CN111393523A - Preparation method of HA blocking agent and antibody detection system - Google Patents

Abstract

The invention belongs to the technical field of biomedicine, and discloses a preparation method of an HA blocking agent and an antibody detection system, wherein an antibody is purified by using ammonium sulfate with the concentration of 33-50%; adding glutaraldehyde into the obtained purified antibody for crosslinking; recovering and storing the antibody after dialysis; mixing the sample, the detection microspheres, the heterophilic antibody interference indication microspheres and the second antibody for marking the detectable signal to form a reaction system; and detecting the detectable signal on the microspheres in the quaternary complex of the second antibody-antigen-first antibody-microspheres in the reaction system, and comparing the detectable signal with a standard value or a standard curve to determine the existence and/or quantity of the target antigen in the reaction system. The HA blocking agent prepared by the invention HAs strong anti-interference capability, completely eliminates HA interference, does not interfere immunoassay, effectively reduces the incidence rate of false positive, and improves the accuracy and precision rate of immunoassay; the preparation method is simple, easy to control and low in cost.

Description

Preparation method of HA blocking agent and antibody detection system

Technical Field

The invention belongs to the technical field of biomedicine, and particularly relates to a preparation method of an HA blocking agent and an antibody detection system.

Background

Currently, the closest prior art: blockers, also called receptor antagonists, refer to a class of ligand substances that bind to the receptor and prevent the effect of an agonist. Antagonists have affinity for the corresponding receptor but no potency, thereby inhibiting the effect of agonists on the receptor. Antagonists may bind to the active site of the receptor, to allosteric sites, or even to sites that are not normally bioregulatory alone. Antagonists and receptors have different binding properties, so that the binding time is long or short, and the reversibility of binding is different. Antagonists are classified into competitive antagonists and non-competitive antagonists. Most drug antagonists compete with endogenous ligands or substrates for receptor binding sites to achieve their effect. Receptors are large protein molecules that can be activated by ligand binding. The receptor may be bound to the cell membrane or may be present inside the cell, such as in the nucleus or mitochondria. Receptors bind ligands non-covalently at the active site, and there may be multiple sites on a single receptor that bind different ligands. Both ligands and receptors can modulate receptor activity upon binding at the active site or allosteric site. Antagonists may be effective by binding to the active site, allosteric site, or separately to a site not normally involved in biological regulation. Tropism antibodies (HA) in serum have been shown to cause false positive or false negative interference in immunoassays, and the addition of blockers to immunoassay conjugates can eliminate the interference of HA. However, in the antibody detection, the source of HA in the sample is unknown, and the HA can not be accurately manipulated aiming at the binding site of HA, so that a blocking agent for completely eliminating HA interference does not exist at present.

The existing patent CN201811323185.4 heterophilic antibody blocker HBR-6 and the preparation method thereof comprise the amino acid sequence and the nucleotide sequence of the variable region, wherein 3 CDR sequences of the heavy chain are respectively Phe-Thr-Ala-Asp-Thr-Ser-Ser, CDR2 Glu-Asp-Asp-Pro, CDR3 is Val-L eu-Asp-Ser-Asp-Gly-Ser-Tyr, and 3 CDR sequences of the light chain are respectively CDR1 Asn-Ile-Pro-L ys-L eu-L eu-Ile-Tyr, CDR2 is Ile-Val-Met-Thr, CDR3 is Gln-Gly-Glu-Asp-Ile-Ala-Thr-Tyr-Arg, and the prepared heterophilic antibody blocker has poor affinity and anti-interference, strong capacity and is not suitable for large-scale industrial production.

The existing patent CN201811323396.8 heterophilic antibody blocker HBT-1 and the preparation method thereof comprise an amino acid sequence and a nucleotide sequence of a variable region, wherein 3 CDR sequences of a heavy chain are respectively Val-Gln-Ser-Ser-Ile-Thr, CDR2 Gln-Asp-Asp-Tyr-Ala-Tyr-Thr-L eu-Tyr, CDR3 Val-Tyr-L ys-L eu, and 3 CDR sequences of a light chain are respectively CDR 6 Gly-Asp-Val-L eu-Ser-L ys-Ser-Cys, CDR 2L ys-Asp-Arg-Phe, CDR3 His-Ile-Ile-Ser-483-Ser.

In summary, the problems of the prior art are as follows: the existing HA blocking agent HAs low affinity, can not completely eliminate HA interference, and can affect an immunoassay result; the preparation process of the HA blocking agent is complex and difficult to popularize and use.

The difficulty of solving the technical problems is as follows: currently there are commercially available HA blockers on the market such as: IIR, NS-ABT, Heteroblock, MAB33 and polyMAB33 have poor affinity, and the effect of reducing interference by combining with HA is not obvious, so that error results appear; in addition, the existing preparation method of the HA blocking agent is complex, is not suitable for large-scale production and is difficult to popularize and use.

The significance of solving the technical problems is as follows: after the problems are solved, the HA blocking agent can completely eliminate HA interference, so that the interference of the combination of residual HA and fragments of animal immunoglobulin on an experiment is avoided, and the accuracy of sample detection is improved; the improvement of the preparation method can reduce the complexity of the preparation of the HA blocking agent, and is convenient for popularization and use. The prepared HA blocking agent can block more false positives than the traditional blocking method; and the application range is wide, and the antibody can block all species (anti-rabbitt, anti-coat, andanti-mouse) and be used as a general reagent, thereby providing more convenience for detection.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a preparation method of an HA blocking agent and an antibody detection system.

The invention is realized in such a way that a preparation method of the HA blocking agent comprises the following steps:

firstly, resuspending and dissolving the precipitate by phosphate buffered saline solution with the volume which is twice that of the original ascites and is 0.01MpH7.4, and dialyzing by phosphate buffered saline solution with the volume of 0.01 MpH7.4; discarding the supernatant, removing the dialysis bag, transferring the antibody to a beaker, and pouring saturated ammonium sulfate solution with the volume of the original ascites into a measuring cylinder under the stirring state, wherein the saturation degree of the ammonium sulfate is 33%; stirring, mixing, packaging into a centrifugal cup, balancing, centrifuging, and removing supernatant to obtain purified antibody;

secondly, pouring the obtained antibody into a beaker, and carrying out ice bath; placing the ice bath on a magnetic stirrer, and adding 25% glutaraldehyde while stirring to a final concentration of 0.4 mg/ml;

thirdly, filtering by using slow filter paper, detecting the concentration of the antibody by using an ultraviolet spectrophotometer OD280nm, diluting by using PBS buffer solution, adding glycerol, adding Proclin300, and storing;

and fourthly, dripping the HBR diluent into a culture medium containing an HA sample to be tested, adding 100u L of the HBR diluent into each hole, culturing in the environment with 95% of air, 5% of carbon dioxide and 70-80% of humidity at 37 ℃ for 2-3 hours, mixing the sample, the detection microspheres, the heterophilic antibody interference indication microspheres and a second antibody labeled with a detectable signal to form a reaction system, detecting the detectable signal on the microspheres in the second antibody-antigen-first antibody-microsphere quaternary complex in the reaction system, and comparing the detectable signal with a standard value or a standard curve to determine the existence and/or quantity of the target antigen in the reaction system.

