-2.5 h; magnetically separating, discarding the supernatant, then taking a blocking solution mother solution (5% BSA) to block the system, and carrying out oscillation reaction for 1.5-2.5 h at room temperature; magnetically separating, discarding supernatant;
4) Re-dispersing the magnetic particles by using a washing buffer TBS-T, oscillating for 5-10 min, magnetically separating, discarding the supernatant, and repeating for three times; washing once with magnetic particle storing liquid, re-dispersing antigen coupled magnetic particle in the magnetic particle storing liquid and storing at 2-8 deg.c for further use.
(2) The preparation method of the acridine sulfonamide marked sheep anti-human IgG antibody comprises the following steps:
placing 0.2mg of goat anti-human IgG antibody into a glassware, and diluting the goat anti-human IgG antibody to 0.5-1 mg/mL with 0.1M CBS (pH 9.0-12.0) buffer solution; adding 9.1 mu L of NSP-SA-NHS solution with the concentration of 2mg/mL into the antibody diluent, uniformly mixing, and keeping the mixture at the room temperature for 60-90 min in a dark place;
then adding 1.33 mu L of 10% lysine solution, uniformly mixing, and keeping away from light at room temperature for 30-40 minutes; dialyzing with 0.02M PBS buffer solution at 4deg.C overnight to obtain acridine sulfonamide labeled antibody mother liquor;
the working concentration of the acridine sulfonamide marked goat anti-human IgG antibody is 0.5-1 mug/mL.
Example 2
The present embodiment provides two different detection steps, specifically including:
(1) Two-step two-wash method: adding 40 mu L of magnetic bead labeled antigen into 100 mu L of reference sample, incubating for 10min, and washing; adding 100 mu L of acridine sulfonamide labeled antibody, incubating for 10min, and washing; and adding an excitation liquid to detect a luminescence value.
(2) A two-step one-step cleaning method: adding 40 mu L of magnetic bead labeled antigen into 100 mu L of reference sample for incubation, and adding 100 mu L of acridine sulfonamide labeled antibody for incubation for 10min for washing; and adding an excitation liquid to detect a luminescence value. The detection area was indexed and the results are shown in table 1:
the reference sample is a standard sample, and is a sample to be detected used in the research and development process of the kit, and the numbers are N1, N2, N3, P1, P2 and P3; as can be seen from the above table, the two-step two-washing method was selected as the operation step because the degree of distinction between the two-step two-washing method is large.
Example 3
The present example provides a reagent kit for detecting a Mycobacterium tuberculosis antibody based on magnetic particle chemiluminescence, which is different from example 2 in that the working concentration of acridine sulfonamide labeled goat anti-human IgG is 0.5-1. Mu.g/mL, and can be, for example, 0.5. Mu.g/mL, 0.75. Mu.g/mL, 0.8. Mu.g/mL, 0.9. Mu.g/mL or 1. Mu.g/mL.
Likewise, the different schemes provided in example 1 were tested for discrimination and are not listed here for the sake of brevity and conciseness.
Test example 1: precision evaluation
Testing for 20 days, taking high, medium and low value samples, detecting with 2 machine types, repeating each sample 3 times per day, and storing samples with proper method at intervals of not less than 2 hours. The repeatability, indoor precision and batch-to-batch precision of each sample were calculated.
The test results are shown in table 2 below:
from the analysis results of the data in the table, the repeatability, the indoor precision and the inter-batch precision CV of the positive sample, the strong positive sample and the positive quality control product in the detection of the model SMART 6500 are all less than or equal to 10 percent, and the requirements (the repeatability CV is less than 10 percent and the inter-batch CV is less than 15 percent) are met; the positive detection rate of the critical positive sample is more than or equal to 95 percent, which meets the requirements; the negative detection rate of the negative sample and the negative quality control product is 100%, which meets the requirements;
likewise, the detection result of the model SMART 500S is also satisfactory, and is not listed here for the sake of brevity and conciseness.
Test example 2: influence of substances which may cause interference on the detection result
Hemoglobin in clinical samples is mainly the sample that undergoes hemolysis, including pathological hemolysis and technical hemolysis. Non-dominant hemolysis means that the hemoglobin content is less than 0.5mg/mL, which hemolysis is not visible to the naked eye. The concentration of slightly hemolyzed hemoglobin is 0.5-3mg/mL, the concentration of moderately hemolyzed hemoglobin is 3.1-5mg/mL, and the concentration of severely hemolyzed hemoglobin is > 5mg/mL. Experiments prove that when the concentration of hemoglobin reaches 7mg/mL, the absolute value of the relative deviation of the concentration values of the adjacent negative, the adjacent positive and the high-value positive is not more than 10%, so that the detection result is not interfered.
