CN116217720A

CN116217720A - anti-HbA 1c nano antibody and preparation method and application thereof

Info

Publication number: CN116217720A
Application number: CN202310055153.5A
Authority: CN
Inventors: 李泓彦; 胡利华; 范海艳; 赖燕晖; 梁树旺; 李琦
Original assignee: Shenzhen Aivd Biotechnology Co ltd
Current assignee: Shenzhen Aivd Biotechnology Co ltd
Priority date: 2023-02-03
Filing date: 2023-02-03
Publication date: 2023-06-06

Abstract

The invention relates to the technical field of antibody preparation, in particular to an anti-HbA 1c nano antibody and a preparation method and application thereof, wherein the preparation method comprises the following steps: s1, constructing a nanobody phage display library; s2, screening HbA1c nanobody based on the nanobody phage display library and verifying prokaryotic expression; step S1 comprises the steps of: preparing high-activity immune antigen and immunizing target animals; isolating peripheral blood lymphocytes of the target animal to extract RNA, and obtaining cDNA through reverse transcription; and carrying out two rounds of PCR amplification by taking the cDNA as a template to obtain a nano antibody fragment, and connecting the nano antibody fragment with a display carrier to construct a nano antibody gene library and a nano antibody display library. The antibody prepared by the invention has high specific antigen recognition capability, high affinity and unique antigen determinant recognition site, and can obtain excellent detection efficiency in the immune detection of HbA1c antigen, especially in a double-antibody sandwich method.

Description

anti-HbA 1c nano antibody and preparation method and application thereof

Technical Field

The invention relates to the technical field of antibody preparation, in particular to a nano antibody for resisting HbA1c, and a preparation method and application thereof.

Background

Hamers-Casterman et al, university of Brussell, 1993, first reported that there was another type of antibody in camel blood in addition to the traditional IgG antibody, which, unlike the traditional mammalian antibody IgG molecular structure, lacks both the light chain of the traditional antibody and the CH1 region of the heavy chain constant region, known as the heavy chain antibody (Heavy chain antibody, hcAb). The antigen binding site, i.e. the variable region, also called VHH region, is directly cloned and recombinantly expressed by molecular biology techniques to obtain the variable region fragments described above, known as single domain antibodies, typically consisting of only 110-130 amino acids and having a molecular weight of only 12-15kDa, and therefore also known as nanobodies. Nanobodies are much smaller than traditional whole antibodies (150-160 kDa) and their Fab fragments (about 50 kDa), but have a similar or higher specific antigen affinity than traditional antibodies. The inherent characteristics of small molecular weight, stable physicochemical characteristics, high affinity and easy recombinant expression preparation of the single-domain antibody make the single-domain antibody become attractive after being discovered, and through more than 20 years of research, the single-domain antibody has positive progress in the fields of immune research, diagnostic detection, medical and biological imaging, therapeutic antibody development and the like.

Glycosylated hemoglobin (HbA 1 c) is a product of binding of hemoglobin in red blood cells in blood to blood glucose, and is proportional to the concentration of glucose in blood, and reflects the blood glucose level 120 days ago. And glycosylated hemoglobin is not affected by blood drawing time, fasting state, insulin use, and other factors that can cause short fluctuations in blood glucose levels. Glycosylated hemoglobin consists of HbA1a, hbA1b and HbA1c, wherein HbA1c accounts for about 70%, and has a relatively stable structure, and is clinically used as a monitoring index for diabetes control, and the concentration of the glycosylated hemoglobin is expressed as a percentage of adult hemoglobin. The glycosylated hemoglobin has strong detection specificity, good repeatability and high sensitivity, and has important significance for screening and monitoring diabetes.

With the continuous improvement of the living standard of people and the rapid aging process of population, the total number of diabetics is rapidly expanding, the diabetics and potential diabetics in various countries are rapidly increasing, and the population of the global diabetics reaches 3.5 hundred million people by 2030 in the current form. However, the current mainstream method for measuring HbA1c is direct latex immunoturbidimetry, which requires a corresponding antibody, and has certain drawbacks in terms of sensitivity and operation stability.