Further, the ammonium sulfate purification antibody in the first step specifically comprises the following steps:

step one, dissolving frozen ascites in a warm bath at 37 ℃ in a sterile environment, filtering with absorbent gauze, measuring the volume of the filtered ascites, adding phosphate buffer solution with the same volume as 0.01MpH7.4, and uniformly mixing;

step two, 1/10 parts of Tris-HCl with pH8.0 is added into 1 part of ascites, and the mixture is stirred gently; gradually dropwise adding an ammonium sulfate solution until the saturation degree is 50%; stirring for 1 hour, centrifuging at 10000r/min at 4 ℃ for 20 minutes, removing supernatant, resuspending and precipitating twice with ammonium sulfate with the same volume, centrifuging again, and removing supernatant;

thirdly, resuspending and dissolving the obtained precipitate by phosphate buffer solution with the volume which is twice that of the original ascites and 0.01MpH7.4, and dialyzing by phosphate buffer solution with the volume which is 0.01MpH7.4, wherein the solution is changed for three times, and the solution changing time is not less than 4 hours;

step four, discarding the supernatant, removing the dialysis bag, transferring the antibody to a beaker, and pouring a saturated ammonium sulfate solution with the volume of the original ascites into the beaker at a constant speed by using a measuring cylinder under a stirring state, wherein the saturation degree of the ammonium sulfate is 33%; stirring for 1 hr, mixing, packaging into a centrifugal cup, balancing, centrifuging at 10000r/min at 4 deg.C for 20 min, and discarding the supernatant.

Further, the fourth step is followed by: dissolving the collected precipitate with 0.01MpH7.4 phosphate buffered saline solution of original ascites volume 1/2, dialyzing with 0.01MpH7.4 phosphate buffered saline solution, and changing the solution for 3 times for not less than 4 hr; antibody was collected and observed for precipitation: if the precipitate exists, filtering the precipitate by using quick filter paper to remove the precipitate, and measuring the volume of the antibody; if no precipitate is present, the antibody volume is directly measured, the antibody is gently mixed, the protein content of A280nm is determined using a micro-UV spectrophotometer, and the measurement is recorded diluted to 17mg/ml with 0.01MpH7.4 phosphate buffered saline, the antibody concentration is determined using a micro-UV spectrophotometer in the A280 mode, and the measurement result is recorded.

Further, the glutaraldehyde crosslinking in the second step specifically comprises the following steps:

step one, pouring the obtained antibody into a beaker, and carrying out ice bath for 1 h; placing the ice bath on a magnetic stirrer, keeping the ice amount to be over the antibody liquid level, adding 25% glutaraldehyde while stirring to a final concentration of 0.4mg/ml, wherein the adding time is 20 s;

adding 2M glycine to a final concentration of 0.01M under stirring, and stirring for 30 min;

step three, dialyzing the sample by using 0.01MpH7.4 phosphate buffer saline solution, wherein the fluid is changed twice, and the fluid changing time is not less than 6 hours;

and step four, the dialysis bag is removed, the sample is centrifuged at 5500r/min at 4 ℃ for 18min, the supernatant is taken, an ultraviolet spectrophotometer is used for measuring the antibody concentration, and the measurement result is recorded.

Further, in the third step, the filtration is performed through slow filter paper, the concentration of the antibody is detected by using an ultraviolet spectrophotometer OD280nm, the antibody is diluted by using PBS buffer solution, glycerol is added, then Proclin300 is added, and the storage specifically comprises the following steps: filtering with slow filter paper, detecting antibody concentration with ultraviolet spectrophotometer OD280nm, diluting with 0.01MpH7.4 PBS buffer solution to 10mg/ml, adding glycerol 2% of the total volume, adding Proclin300 0.1% of the total volume, and storing at-20 deg.C.

Further, the fourth step comprises the steps of placing the frozen HBR at room temperature for 15 minutes for unfreezing, stirring the unfrozen HBR until the texture is uniform to obtain HBR binding solution, enabling the HBR concentration to be 0.040mg/ml-1.0mg/ml, supplementing the HBR binding solution to 100ml with dilution buffer, stirring and uniformly mixing the HBR binding solution to obtain HBR diluent, dropping the HBR diluent into a culture medium containing an HA sample to be tested, adding 100u L of HBR diluent into each hole, and culturing the HBR diluent in an environment with 95% of air, 5% of carbon dioxide, 70% to 80% of humidity and 37 ℃ for 2 to 3 hours.

Further, the reaction system in the fourth step specifically includes:

the detection microsphere is a first antibody-microsphere binary complex represented by formula I:

Anti1X-bead(I)

wherein X represents a target antigen, anti1X represents a first antibody against the target antigen X, bead represents a microsphere, and the binding mode between the first antibody and the microsphere is represented.

Further, the second antibody and the first antibody in the fourth step can be simultaneously combined to different epitopes of the target antigen to form a quaternary complex of the second antibody-antigen-first antibody-microsphere;

the heterophilic antibody interference indicating microsphere is a heterophilic interference indicator-microsphere binary complex represented by formula II:

Z-bead″(II)

wherein Z represents an heterophilic interference indicator, bead "represents a different microsphere distinguishable from bead, and-represents the binding mode between the heterophilic interference indicator and the microsphere, and forms a second antibody-heterophilic interference indicator-microsphere quaternary complex in the presence of heterophilic antibody in a sample.