The bilirubin range in normal human serum is 2-8mg/L (bilirubin range in infants for one week is 10-120 mg/L), and the highest interference detection concentration is 150mg/L according to the third edition of EP 07. As shown in the table, when the bilirubin concentration in the sample reaches 300mg/L, the absolute value of the relative deviation of the concentration values of the adjacent negative, the adjacent positive and the high-value positive is not more than 10%, so that the bilirubin concentration in the sample has no interference on the detection result. The test results are shown in table 3 below:
the normal ceiling of triglycerides in clinical samples was 1.7mmoL/L, and the highest interference detection concentration according to the third edition of EP 07 was 2.83mmoL/L. As seen from the table, when the concentration of triglyceride in the sample reaches 7.5mmoL/L, the absolute value of the relative deviation of the concentration values of the adjacent negative, the adjacent positive and the high-value positive is not more than 10%, so that the detection result is not interfered.
Test example 3: detecting samples sampled in different time periods
The stability of an In Vitro Diagnostic (IVD) agent reflects its ability to maintain consistent performance characteristics over time. The evaluation experiment steps were as follows: the detection reagents of the antibodies of the mycobacterium tuberculosis are respectively stored at 4 ℃ and 37 ℃, the detection reagents of the IgG antibodies of the research are respectively taken out on days 1, 2, 4 and 6, negative samples 1# and positive samples 10# are respectively detected, and each sample is repeatedly detected for 4 times. The trend of the reagent was determined by calculating the I value at 4℃and 37℃and the results are shown in Table 4 below:
as can be seen from the above table, the Mycobacterium tuberculosis antibody detection kit is relatively stable at both 4℃and 37℃as a result of I-value analysis at 4℃and 37 ℃.
In conclusion, the invention selects carboxyl magnetic particles to mark antigen and acridine sulfonamide to mark sheep anti-human IgG, on one hand, false positive factors in blood samples are effectively reduced by adjusting magnetic particle coupling, closing conditions and system stabilizer composition; on the other hand, by adopting a means of mixing three antigens (16 KD,38KD and Ag 85A) of the mycobacterium tuberculosis specific protein, the detection specificity is improved, the sensitivity and precision of the detection kit are high, and compared with manual operation, the automatic device has the advantages of high precision, more accurate sample adding time and reaction time, great improvement of the detection accuracy and repeatability, simple system and low background value. Compared with luminol and its derivative and other luminescent material, the main system has small acridinium ester molecular weight, easy combination with protein, high labeling stability, great luminous intensity, fast release, no need of catalyst and reinforcing agent, and capacity of emitting light in H2O 2-containing alkaline environment, low cost, simple operation and high speed. The system is combined with the full-automatic chemiluminescence determinator, an operator only needs to complete the loading and setting procedures, and the rest is automatically completed by the determinator; on the other hand, the system detection speed is faster, and the result can be obtained after 40 min.
The foregoing invention has been generally described in great detail, but it will be apparent to those skilled in the art that modifications and improvements can be made thereto. Accordingly, it is intended to cover modifications or improvements within the spirit of the inventive concepts.
Claims (9)
1. The mycobacterium tuberculosis antibody detection kit based on the magnetic particle chemiluminescence method is characterized in that: comprises antigen-marked magnetic particles, acridine sulfonamide-marked antibodies, sample diluent and quality control substances;
the magnetic particle labeled mycobacterium tuberculosis specific antigen comprises 16KD,38KD and Ag85A.
2. The mycobacterium tuberculosis antibody detection kit based on the magnetic particle chemiluminescence method of claim 1, wherein: the mass ratio of the protein 16KD,38KD and Ag85A in the antigen marked by the carboxyl magnetic beads is (1-3): 1-3.
3. The mycobacterium tuberculosis antibody detection kit based on the magnetic particle chemiluminescence method of claim 1, wherein: the sample diluent comprises any one or a combination of at least two of bovine serum albumin, a rheumatoid factor adsorbent, a bacteriostatic agent, urea or a blocking agent.