Disclosure of Invention

The invention aims to provide a screening method of a force nano antibody, thereby rapidly obtaining a high-affinity nano antibody

In order to solve the technical problems, the aim of the invention is realized by the following technical scheme: the preparation method of the anti-HbA 1c nano antibody comprises the following steps:

s1, constructing a nanobody phage display library;

s2, screening HbA1c nanobody based on the nanobody phage display library and verifying prokaryotic expression;

the step S1 includes the following steps:

a1, preparing a high-activity immune antigen and immunizing a target animal;

a2, separating peripheral blood lymphocytes of the target animal to extract RNA, and obtaining cDNA through reverse transcription;

a3, carrying out two-round PCR amplification by taking the cDNA as a template to obtain a nano antibody fragment, and connecting the nano antibody fragment with a display carrier to construct a nano antibody gene library and a nano antibody display library.

Preferably, the amino acid sequence of the high-activity immune antigen is shown as SEQ ID NO.1, after gene synthesis, the high-activity immune antigen is shown as SEQ ID NO.2, the high-activity immune antigen is synthesized and constructed on a mammal expression vector, and is transiently transfected into 293F cells to be expressed by PEI, and then the antigen is purified by His tag.

Preferably, in the step A1, the high activity immune antigen is mixed with freund's adjuvant according to 1: after mixing according to the proportion of 1, the immunized animals are immunized by subcutaneous multipoint injection at the back of the animal at a dose of 0.5 mg/time, the total immunization is 4 times, the immunization interval is 2 weeks, and the target animal is alpaca.

Preferably, in the step A3, the specific method for obtaining the nanobody fragment by performing two rounds of PCR amplification using the cDNA as a template is as follows: 10ml of peripheral blood of immunized animals is taken, PBMC is separated, RNA is extracted, cDNA is obtained through reverse transcription, and the primers used are random primers shown as SEQ ID NO. 3. And performing PCR amplification by taking the obtained cDNA as a template to obtain a nano antibody gene library, wherein primers used in two rounds of PCR are SEQ ID NO.4 and SEQ ID NO.5, and SEQ ID NO.6 and SEQ ID NO.7 respectively.

Preferably, in step A3, the display vector is a pad1-10b phage display vector.

Preferably, in the step A3, the nanobody fragment of the target animal is connected with the display carrier, then competent cells SS320 are transformed to prepare a nanobody gene library, and the nanobody library is infected by helper phage M13K07 to prepare a nanobody display library.

Preferably, in the step S2, the nanobody prokaryotic expression is: the screened high-affinity nano antibody sequence is amplified by PCR, is connected with a pET32a carrier, is transformed into TG1 competent cells, is transformed into BL21 (DE 3) by constructed plasmids, is subjected to IPTG induced expression, is subjected to His tag mediated Ni-agarose affinity chromatography, and is subjected to fine purification by superdex200 gel medium.

Preferably, in the step S2, the nanobody prokaryotic expression is: the high affinity nano antibody sequence is amplified by PCR, connected with pET32a carrier, transformed into SS320 competent cells, and then the constructed plasmid is transformed into Origami B (DE 3), and is induced to express by IPTG, and His tag mediated Ni-agarose affinity chromatography is used.

Preferably, in the step S2, the validation of the nanobody is as follows: the enzyme-labeled plate was coated with HbA1c antigen, blocked with 3% BSA, and washed with PBST. 100ul of nano antibodies diluted in different concentrations are added, incubated for 12 hours at room temperature, washed for 5 times by PBST, reacted for 30min by adding alpaca-HRP, washed for 5 times by PBST, developed for 30min by adding TMB, and detected on an enzyme-labeled instrument after termination.

The invention provides an anti-HbA 1c nano antibody and an application method thereof, which have the following beneficial effects:

1. the anti-HbA 1c nanobody and the application method thereof can fully exert the superior performance of the nanobody, have high specific antigen recognition capability, high affinity and unique epitope recognition sites, and can obtain excellent detection efficiency in the immunodetection of HbA1c antigen, in particular to a double-antibody sandwich method.