Further, the fourth step further includes: detecting a detectable signal on the microsphere in a second antibody-heterophilic interference indicator-microsphere quaternary compound in a reaction system to obtain a signal value of heterophilic antibody interference indicator microsphere, comparing the signal value with the signal value of heterophilic antibody interference indicator microsphere without heterophilic antibody interference effect, namely a normal value, and judging that the determination result of the target antigen is unreliable when the measured value of the heterophilic antibody interference indicator microsphere is 1.5 times greater than the normal value of the heterophilic antibody interference indicator microsphere; and if the measured value of the heterophilic antibody interference indication microsphere is less than or equal to 1.5 times of the normal value of the heterophilic antibody interference indication microsphere, judging that no heterophilic antibody interference exists in the detected sample.

Another object of the present invention is to provide an antibody detection system used in the method for preparing the HA blocking agent, wherein the antibody detection system comprises:

the temperature control module is connected with the detection control center and used for regulating and controlling the temperature of the detection environment by using the heater and the radiator;

the humidity control module is connected with the detection control center and used for controlling the detection environment humidity by utilizing the humidifier and the dehumidifier;

the dilution module is connected with the detection control center and used for diluting the HBR to a specified concentration by using water;

the stirring module is connected with the detection control center and used for uniformly stirring the HBR or the HBR diluent by using a stirrer;

the detection control center is connected with the temperature control module, the humidity control module, the dilution module, the stirring module, the time control module, the dripping module and the detection module and is used for presetting relevant temperature, humidity, time and concentration parameters and regulating and controlling each module to complete corresponding functions;

the time control module is connected with the detection control center and used for controlling the time of the detection process by utilizing the timer;

the dropwise adding module is connected with the detection control center and is used for dropwise adding the diluted HBR solution into a culture medium containing an HA sample;

and the detection module is connected with the detection control center and is used for performing antigen detection by using the detection microspheres and the heterophilic antibody interference indication microspheres.

In summary, the advantages and positive effects of the invention are: the HA blocking agent prepared by the invention HAs strong anti-interference capability, can completely eliminate HA interference, does not interfere immunoassay, effectively reduces the incidence rate of false positive, and can improve the accuracy and precision rate of immunoassay; meanwhile, the preparation method is simple, the process is easy to control, and various reagents are common laboratory reagents, so that the cost is low; and the preparation environment at normal temperature is kept, the stability of the analyte and the corresponding blocking agent is ensured, and the method is safe and reliable.

Meanwhile, the invention also provides a corresponding test method, which proves that the blocking agent prepared by the invention has excellent performance and can realize the purposes of eliminating interference and improving the accuracy of immunoassay. The HA blocking agent and the preparation method thereof provided by the invention screen the HA blocking agent, obtain the variable regions of the heavy chain and the light chain of the HA blocking agent, obtain the unique nucleic acid common sequence after sequencing and have good affinity.

Drawings

FIG. 1 is a flow chart of the preparation method of the HA blocking agent provided by the embodiment of the invention.

FIG. 2 is a flow chart of a method for purifying antibodies provided in an embodiment of the invention.

FIG. 3 is a schematic diagram of the structure of an antibody detection system provided in the embodiments of the present invention;

in the figure: 1. a temperature control module; 2. a humidity control module; 3. a dilution module; 4. a stirring module; 5. detecting a control center; 6. a time control module; 7. a dropping module; 8. and a detection module.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

The present invention will be described in detail below with reference to the accompanying drawings.

The HA blocking agent provided by the embodiment of the invention comprises a heavy chain and a light chain; the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 1 is shown in the specification; the nucleotide sequence of the heavy chain variable region is shown as SEQ ID NO: 2 is shown in the specification; the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 3 is shown in the specification; the nucleotide sequence of the light chain variable region is shown as SEQ ID NO: 4, respectively.

The CDR sequences of the 3 complementarity determining regions of the heavy chain are respectively CDR 1: VQSSIT, CDR 2: QDDYAYT L Y and CDR 3: VYK L.

The CDR sequences of the 3 complementarity determining regions of the light chain are respectively CDR1, GDV L SKSC, CDR2, KDRF and CDR3, HIISTS.

The gene sequence of the HA blocker provided by the embodiment of the invention specifically comprises:

SEQ ID NO: 1: heavy chain variable region amino acid sequence:

EVQLQQSGAELMKPGASVKISCKATGYTFSSYWIEWVKQRPGHGLEWSANILPGSGSPNYNEKFKGKDTFTADTSSNTAYMVQSSITSEDSAVYYCARRGRGFPYWGQGTLVTVSA

AKTTAPSVYPLAPVCGDTTGSSVTLRCLVKGYSPEPVTLPWNSGSLSSGVHTLPAVLQSGLNTLSSSLTVTSSTSPSQSITRNVANPASSPKVDKKIEPRGPTIKPCPRCKCPAPNLLGGPSVFIGPPKIKNVLMISKSPIVTYVVVDKSEDDPDVQISWFVGNVEVHTAQDDYAYTLYHSTLRAVSALPIQAQDWMPGKEFKCKDNNKDLAAPIRRTISKAKGSVRAPQVYVTPPREEERTKKQVTRTCMVRDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFVYKLLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSR；

SEQ ID NO: 2: heavy chain variable region nucleotide sequence:

GAGGTGCAGCTGCAGCAGAGCGGCGCCGAGCTGATGAAGCCCGGCGCCAGCGTGAAGATCAGCTGCAAGGCCACCGGCTACACCTTCAGCAGCTACTGGATCGAGTGGGTGAAGCAGAGACCCGGCCACGGCCTGGAGTGGAGCGCCAACATCCTGCCCGGCAGCGGCAGCCCCAACTACAACGAGAAGTTCAAGGGCAAGGACACCTTCACCGCCGACACCAGCAGCAACACCGCCTACATGGTGCAGAGCAGCATCACCAGCGAGGACAGCGCCGTGTACTACTGCGCCAGAAGAGGCAGAGGCTTCCCCTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCGCCGCCAAGACCACCGCCCCCAGCGTGTACCCCCTGGCCCCCGTGTGCGGCGACACCACCGGCAGCAGCGTGACCCTGAGATGCCTGGTGAAGGGCTACAGCCCCGAGCCCGTGACCCTGCCCTGGAACAGCGGCAGCCTGAGCAGCGGCGTGCACACCCTGCCCGCCGTGCTGCAGAGCGGCCTGAACACCCTGAGCAGCAGCCTGACCGTGACCAGCAGCACCAGCCCCAGCCAGAGCATCACCAGAAACGTGGCCAACCCCGCCAGCAGCCCCAAGGTGGACAAGAAGATCGAGCCCAGAGGCCCCACCATCAAGCCCTGCCCCAGATGCAAGTGCCCCGCCCCCAACCTGCTGGGCGGCCCCAGCGTGTTCATCGGCCCCCCCAAGATCAAGAACGTGCTGATGATCAGCAAGAGCCCCATCGTGACCTACGTGGTGGTGGACAAGAGCGAGGACGACCCCGACGTGCAGATCAGCTGGTTCGTGGGCAACGTGGAGGTGCACACCGCCCAGGACGACTACGCCTACACCCTGTACCACAGCACCCTGAGAGCCGTGAGCGCCCTGCCCATCCAGGCCCAGGACTGGATGCCCGGCAAGGAGTTCAAGTGCAAGGACAACAACAAGGACCTGGCCGCCCCCATCAGAAGAACCATCAGCAAGGCCAAGGGCAGCGTGAGAGCCCCCCAGGTGTACGTGACCCCCCCCAGAGAGGAGGAGAGAACCAAGAAGCAGGTGACCAGAACCTGCATGGTGAGAGACTTCATGCCCGAGGACATCTACGTGGAGTGGACCAACAACGGCAAGACCGAGCTGAACTACAAGAACACCGAGCCCGTGCTGGACAGCGACGGCAGCTACTTCGTGTACAAGCTGCTGAGAGTGGAGAAGAAGAACTGGGTGGAGAGAAACAGCTACAGCTGCAGCGTGGTGCACGAGGGCCTGCACAACCACCACACCACCAAGAGCTTCAGCAGATGA；

SEQ ID NO: 3: light chain variable region amino acid sequence:

DIVMTQTPSSLSASLGDVLSKSCHASQNIKVWLSWYQQKPGNIPKLLIYKTSNLHTGVSSRFSGSGSGTAFTLTISSLQGEDIATYYCQQGQSYPFTFGGGTKLEIKRADVAPTASIFPPSSEQLTSGGASVVCFLNKDRFKDSNVKCEIDGSERQNGVLNPWTDQDSKHSTYSTSSTLTLTKDEYERHNSYTTEATHIISTSPIVRSFNRNEGIRDRADLIG；

SEQ ID NO: 4: light chain variable region nucleotide sequence:

GACATCGTGATGACCCAGACCCCCAGCAGCCTGAGCGCCAGCCTGGGCGACGTGCTGAGCAAGAGCTGCCACGCCAGCCAGAACATCAAGGTGTGGCTGAGCTGGTACCAGCAGAAGCCCGGCAACATCCCCAAGCTGCTGATCTACAAGACCAGCAACCTGCACACCGGCGTGAGCAGCAGATTCAGCGGCAGCGGCAGCGGCACCGCCTTCACCCTGACCATCAGCAGCCTGCAGGGCGAGGACATCGCCACCTACTACTGCCAGCAGGGCCAGAGCTACCCCTTCACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGAGAGCCGACGTGGCCCCCACCGCCAGCATCTTCCCCCCCAGCAGCGAGCAGCTGACCAGCGGCGGCGCCAGCGTGGTGTGCTTCCTGAACAAGGACAGATTCAAGGACAGCAACGTGAAGTGCGAGATCGACGGCAGCGAGAGACAGAACGGCGTGCTGAACCCCTGGACCGACCAGGACAGCAAGCACAGCACCTACAGCACCAGCAGCACCCTGACCCTGACCAAGGACGAGTACGAGAGACACAACAGCTACACCACCGAGGCCACCCACATCATCAGCACCAGCCCCATCGTGAGAAGCTTCAACAGAAACGAGGGCATCAGAGACAGAGCCGACCTGATCGGCTGA。

as shown in fig. 1, the preparation method of the HA blocking agent provided by the embodiment of the present invention includes the following steps:

s101: adding 33% -50% ammonium sulfate solution into antibody, filtering, mixing, stirring, centrifuging, dissolving, dialyzing, stirring, centrifuging, dissolving, filtering, etc. to purify antibody.

S102: slowly adding glutaraldehyde and glycine into the purified antibody obtained in the step S101 and stirring; and adding phosphate buffer salt solution for dialysis and centrifugation to perform crosslinking of the antibody.

S103: after completion of dialysis, the antibody was recovered and filtered through a slow filter while detecting the antibody concentration by using an ultraviolet spectrophotometer OD280nm, diluted to 10mg/ml with 0.01M PBS buffer solution having a pH of 7.4, added with 2% glycerol in total volume, and then added with 0.1% Proclin300 in total volume and stored at-20 ℃.

S104: and (4) detecting the antibody obtained in the step (S103) by using a detection reaction system.

As shown in fig. 2, in step S101, the ammonium sulfate purified antibody provided in the embodiment of the present invention specifically includes the following steps:

s201: dissolving frozen ascites in sterile environment at 37 deg.C, filtering with absorbent gauze, measuring the volume of the filtered ascites, adding phosphate buffered saline solution with equal volume of pH7.40.01M, and mixing.

S202: 1/10 parts of Tris-HCl (pH8.0) is added into 1 part of ascites, and the mixture is stirred gently; gradually dropwise adding an ammonium sulfate solution until the saturation degree is 50%; stirring for 1 hour, centrifuging at 10000r/min at 4 ℃ for 20 minutes, removing the supernatant, resuspending the precipitate twice with equal volume of ammonium sulfate, centrifuging again, and removing the supernatant.

S203: the obtained precipitate was resuspended and dissolved in pH7.40.01M phosphate buffered saline twice the volume of the original ascites, and dialyzed against pH7.40.01M phosphate buffered saline, during which the fluid was changed three times for not less than 4 hours.

S204: discarding the supernatant, removing the dialysis bag, transferring the antibody to a beaker, slowly pouring saturated ammonium sulfate solution with the volume of the original ascites into the beaker by using a measuring cylinder at a constant speed under the condition of slow stirring, wherein the saturation degree of the ammonium sulfate is about 33%; stirring for 1 hr, mixing, packaging into a centrifugal cup, balancing, centrifuging at 10000r/min at 4 deg.C for 20 min, and discarding the supernatant.

S205: dissolving the collected precipitate with pH7.40.01M phosphate buffered saline solution of original ascites volume 1/2, and dialyzing with pH7.40.01M phosphate buffered saline solution for 3 times with the change of the solution not less than 4 hr.