4. The mycobacterium tuberculosis antibody detection kit based on the magnetic particle chemiluminescence method of claim 1, wherein: the quality control product comprises a positive quality control product and a negative quality control product, wherein the positive quality control product is a buffer solution containing humanized mycobacterium tuberculosis antibodies.
5. The mycobacterium tuberculosis antibody detection kit based on the magnetic particle chemiluminescence method of claim 1, wherein: the mycobacterium tuberculosis antibody detection kit also comprises an excitation liquid and a washing liquid.
6. The mycobacterium tuberculosis antibody detection kit based on the magnetic particle chemiluminescence method of claim 1, wherein: the preparation method of the kit comprises the following steps:
step one, preparing a magnetic particle marked mycobacterium tuberculosis specific protein antigen:
(1) Taking carboxyl magnetic particles, magnetically separating (1-2 min), and discarding the supernatant; then, taking a coupling buffer MES (pH=5.0-7.0) solution to redisperse the magnetic particles, oscillating for 5-10 min, magnetically separating, discarding the supernatant, and repeating for three times;
(2) Re-dispersing the magnetic particles by using a coupling buffer MES (pH=5.0-7.0), adding a coupling activating reagent EDC, and oscillating for 30-50 min at room temperature; magnetically separating, discarding supernatant;
(3) Adding antigen and diluting with MES coupling buffer solution, and vibrating at room temperature for 1.5-2.5 h; magnetically separating, discarding the supernatant, then taking a blocking solution mother solution (5% BSA) to block the system, and carrying out oscillation reaction for 1.5-2.5 h at room temperature; magnetically separating, discarding supernatant;
(4) Re-dispersing the magnetic particles by using a washing buffer TBS-T, oscillating for 5-10 min, magnetically separating, discarding the supernatant, and repeating for three times; washing the mixture once by using magnetic particle storage liquid, and redispersing antigen coupling magnetic particles in the magnetic particle storage liquid for standby at 2-8 ℃;
step two, preparing an acridine sulfonamide marked goat anti-human IgG antibody:
placing 0.2mg of goat anti-human IgG antibody into a glassware, and diluting the goat anti-human IgG antibody to 0.5-1 mg/mL with 0.1M CBS (pH 9.0-12.0) buffer solution; adding 2mg/mL NSP-SA-NHS solution into the antibody diluent, uniformly mixing, and keeping away from light at room temperature for 60-90 min;
adding 10% lysine solution, mixing uniformly, and placing at room temperature for 30-40 minutes in a dark place; dialyzing with 0.02M PBS buffer solution at 4deg.C overnight to obtain acridine sulfonamide labeled antibody mother liquor;
wherein the working concentration of the acridine sulfonamide marked goat anti-human IgG antibody is 0.5-1 mug/mL;
and thirdly, packaging the magnetic particle labeled mycobacterium tuberculosis specific protein antigen, the acridine sulfonamide labeled goat anti-human IgG, the sample diluent and the quality control product respectively to obtain the mycobacterium tuberculosis antibody detection kit.
7. The mycobacterium tuberculosis antibody detection kit based on the magnetic particle chemiluminescence method of claim 1, wherein: the using method of the kit comprises the following steps: adding the sample to be detected and the magnetic particles marked by the antigen into a reaction cup, incubating and magnetically washing, adding the acridine sulfonamide marked antibody, incubating and magnetically washing for the second time, and detecting to obtain a detection result.
8. The mycobacterium tuberculosis antibody detection kit based on the magnetic particle chemiluminescence method of claim 7, wherein: the sample to be tested is serum or plasma;
diluting a sample to be tested by a sample diluent;
the dilution factor of the sample to be measured is 1 (90-150);
the antigen-labeled carboxyl magnetic beads have the quantity of 40 mug/mL and the antigen-labeled carboxyl magnetic beads have the quantity of 40-60 mug/mL;
the working concentration of the acridine sulfonamide marked sheep anti-human IgG is 0.5-1 mug/mL;
the first incubation time is 5-15 min;
the second incubation time is 5-15 min.
9. The mycobacterium tuberculosis antibody detection kit based on the magnetic particle chemiluminescence method of claim 8, wherein: the index I in the detection result is more than or equal to 1.0, and the sample to be detected is positive;
and if the index I in the detection result is smaller than 1.0, the sample to be detected is negative.
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