2. HbA1c detection kit (fluorescence immunochromatography) prepared by using the anti-HbA 1c nano antibody can meet the following performance requirements:

1) Linear range: within the range of 4 to 14 percent, the value of the clinical correlation coefficient r is more than or equal to 0.990.

2) Accuracy and precision: the relative error is within + -12%.

3) Minimum detection limit: less than or equal to 4 percent.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a schematic diagram showing the preparation of specific antigens according to the present invention

FIG. 2 is a schematic diagram showing the first round of PCR results of the nanobody of the invention;

FIG. 3 is a schematic diagram showing the second round PCR results of the nanobody of the invention;

FIG. 4 is a schematic diagram of the expression and purification of the nanobody of the invention;

FIG. 5 is a schematic diagram showing the identification of nanobody activity according to the present invention;

FIG. 6 is a schematic diagram of the detection of clinical relevance of nanobody kit of the invention;

FIG. 7 is a schematic diagram of the structure of the nanobody test paper of the invention.

Detailed Description

The technical solutions of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

It should be understood that the terms "comprises" and "comprising," when used in this specification and the appended claims, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof.

It is also to be understood that the terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in this specification and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

It should be further understood that the term "and/or" as used in the present specification and the appended claims refers to any and all possible combinations of one or more of the associated listed items, and includes such combinations.

Furthermore, the terms "substantially," "essentially," and the like, are intended to be limited to the precise form disclosed herein and are not necessarily intended to be limiting. For example: the term "substantially equal" does not merely mean absolute equal, but is difficult to achieve absolute equal during actual production and operation, and generally has a certain deviation. Thus, in addition to absolute equality, "approximately equal to" includes the above-described case where there is a certain deviation. In other cases, the terms "substantially", "essentially" and the like are used in a similar manner to those described above unless otherwise indicated.

Glycosylated hemoglobin (HbA 1 c) is the product of the combination of hemoglobin in red blood cells and carbohydrates (mainly glucose) in serum by a non-enzymatic reaction. The non-enzymatic reaction forming the glycosylated hemoglobin has the characteristics of being continuous, slow and irreversible, so that the content of the glycosylated hemoglobin is determined by the past blood glucose concentration rather than the instant blood glucose concentration, and is irrelevant to factors such as whether the patient is empty or not, whether insulin is injected or not, whether the hypoglycemic agent is taken or not before detection. It is generally believed that the glycosylated hemoglobin concentration effectively reflects the average blood glucose level over the past 8-12 weeks. Glycosylated hemoglobin consists of HbA1a, hbA1b and HbA1c, wherein HbA1c accounts for about 70%, and has a relatively stable structure, and is clinically used as a monitoring index for diabetes control, and the concentration of the glycosylated hemoglobin is expressed as a percentage of adult hemoglobin.

Glycosylated hemoglobin level can be used as diagnostic detection means for diabetes. The Chinese guidelines for preventing and treating type 2 diabetes (2020 edition) formally incorporate the glycosylated hemoglobin into the diabetes diagnosis standard for the first time, and the glycosylated hemoglobin is more than or equal to 6.5 percent and can be used as the basis for diagnosing diabetes. However, a result of less than 6.5% does not exclude the possibility of diabetes, and reference should also be made to the result of the glucose assay. If glycosylated hemoglobin is >9.0%, this indicates that the patient is continuously present with hyperglycemia.

Currently, the main method for measuring HbA1c is a direct latex immunoturbidimetry, and the corresponding antibody is required, so that certain defects exist in sensitivity and operation stability. Therefore, the invention provides a nano antibody for resisting HbA1c and a preparation method thereof. The nano antibody for resisting HbA1c has the advantages of high affinity, unique epitope recognition site and the like, and solves the problem that the existing HbA1c monoclonal antibody has certain defects in sensitivity and specificity.

Therefore, the invention provides a preparation method of a nano antibody for resisting HbA1c, which comprises the following steps:

s1, constructing a nanobody phage display library;

the step S1 includes the following steps:

a1, preparing a high-activity immune antigen and immunizing a target animal;

a3, carrying out two rounds of PCR amplification by taking the cDNA as a template to obtain a nano antibody fragment, and connecting the nano antibody fragment with a display carrier to construct a nano antibody gene library and a nano antibody display library.