S206: antibody was collected and observed for precipitation: if the precipitate exists, filtering the precipitate by using quick filter paper to remove the precipitate, and measuring the volume of the antibody; if no precipitate is present, the antibody volume is directly measured, the antibody is gently mixed, the protein content of A280nm is determined using a micro-UV spectrophotometer, and the measurement is recorded.

S207: the solution was diluted to 17mg/ml with pH7.40.01M phosphate buffered saline, the antibody concentration was determined using a micro-UV spectrophotometer (A280 mode assay), and the results were recorded.

In step S102, the glutaraldehyde crosslinking provided in the embodiment of the present invention specifically includes the following steps:

(1) pouring the antibody obtained in the step S101 into a beaker, and carrying out ice bath for 1 h; the ice bath was placed on a magnetic stirrer, keeping the amount of ice above the antibody level, and 25% glutaraldehyde was added with rapid stirring to a final concentration of 0.4mg/ml for 20 s.

(2) Under rapid stirring, 2M glycine was added uniformly to a final concentration of 0.01M, and the mixture was stirred slowly for 30 min.

(3) Dialyzing the sample with phosphate buffered saline solution of pH7.40.01M, wherein the fluid is changed twice, and the fluid changing time is not less than 6 hours.

(4) The dialysis bag is disassembled, the sample is 5500r/min, centrifuged at 4 ℃ for 18min, the supernatant is taken, the antibody concentration is determined by using an ultraviolet spectrophotometer, and the measurement result is recorded.

In step S104, the antibody detection provided in the embodiment of the present invention specifically includes:

(1) unfreezing frozen HBR at room temperature for 15 minutes, and stirring until the texture is uniform after unfreezing to obtain HBR binding solution (the HBR concentration is 0.040mg/ml-1.0 mg/ml); and (3) complementing the HBR binding solution to 100ml by using a dilution buffer solution, stirring and uniformly mixing to obtain the HBR dilution solution.

(2) And dropwise adding the HBR diluent into a culture medium containing an HA sample to be tested, adding 100u L of the HBR diluent into each hole, and culturing for 2-3 hours in an environment with air of 95%, carbon dioxide of 5%, humidity of 70-80% and temperature of 37 ℃.

(3) And (3) detecting the sample obtained in the step (2).

In step (3), the method for detecting a sample provided in the embodiment of the present invention specifically includes:

(a) the sample, the detection microspheres, the heterophilic antibody interference indication microspheres and the second antibody labeled with the detectable signal are mixed to form a reaction system.

Wherein the detection microsphere is a first antibody-microsphere binary complex represented by formula I:

Anti1X-bead(I)

in the formula, "X" represents a target antigen, "anti 1X" represents a first antibody against the target antigen "X," bead "represents a microsphere, and" - "represents a binding mode between the first antibody and the microsphere.

The second antibody and the first antibody can simultaneously bind to different epitopes of the target antigen, thereby forming a quaternary complex of "second antibody-antigen-first antibody-microsphere".

The heterophilic antibody interference indicator microsphere is a heterophilic interference indicator-microsphere binary complex represented by formula II:

Z-bead″(II)

wherein "Z" represents an heterophilic interference indicator, "bead" represents a different microsphere that is distinguishable from "bead," and "-" represents the manner of binding between the heterophilic interference indicator and the microsphere, such that a "second antibody-heterophilic interference indicator-microsphere" quaternary complex is formed in the presence of heterophilic antibody in a sample.

(b) And detecting the detectable signal on the microspheres in the quaternary complex of the second antibody-antigen-first antibody-microspheres in the reaction system, and comparing the detectable signal with a standard value or a standard curve so as to determine the existence and/or quantity of the target antigen in the reaction system.

Detecting a detectable signal on the microsphere in the quaternary complex of the second antibody, the heterophilic interference indicator and the microsphere in the reaction system to obtain a signal value of the heterophilic antibody interference indicator microsphere, comparing the signal value with a signal value, namely a normal value, of the heterophilic antibody interference indicator microsphere when the heterophilic antibody interference indicator has no heterophilic antibody interference effect, and judging that the determination result of the target antigen is unreliable when the measured value of the heterophilic antibody interference indicator microsphere is 1.5 times greater than the normal value of the heterophilic antibody interference indicator microsphere; and if the measured value of the heterophilic antibody interference indication microsphere is less than or equal to 1.5 times of the normal value of the heterophilic antibody interference indication microsphere, judging that no heterophilic antibody interference exists in the detected sample.

As shown in fig. 3, the antibody detection system provided in the embodiment of the present invention specifically includes:

temperature control module 1: the detection control center 5 is connected with a heater and a radiator for regulating and controlling the temperature of the detection environment;

the humidity control module 2: the detection control center 5 is connected with the humidifier and the dehumidifier and is used for controlling the humidity of the detection environment;

a dilution module 3: is connected with the detection control center 5 and is used for diluting the HBR to a specified concentration by using water;

and (4) a stirring module: is connected with the detection control center 5 and is used for uniformly stirring the HBR or HBR diluent by using a stirrer;

the detection control center 5: the device is connected with a temperature control module 1, a humidity control module 2, a dilution module 3, a stirring module 4, a time control module 6, a dripping module 7 and a detection module 8, and is used for presetting relevant temperature, humidity, time and concentration parameters and regulating and controlling each module to complete corresponding functions;

the time control module 6: the detection control center 5 is connected with the detection control center and is used for controlling the time of the detection process by using a timer;

and a dropping module 7: is connected with the detection control center 5 and is used for dripping the diluted HBR solution into a culture medium containing an HA sample;

the detection module 8: is connected with the detection control center 5 and is used for antigen detection by utilizing the detection microspheres and the heterophilic antibody interference indication microspheres.

The technical effects of the present invention are further illustrated by the following experiments:

the HA blocking agent in the experiment verifies the accuracy of the immunodetection result, and a glomerular sclerosis rat model is established for verifying the effect of the HA blocking agent on HA in an immunodetection conjugate.

Experiment one

1 course of the experiment

1.1 model establishment:

12 male SD rats were randomly divided into a normal control group (sham) and a model group (8 animals each), and the experiment was started after adaptive feeding in an SPF-grade animal room for one week, during which time the animals were freely fed water and naturally irradiated with light. A rat glomerulosclerosis model is constructed by combining left nephrectomy with doxorubicin hydrochloride injection to the tail vein of a rat.