The amino acid sequence of the high-activity immune antigen is shown as SEQ ID NO.1, after gene synthesis, the high-activity immune antigen is synthesized and constructed on a mammal expression vector as shown as SEQ ID NO.2, and is transiently transfected into 293F cells for expression by PEI, and then the antigen is purified by His tag.

In the step A1, the high-activity immune antigen and Freund's adjuvant are mixed according to the following ratio of 1: after mixing according to the proportion of 1, the immunized animals are immunized by subcutaneous multipoint injection at the back of the animal at a dose of 0.5 mg/time, the total immunization is 4 times, the immunization interval is 2 weeks, and the target animal is alpaca. In the step A3, the specific method for obtaining the nano antibody fragment by two PCR amplifications by taking cDNA as a template is as follows: 10ml of peripheral blood of immunized animals is taken, PBMC is separated, RNA is extracted, cDNA is obtained through reverse transcription, and the primers used are random primers shown as SEQ ID NO. 3. And performing PCR amplification by taking the obtained cDNA as a template to obtain a nano antibody gene library, wherein primers used in two rounds of PCR are SEQ ID NO.4 and SEQ ID NO.5, and SEQ ID NO.6 and SEQ ID NO.7 respectively; and the display vector is a pad1-10b phage display vector; in addition, in the step A3, the nano antibody fragment of the target animal is connected with a display carrier, then competent cells SS320 are transformed to prepare a nano antibody gene library, and then the nano antibody library is infected by an auxiliary phage M13K07 to prepare a nano antibody display library.

In step S2, nanobody prokaryotic expression is: the screened high-affinity nano antibody sequence is amplified by PCR, is connected with a pET32a carrier, is transformed into TG1 competent cells, is transformed into BL21 (DE 3) by constructed plasmids, is subjected to IPTG induced expression, is subjected to His tag mediated Ni-agose affinity chromatography, and is subjected to fine purification by superdex200 gel medium; in addition, the validation of nanobodies was: the enzyme-labeled plate was coated with HbA1c antigen, blocked with 3% BSA, and washed with PBST. 100ul of nano antibodies diluted in different concentrations are added, incubated for 12 hours at room temperature, washed for 5 times by PBST, reacted for 30min by adding alpaca-HRP, washed for 5 times by PBST, developed for 30min by adding TMB, and detected on an enzyme-labeled instrument after termination.

The relevant sequences in this text are as follows:

SEQ ID NO.1:

MVSADKTNVKAAWGKVGAHAGYGAARMSTTKTYHDSHGSAVKGHGKKVADATNAVAHVDDMNASASDHAHKRVDVNKSHCVTAAHATAVHASDKASVSTVTSKYR

SEQ ID NO.2:

ATGGTGTTGTCCCCAGCCGATAAGACAAATGTGAAAGCAGCCTGGGGCAAGGTAGGCGCACATGCTGGTGAGTACGGCGCAGAAGCCCTCGAGCGAATGTTTTTGAGTTTTCCCACCACTAAGACATATTTTCCCCATTTCGACCTGTC CCATGGATCTGCACAGGTTAAGGGGCATGGGAAGAAGGTGGCTGATGCCCTGACCAATGCAGTGGCGCATGTGGACGACATGCCCAACGCCCTTAGCGCCTTGAGCGACTTGCATGCTCACAAGCTGAGAGTTGATCCGGTAAATTTTAAGCTGCTGAGCCACTGCCTTCTGGTCACTCTCGCAGCCCACCTTCCTGCCGAGTTTACACCTGCTGTGCACGCTAGCCTGGATAAGTTTCTGGCCAGCGTGTCCACCGTCCTCACAAGTAAGTATCGGGGAGGGAGTTGA

SEQ ID NO.3:

5’-NNNNNN-3’

SEQ ID NO.4:

5’-GTCCTGGCTGCTCTTCTACAAGG-3’

SEQ ID NO.5:

5’-GGTACGTGCTGTTGAACTGTTCC-3’

SEQ ID NO.6:

5’-GATGTGCAGCTGCAGGAGTCTGGRGGAGG-3’

SEQ ID NO.7:

5’-CTAGTGCGGCCGCTGGAGACGGTGACCTGGGT-3’

the following will make specific description in connection with the examples

Example 1

HbA1c antigen preparation and alpaca immunization, comprising the following steps:

1. HbA1c antigen protein sequence was retrieved by NCBI as shown in SEQ ID NO. 1. Through codon optimization, the sequence is shown as SEQ ID NO.2, the sequence is synthesized on a vector pTT5 by a gene synthesis company, plasmids are extracted by a plasmid big extraction kit (OMEGA, D6924-04), the concentration of the plasmids is detected by an ultraviolet spectrophotometer, agarose gel detection is carried out, and the plasmid is frozen at-20C for standby

2. Resuscitating HEK293 cells with serum-free medium (KOP 293, Ry) until the density reaches 4-6×106cells/ml, passaging according to 0.5×106cells/ml, adapting to 3 passages, passaging according to 2.0×106cells/ml, measuring antigen plasmid according to 1ug/ml, and mixing with appropriate amount of serum-free medium (KPM, Ry); weighing PEI (1 mg/ml) according to 3ug/ml, mixing with equal volume of serum-free culture medium (KPM, Rayleigh) and adding PEI solution into plasmid, mixing, standing for 15min, adding into cells, and culturing in shaking table (37C, 5% CO2, 80 rpm)

3. After 24h of transfection, 50xFT-Feed,0.6% TA-293,5% glucose solution was added followed by 4-6 days of incubation to a cell density of around 60%. Centrifuging at 1000rpm at room temperature for 10min, collecting supernatant

(4) HbA1c antigen was purified with Ni Sepharose Excel (cytova, 173712) packing, dialyzed against PBS and ultrafiltered, the protein concentration was detected by spectrophotometry and the protein purity was detected by SDS-PAGE

(5) Antigen of interest and Freund's adjuvant were mixed according to 1: after mixing according to the proportion of 1, carrying out subcutaneous multipoint injection immunization on adult alpaca according to the dosage of 0.5 mg/time, carrying out immunization for 4 times at the interval of 2 weeks, taking immune serum for the fourth time, and ELISA detection of serum titer

Example 2

Preparation of nanobody phage display library

1. Collecting peripheral blood of immune alpaca, centrifuging at 2000rpm for 5min, separating PMBC, collecting 1ml PBMC, adding Trizol reagent, and extracting total RNA

2. Reverse transcription was performed using the extracted RNA as template, according to the requirements of PrimerScript II Rtase reverse transcription kit, with primer sequences as follows: the cDNA was prepared and stored at-20 ℃.

3. The cDNA is used as a template, primestar-HS is used for carrying out first round amplification, and the sequences of the upstream primer and the downstream primer are shown as SEQ ID NO.4 and SEQ ID NO. 5. Reaction conditions: 98 ℃ for 3 minutes; 94℃for 50 seconds, 55℃for 30 seconds, 68℃for 40 seconds, and 40 cycles; extension was carried out at 72℃for 10 minutes. 1% agarose gel electrophoresis, DNA recovery and concentration determination.

4. The recovered DNA is used as a template for the second round of amplification, and the sequences of the upstream primer and the downstream primer are shown as SEQ ID NO.6 and SEQ ID NO.7. Reaction conditions: 98 ℃ for 3 minutes; 94℃for 50 seconds, 55℃for 30 seconds, 68℃for 40 seconds, and 40 cycles; extension was carried out at 72℃for 10 minutes. 1% agarose gel electrophoresis, recovery of DNA fragment of about 450bp, concentration measurement, and short-term preservation at-20 ℃.

5. The VHH fragment with about 450bp obtained by the second round of PCR amplification is connected with a panl-10 b phage display vector, and is electrically converted into SS320 competence, so as to prepare a nanobody gene library. The nano-antibody library capacity was measured to be about 2×109. 12 colonies were randomly selected and identified by PCR, and all of the 12 colonies contained VHH fragments with a VHH fragment insertion rate of approximately 100%. Further adopting helper phage VCSM13 to infect SS320 nano antibody gene library to prepare phage display library.