The specific method comprises the following steps: with 10% chloral hydrate (in 0.3ml 100 g)^-1Conversion) abdominal cavity injection anesthesia rat, fix on the back, abdominal skin preparation, iodophor disinfection, under aseptic condition, make a longitudinal incision about 3cm along the left 1cm of abdominal midline, separate skin, tissue layer by layer, expose left side kidney, ligate renal artery and renal vein together with No. 4-0 operation suture line in the position close to the hilum of the kidney, and keep away from ligature department 0.5cm and carry out secondary ligation again, and stop bleeding with hemostatic forceps here, in the middle of two ligations, cut off renal vein and renal artery together with the operation scissors, if there is not the condition of bleeding oozing blood, loosen hemostatic forceps, free the kidney and do not damage adrenal gland, ligate left side ureter simultaneously. Then 1ml of cefazolin sodium pentahydrate (1 g.100 ml) is added^-1Double distilled water) was applied to the operative group for local infection resistance, followed by sequential suture of the muscle layer and the skin layer, and closure of the abdominal cavity. The operation is carried out according to the operation steps in the sham operation group, only the left kidney is dissociated and touches the kidney capsule, the kidney is not resected, ligation and resection are not carried out on the kidney, the operation is continuously carried out for three days, 1ml of cefazolin sodium pentahydrate (1 g.100 ml-1 double distilled water) is injected into the abdominal cavity every day, and meanwhile, the wound is disinfected by iodophor at the operation suture position to prevent infection. Meanwhile, doxorubicin hydrochloride injections 5 mg/kg each were injected from the tail vein on days 7 and 14 after the left kidney of the rat was excised^-1。

1.2 establishment of evaluation method of glomerulosclerosis rat model

1) And (3) representing the metabolic profile by using a multivariate statistical analysis method, performing pattern recognition on the data by using a Principal Component Analysis (PCA) method, and inspecting the separation condition of each group of data profiles.

The method comprises the steps of collecting urine of a rat model on the 7 th day, the 10 th day and the 14 th day after a rat operation respectively, firstly conducting L C-MS analysis on the urine of the rat model collected on the 7 th day, the 10 th day and the 14 th day after the rat operation respectively to obtain a L C-MS spectrum of the rat model, then conducting multivariate statistical analysis on a L C-MS spectrum integral data matrix of the rat model to obtain a contour map of the rat model, further conducting contour dynamic analysis on the contour map of the rat model to obtain a contour dynamic change trend map of the rat model, wherein the degree of deviation of a model group from a normal control group is different at different time points, the deviation degree is the largest at the 14 th day after the model copying operation, and the fact that a metabolic regulation network is changed remarkably on the 14 th day after the operation proves that the glomerular sclerosis model is copied successfully.

2) The urine profile of the model group at the 14 th day after operation is further analyzed by using partial least squares-discriminant analysis (P L S-DA) on the basis of PCA dynamic analysis, and a urine profile map of the model group at the 14 th day after operation is obtained, then a response displacement test is used for verifying the model (R2 value represents the interpretation capability of the model, Q2 value represents the prediction capability of the model, Q2>0.5 indicates that the model has better prediction capability and is effective, Q2>0.9 indicates that the model has prominent effect), the response displacement test results of the model are that R2 is 0.54 and Q2 is-0.192, the response displacement test results of Q2<0 indicates that the modeling is successful and no overfitting occurs, sample points in each group in the score map tend to be concentrated, the normal control group is further separated from the model group, the response displacement test indicates that the model has better fitting degree, the construction of the P L S-DA model is successful, the final construction of the P L S-DA model is verified, the analysis results are described successfully, and the variation of the metabolic load of the kidney glomerular sclerosis is likely to be found by using a statistical variable of a metabolic load, and a variable of a kidney factor group (VIP) is likely to form a statistical variable of a model.

L C-MS analysis of urine collected from the rat model on day 14 gave an integral of the L C-MS profile of the rat model, giving a change in the content of 15 biomarkers:

3) firstly, analyzing the dynamic change trend graph of the contour obtained in the step 1), and compared with the dynamic change trend graph on the 0 th day, the dynamic change trend graph has the maximum deviation degree when the model is constructed on the 14 th day; then, the analysis step 2) gave a change in the content of 15 biomarkers.

1.3HA blocking agent addition

2 biological assay results

The rat kidney of the model group is obviously reduced, fine particles are on the surface, the color is pale, the capillary vessel of the renal tubule is collapsed, mesangial cells and stroma are obviously proliferated, the wall of the renal glomerulus sac is thickened, interstitial cells are increased, fibers are increased, the renal tubule at the far end is expanded, inflammatory cell infiltration is frequently accompanied, and the characteristic of glomerular sclerosis is very obvious.

Experiment two

1 course of the experiment

1.1 model establishment:

12 male SD rats were randomly divided into a normal control group (sham) and a model group (8 animals each), and the experiment was started after adaptive feeding in an SPF-grade animal room for one week, during which time the animals were freely fed water and naturally irradiated with light. A rat glomerulosclerosis model is constructed by combining left nephrectomy with doxorubicin hydrochloride injection to the tail vein of a rat.

The specific method comprises the following steps: with 10% chloral hydrate (in 0.3ml 100 g)^-1Conversion) abdominal cavity injection anesthesia rat, fix on the back, abdominal skin preparation, iodophor disinfection, under aseptic condition, make a longitudinal incision about 3cm along the left 1cm of abdominal midline, separate skin, tissue layer by layer, expose left side kidney, ligate renal artery and renal vein together with No. 4-0 operation suture line in the position close to the hilum of the kidney, and keep away from ligature department 0.5cm and carry out secondary ligation again, and stop bleeding with hemostatic forceps here, in the middle of two ligations, cut off renal vein and renal artery together with the operation scissors, if there is not the condition of bleeding oozing blood, loosen hemostatic forceps, free the kidney and do not damage adrenal gland, ligate left side ureter simultaneously. Then 1ml of cefazolin sodium pentahydrate (1 g.100 ml) is added^-1Double distilled water) was applied to the operative group for local infection resistance, followed by sequential suture of the muscle layer and the skin layer, and closure of the abdominal cavity. The operation is carried out according to the operation steps in the sham operation group, only the left kidney is dissociated and touches the kidney capsule, the kidney is not resected, ligation and resection are not carried out on the kidney, the operation is continuously carried out for three days, 1ml of cefazolin sodium pentahydrate (1 g.100 ml-1 double distilled water) is injected into the abdominal cavity every day, and meanwhile, the wound is disinfected by iodophor at the operation suture position to prevent infection. Meanwhile, doxorubicin hydrochloride injections 5 mg/kg each were injected from the tail vein on days 7 and 14 after the left kidney of the rat was excised^-1。

1.2 establishment of evaluation method of glomerulosclerosis rat model

1) And (3) representing the metabolic profile by using a multivariate statistical analysis method, performing pattern recognition on the data by using a Principal Component Analysis (PCA) method, and inspecting the separation condition of each group of data profiles.