Example 3

In this example, hbA1c nanobody was screened and expression verified

1. The immune tubes were coated with HbA1c antigen, blocked with 3% BSA, and washed with PBST. 100uL phage were added, incubated at room temperature for 2h, washed 10 times with PBST, and phage binding HbA1c eluted with trypsin. Eluted phage were transfected with SS320 and passed to the next round of screening. After 3 rounds of screening, positive clones were verified by ELISA and sequenced.

2. Positive clones obtained by sequencing were constructed into BamHI and XhoI sites of pET32a by PCR cloning, chemically transferred into an expression strain of Origami B (DE 3), plated, and cultured overnight at 37C. The monoclonal was picked and inoculated into 5mL of LB medium at 37℃and 200rpm for 6-8h, and then activated according to 1:100 were inoculated into 400mL of LB medium, cultured at 37℃until OD600 = 0.6-0.8, induced by adding IPTG at a final concentration of 0.5mM, and induced at 20℃at 200rpm for 16h.6000g, centrifuging at room temperature for 5min, collecting thalli, adding PBS according to the proportion of 10ml/g thalli to resuspend cells, performing ultrasonic disruption (5s on;5s off,15min), and centrifuging at 12000rpm for 30min. The supernatant obtained by centrifugation was filtered through a 0.45um filter, and then added to a nickel column equilibrated with equilibration solution (25mM Tris,300mM NaCl,pH 8.0), washed with a wash buffer (25mM Tris,300mM NaCl,20mM Imidazole,pH 8.0), and eluted with 25mM Tris,300mM NaCl,250mM Imidazole,pH 8.0. Ultrafiltering the collected target protein, and performing secondary purification by superdex200 gel medium to obtain high-purity nanometer antibody

3. The HbA1c antigen was diluted to 1ug/ml with the coating solution, 100 ul/well coated with the ELISA plate, and 4℃overnight. The coating solution was poured off, the ELISA plate was washed 2 times with 200ul of PBST, and the plate was beaten on absorbent paper to ensure complete pouring of the wash solution. Blocking 1 hr with 3% BSA, 200 uL/well, washing the ELISA plate 2 times with PBST, adding 100uL of purified antibody (PBS diluted to 100ng/mL final concentration), incubating for 30min at 37 degrees, washing 5 times with PBST, adding alpaca-HRP, incubating for 30min at 37 degrees. Washed 5 times with PBST, and developed for 10 minutes by adding 100ul of TMB developing solution. 50UL stop solution was added. The values were read on a microplate reader at 405nm/620 nm.

Example 4

HbA1c nanobody performance detection (reagent application)

As shown in fig. 7, the embodiment provides a HbA1C detection kit, which comprises a kit body and HbA1C detection test paper arranged in the kit body, wherein the HbA1C detection test paper comprises a bottom plate 1, a sample pad 2, a binding pad 3, a nitrocellulose membrane 4 and absorbent paper 5 which are connected end to end are sequentially arranged on the bottom plate 1 along the horizontal direction of the bottom plate, a detection line 6 and a quality control line 7 are sequentially arranged on the nitrocellulose membrane 4 along the chromatographic direction, hbA1C nano antibodies are coated on the detection line 6, chicken IgY antigens are coated on the quality control line 7, and a T probe and a C probe are arranged on the binding pad 3;

the T probe is a quantum dot fluorescent marked hemoglobin nano antibody coupling complex;

the C probe is a quantum dot fluorescent marked goat anti-chicken IgY monoclonal antibody coupling complex;

the bottom plate 1 is a PVC bottom plate.

In this example, the sample pad 2 was prepared by the following method:

preparing pretreatment liquid: 20mM BS, 5g/mL trehalose, and 1g/mL BSA; the sample pad 2 was placed in the pretreatment liquid for 1 minute, the soaked sample pad 2 was taken out to be placed in a screen, dried in a 37-degree oven for 24 hours, and the treated sample pad 2 was cut into 23mm.

The detection principle of the HbA1c detection kit provided by the invention is as follows: by adopting a quantum dot fluorescent labeling technology and a sandwich method principle, hbA1C nano antibody and chicken IgY antigen are respectively coated on a detection line 6 and a quality control line 7 area on a nitrocellulose membrane 4, and a quantum dot fluorescent labeled hemoglobin nano antibody coupling complex (T probe) and a quantum dot fluorescent labeled goat anti-chicken IgY monoclonal antibody coupling complex (C probe) are distributed on the nitrocellulose membrane 4. In the detection process, a sample to be detected is added into a sample adding hole, for example, the sample contains a detected object (glycosylated hemoglobin and hemoglobin), the detected object can be combined with a quantum dot fluorescent marked hemoglobin nano antibody coupling compound, the combined object can move onto a detection line 6 on a nitrocellulose membrane 4 through capillary action, hbA1c in the combined object can be captured by HbA1c nano antibodies contained in the combined object to form a quantum dot fluorescent detection line 6 band, and a quantum dot fluorescent marked goat anti-chicken IgY monoclonal antibody coupling compound can continuously move to a quality control line 7 area to be combined with chicken IgY to form a quantum dot fluorescent quality control line 7 band. The concentration of the measured object in the sample is positively correlated with the fluorescence intensity of the quantum dots of the detection line 6, and the concentration of the measured object in the sample can be determined by measuring and calculating by a fluorescence immunoassay analyzer and comparing with a calibration curve.

The preparation method of the HbA1c detection kit comprises the following steps:

coating

Adhering a CN140 nitrocellulose membrane to a designated position of a PVC bottom plate;

diluting HbA1c nano antibody to 1.5mg/mL by using coating liquid, diluting chicken IgY antigen to 0.4mg/mL, coating HbA1c nano antibody at the position of a detection line by using a metal spraying film drawing instrument, coating chicken IgY antigen at the position of a quality control line, drying for 48 hours in a 55-DEG oven after coating, packaging by using a self-sealing bag, packaging by using an aluminum foil bag, placing a certain amount of drying agent in the aluminum foil bag, and sealing by using a heat sealing machine for later use.

Preparation of probes and probe spraying

50 μl of quantum dots were coupled with 10 μg of hemoglobin nanobody in 20mM BS (pH 7.0) buffer for 120 min using EDC and NHS in 20mM MES (pH 6.5) solution to form quantum dot fluorescent labeled hemoglobin nanobody coupled complex (Tprobe). 50 μl of quantum dots were conjugated with 20 μg of goat anti-chicken IgY monoclonal antibody in 20mM BS (pH 7.0) buffer to form a quantum dot fluorescent-labeled goat anti-chicken IgY monoclonal antibody conjugate complex (C probe).

The amounts of T probe and C probe were adjusted, the amount of T probe used was 0.1. Mu.l per person, and the amount of C probe used was 0.02. Mu.l per person.

Qualified test is carried out on the debugged HbA1c probe, and the test results are shown in the following tables 1-4:

table 1: hbA1c clinical relevance detection result

Table 2: hbA1c accuracy measurement result

Table 3: hbA1c detection limit result

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The test results shown in tables 1 to 3 show that the T-probe and the C-probe of this example meet the test standards.

And (3) spraying a probe: according to the debugging dosage of the T probe and the C probe, diluting solution is used for preparing the kit with the dosage of the T probe of 0.1 mu l per person, the dosage of the C probe of 0.02 mu l per person, and then the kit is sprayed on a binding pad by using a metal spraying film-drawing instrument, so that the HbA1C detection kit is obtained.

And (5) attaching a qualified label to the HbA1c detection kit with qualified inspection standard, and warehousing.

The synthetic cannabinoid K2 kit of this example was used for testing, the testing steps included:

(1) 20 blood samples (4-14%) with different HbA1c contents are drawn, and accurate results are tested on a liquid chromatograph;

(2) 10. Mu.L of the blood sample was added to 1mL of HbA1c lysate and shaken well;

(3) Adding a supernatant of the sample lysate to a sample Kong Lidi in the kit, and diffusing the supernatant from a sample pad of HbA1c detection test paper along a chromatographic direction;

(4) The concentration of HbA1c in the sample was determined by measurement calculation using a fluorescence immunoassay and comparison with a calibration curve.