The method comprises the steps of collecting urine of a rat model on the 7 th day, the 10 th day and the 14 th day after a rat operation respectively, firstly conducting L C-MS analysis on the urine of the rat model collected on the 7 th day, the 10 th day and the 14 th day after the rat operation respectively to obtain a L C-MS spectrum of the rat model, then conducting multivariate statistical analysis on a L C-MS spectrum integral data matrix of the rat model to obtain a contour map of the rat model, further conducting contour dynamic analysis on the contour map of the rat model to obtain a contour dynamic change trend map of the rat model, wherein the degree of deviation of a model group from a normal control group is different at different time points, the deviation degree is the largest at the 14 th day after the model copying operation, and the fact that a metabolic regulation network is changed remarkably on the 14 th day after the operation proves that the glomerular sclerosis model is copied successfully.

2) The urine profile of the model group at the 14 th day after operation is further analyzed by using partial least squares-discriminant analysis (P L S-DA) on the basis of PCA dynamic analysis, and a urine profile map of the model group at the 14 th day after operation is obtained, then a response displacement test is used for verifying the model (R2 value represents the interpretation capability of the model, Q2 value represents the prediction capability of the model, Q2>0.5 indicates that the model has better prediction capability and is effective, Q2>0.9 indicates that the model has prominent effect), the response displacement test results of the model are that R2 is 0.54 and Q2 is-0.192, the response displacement test results of Q2<0 indicates that the modeling is successful and no overfitting occurs, sample points in each group in the score map tend to be concentrated, the normal control group is further separated from the model group, the response displacement test indicates that the model has better fitting degree, the construction of the P L S-DA model is successful, the final construction of the P L S-DA model is verified, the analysis results are described successfully, and the variation of the metabolic load of the kidney glomerular sclerosis is likely to be found by using a statistical variable of a metabolic load, and a variable of a kidney factor group (VIP) is likely to form a statistical variable of a model.

L C-MS analysis of urine collected from the rat model on day 14 gave an integral of the L C-MS profile of the rat model, giving a change in the content of 15 biomarkers:

3) firstly, analyzing the dynamic change trend graph of the contour obtained in the step 1), and compared with the dynamic change trend graph on the 0 th day, the dynamic change trend graph has the maximum deviation degree when the model is constructed on the 14 th day; then, the analysis step 2) gave a change in the content of 15 biomarkers.

2 biological assay results

The kidney of the rat in the model group is slightly reduced, fine particles are on the surface, the color is pale, the capillary vessel of the renal tubule is collapsed, mesangial cells and stroma are proliferated, the wall of the renal glomerulus sac is thickened, interstitial cells are increased, fibers are increased, the renal tubule at the far end is expanded, inflammatory cell infiltration is frequently accompanied, and the characteristic of glomerular sclerosis is obvious.

The experimental result shows that the HA interference is completely eliminated by adding the HA blocking agent, the interference of the residual HA and the fragment of the animal immunoglobulin on the experiment is avoided, and the accuracy of sample detection is improved.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

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Claims (10)

1. A preparation method of an HA blocking agent is characterized by comprising the following steps:

firstly, resuspending and dissolving the precipitate by phosphate buffered saline solution with the volume which is twice that of the original ascites and is 0.01MpH7.4, and dialyzing by phosphate buffered saline solution with the volume of 0.01 MpH7.4; discarding the supernatant, removing the dialysis bag, transferring the antibody to a beaker, and pouring saturated ammonium sulfate solution with the volume of the original ascites into a measuring cylinder under the stirring state, wherein the saturation degree of the ammonium sulfate is 33%; stirring, mixing, packaging into a centrifugal cup, balancing, centrifuging, and removing supernatant to obtain purified antibody;

secondly, pouring the obtained antibody into a beaker, and carrying out ice bath; placing the ice bath on a magnetic stirrer, and adding 25% glutaraldehyde while stirring to a final concentration of 0.4 mg/ml;

thirdly, filtering by using slow filter paper, detecting the concentration of the antibody by using an ultraviolet spectrophotometer OD280nm, diluting by using PBS buffer solution, adding glycerol, adding Proclin300, and storing;

and fourthly, dripping the HBR diluent into a culture medium containing an HA sample to be tested, adding 100u L of the HBR diluent into each hole, culturing in the environment with 95% of air, 5% of carbon dioxide and 70-80% of humidity at 37 ℃ for 2-3 hours, mixing the sample, the detection microspheres, the heterophilic antibody interference indication microspheres and a second antibody labeled with a detectable signal to form a reaction system, detecting the detectable signal on the microspheres in the second antibody-antigen-first antibody-microsphere quaternary complex in the reaction system, and comparing the detectable signal with a standard value or a standard curve to determine the existence and/or quantity of the target antigen in the reaction system.

2. The method of claim 1, wherein the purification of the antibody from ammonium sulfate in the first step comprises the steps of:

step one, dissolving frozen ascites in a warm bath at 37 ℃ in a sterile environment, filtering with absorbent gauze, measuring the volume of the filtered ascites, adding phosphate buffer solution with the same volume as 0.01MpH7.4, and uniformly mixing;

step two, 1/10 parts of Tris-HCl with pH8.0 is added into 1 part of ascites, and the mixture is stirred gently; gradually dropwise adding an ammonium sulfate solution until the saturation degree is 50%; stirring for 1 hour, centrifuging at 10000r/min at 4 ℃ for 20 minutes, removing supernatant, resuspending and precipitating twice with ammonium sulfate with the same volume, centrifuging again, and removing supernatant;

thirdly, resuspending and dissolving the obtained precipitate by phosphate buffer solution with the volume which is twice that of the original ascites and 0.01MpH7.4, and dialyzing by phosphate buffer solution with the volume which is 0.01MpH7.4, wherein the solution is changed for three times, and the solution changing time is not less than 4 hours;

step four, discarding the supernatant, removing the dialysis bag, transferring the antibody to a beaker, and pouring a saturated ammonium sulfate solution with the volume of the original ascites into the beaker at a constant speed by using a measuring cylinder under a stirring state, wherein the saturation degree of the ammonium sulfate is 33%; stirring for 1 hr, mixing, packaging into a centrifugal cup, balancing, centrifuging at 10000r/min at 4 deg.C for 20 min, and discarding the supernatant.

3. The method of claim 2, wherein step four is followed by the steps of: dissolving the collected precipitate with 0.01MpH7.4 phosphate buffered saline solution of original ascites volume 1/2, dialyzing with 0.01MpH7.4 phosphate buffered saline solution, and changing the solution for 3 times for not less than 4 hr; antibody was collected and observed for precipitation: if the precipitate exists, filtering the precipitate by using quick filter paper to remove the precipitate, and measuring the volume of the antibody; if no precipitate is present, the antibody volume is directly measured, the antibody is gently mixed, the protein content of A280nm is determined using a micro-UV spectrophotometer, and the measurement is recorded diluted to 17mg/ml with 0.01MpH7.4 phosphate buffered saline, the antibody concentration is determined using a micro-UV spectrophotometer in the A280 mode, and the measurement result is recorded.

4. The process for the preparation of HA blocking agents according to claim 1, characterized in that said glutaraldehyde cross-linking in the second step comprises in particular the following steps:

step one, pouring the obtained antibody into a beaker, and carrying out ice bath for 1 h; placing the ice bath on a magnetic stirrer, keeping the ice amount to be over the antibody liquid level, adding 25% glutaraldehyde while stirring to a final concentration of 0.4mg/ml, wherein the adding time is 20 s;

adding 2M glycine to a final concentration of 0.01M under stirring, and stirring for 30 min;

step three, dialyzing the sample by using 0.01MpH7.4 phosphate buffer saline solution, wherein the fluid is changed twice, and the fluid changing time is not less than 6 hours;

and step four, the dialysis bag is removed, the sample is centrifuged at 5500r/min at 4 ℃ for 18min, the supernatant is taken, an ultraviolet spectrophotometer is used for measuring the antibody concentration, and the measurement result is recorded.

5. The method for preparing the HA blocker according to claim 1, wherein in the third step, the filtering is performed by slow filter paper, the antibody concentration is detected by using an ultraviolet spectrophotometer OD280nm, the PBS buffer solution is used for dilution, glycerol is added, then Proclin300 is added, and the storage specifically comprises the following steps: filtering with slow filter paper, detecting antibody concentration with ultraviolet spectrophotometer OD280nm, diluting with 0.01MpH7.4 PBS buffer solution to 10mg/ml, adding glycerol 2% of the total volume, adding Proclin300 0.1% of the total volume, and storing at-20 deg.C.

6. The method for preparing the HA blocker according to claim 1, wherein the fourth step further comprises thawing frozen HBR for 15 minutes at room temperature, stirring the thawed HBR until the texture is uniform to obtain HBR binding solution, wherein the HBR concentration is 0.040mg/ml-1.0mg/ml, adding the HBR binding solution to 100ml with a dilution buffer solution, stirring and uniformly mixing the solution to obtain HBR dilution solution, dropping the HBR dilution solution into a culture medium containing an HA sample to be tested, adding 100u L of HBR dilution solution into each hole, and culturing the mixture at the temperature of 37 ℃ under the conditions of 95% of air and 5% of carbon dioxide, the humidity of 70% -80%, and the culture time of 2-3 hours.

7. The method for preparing the HA blocking agent according to claim 1, wherein the reaction system in the fourth step specifically comprises:

the detection microsphere is a first antibody-microsphere binary complex represented by formula I:

Anti1X-bead(I)

wherein X represents a target antigen, anti1X represents a first antibody against the target antigen X, bead represents a microsphere, and the binding mode between the first antibody and the microsphere is represented.

8. The method for preparing the HA blocking agent according to claim 1, wherein the second antibody and the first antibody can simultaneously bind to different epitopes of the target antigen in the fourth step to form a quaternary complex of the second antibody-antigen-first antibody-microsphere;

the heterophilic antibody interference indicating microsphere is a heterophilic interference indicator-microsphere binary complex represented by formula II:

Z-bead″(II)

wherein Z represents an heterophilic interference indicator, bead "represents a different microsphere distinguishable from bead, and-represents the binding mode between the heterophilic interference indicator and the microsphere, and forms a second antibody-heterophilic interference indicator-microsphere quaternary complex in the presence of heterophilic antibody in a sample.

9. The method of preparing the HA blocking agent of claim 1, wherein said fourth step further comprises: detecting a detectable signal on the microsphere in a second antibody-heterophilic interference indicator-microsphere quaternary compound in a reaction system to obtain a signal value of heterophilic antibody interference indicator microsphere, comparing the signal value with the signal value of heterophilic antibody interference indicator microsphere without heterophilic antibody interference effect, namely a normal value, and judging that the determination result of the target antigen is unreliable when the measured value of the heterophilic antibody interference indicator microsphere is 1.5 times greater than the normal value of the heterophilic antibody interference indicator microsphere; and if the measured value of the heterophilic antibody interference indication microsphere is less than or equal to 1.5 times of the normal value of the heterophilic antibody interference indication microsphere, judging that no heterophilic antibody interference exists in the detected sample.

10. An antibody detection system used in the method for preparing the HA blocker according to any one of claims 1 to 9, wherein the antibody detection system comprises:

the temperature control module is connected with the detection control center and used for regulating and controlling the temperature of the detection environment by using the heater and the radiator;

the humidity control module is connected with the detection control center and used for controlling the detection environment humidity by utilizing the humidifier and the dehumidifier;

the dilution module is connected with the detection control center and used for diluting the HBR to a specified concentration by using water;

the stirring module is connected with the detection control center and used for uniformly stirring the HBR or the HBR diluent by using a stirrer;

the detection control center is connected with the temperature control module, the humidity control module, the dilution module, the stirring module, the time control module, the dripping module and the detection module and is used for presetting relevant temperature, humidity, time and concentration parameters and regulating and controlling each module to complete corresponding functions;

the time control module is connected with the detection control center and used for controlling the time of the detection process by utilizing the timer;

the dropwise adding module is connected with the detection control center and is used for dropwise adding the diluted HBR solution into a culture medium containing an HA sample;

and the detection module is connected with the detection control center and is used for performing antigen detection by using the detection microspheres and the heterophilic antibody interference indication microspheres.

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