The test results are shown in table 4:

table 4: hbA1c clinical correlation difference results

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The detection kit provided by the invention can accurately detect the concentration of HbA1c in blood, greatly improves the convenience of HbA1c detection and improves the detection efficiency by combining the test results of tables 1-4.

While the invention has been described with reference to certain preferred embodiments, it will be understood by those skilled in the art that various changes and substitutions of equivalents may be made and equivalents will be apparent to those skilled in the art without departing from the scope of the invention. Therefore, the protection scope of the invention is subject to the protection scope of the claims.

Claims

1. The preparation method of the anti-HbA 1c nano antibody is characterized by comprising the following steps of:

s1, constructing a nanobody phage display library;

the step S1 includes the following steps:

a1, preparing a high-activity immune antigen and immunizing a target animal;

2. The method for preparing the anti-HbA 1c nanobody according to claim 1, wherein: the amino acid sequence of the high-activity immune antigen is shown as SEQ ID NO.1, after gene synthesis, the high-activity immune antigen is synthesized and constructed on a mammal expression vector, and is transiently transfected into 293F cells for expression through PEI, and then the antigen is purified through His tags.

3. The method for preparing the anti-HbA 1c nanobody according to claim 1, wherein: in the step A1, the high-activity immune antigen and Freund's adjuvant are mixed according to the following ratio of 1: after mixing according to the proportion of 1, the immunized animals are immunized by subcutaneous multipoint injection at the back of the animal at a dose of 0.5 mg/time, the total immunization is 4 times, the immunization interval is 2 weeks, and the target animal is alpaca.

4. The method for preparing the anti-HbA 1c nanobody according to claim 1, wherein in the step A3, the specific method for obtaining the nanobody fragment by performing two PCR amplifications using the cDNA as a template is as follows: 10ml of peripheral blood of immune animals is taken, PBMC is separated, RNA is extracted, cDNA is obtained through reverse transcription, the used primers are random primers, as shown in SEQ ID NO.3, and PCR amplification is carried out by taking the obtained cDNA as a template, so that a nano antibody gene library is obtained, and the primers used in two rounds of PCR are SEQ ID NO.4 and SEQ ID NO.5, and SEQ ID NO.6 and SEQ ID NO.7 respectively.

5. The method for preparing the anti-HbA 1c nanobody according to claim 1, wherein: in the step A3, the display vector is a pad1-10b phage display vector.

6. The method for preparing the anti-HbA 1c nanobody according to claim 1 or 5, wherein: in the step A3, the nano antibody fragment of the target animal is connected with the display carrier, then competent cells SS320 are transformed to prepare a nano antibody gene library, and the nano antibody library is infected by an auxiliary phage M13K07 to prepare a nano antibody display library.

7. The method of claim 1, wherein in step S2, the nanobody is expressed as a prokaryote: the high affinity nano antibody sequence is amplified by PCR, connected with pET32a carrier, transformed into TG1 competent cells, then the constructed plasmid is transformed into BL21 (DE 3), and the expression is induced by IPTG, and then the His-tag mediated Ni-agose affinity chromatography is used, and then the fine purification is carried out by superdex200 gel medium.

8. The method of claim 1, wherein in step S2, the nanobody is expressed as a prokaryote: the high affinity nano antibody sequence is amplified by PCR, connected with pET32a carrier, transformed into SS320 competent cells, and then the constructed plasmid is transformed into Origamine B (DE 3), and is induced to express by IPTG, and Ni-Agorose affinity chromatography mediated by His tag is used.

9. The method of claim 6, wherein in the step S2, the validation of the nanobody is: the HbA1c antigen is used for coating an enzyme-labeled plate, 3% BSA is used for blocking, PBST is used for washing, 100ul nanometer antibodies diluted in different concentrations are added, the room temperature is used for 12 hours, PBST is used for washing 5 times, alpaca-HRP is added for reaction for 30min, PBST is used for washing 5 times, TMB is added for color development for 30min, and detection is carried out on an enzyme-labeled instrument after termination.

10. A nanobody against HbA1c prepared by the preparation method of claim 1.