A reagent, kit, and preparation method for detecting alanine aminotransferase (ALT).

Abstract

This invention relates to an alanine aminotransferase (ALT) detection reagent and its preparation method, belonging to the field of medical testing. The ALT detection reagent comprises nanobodies AC1 and AT1 binding to different ALT binding sites. Nanobodies AC1 are biotinylated and immobilized on an avidin ELISA plate as a capture antibody, while nanobodies AT1 serve as the detection antibody. The ALT detection reagent and method prepared based on these nanobodies can meet the needs for rapid and accurate ALT detection.

Description

Technical Field

[0001] This invention belongs to the field of medical testing, specifically relating to a nanobody that can specifically identify and bind to alanine aminotransferase (ALT), an ALT detection reagent based on the nanobody, and a method for preparing the same. Background Technology

[0002] Alanine aminotransferase (ALT), also known as alanine aminotransferase, is a key enzyme in the liver and other tissues such as muscle. ALT plays an important role in monitoring liver health; elevated ALT levels may indicate liver disease or damage. Liver diseases such as hepatitis, cirrhosis, fatty liver, and drug- or alcohol-induced liver damage can all cause elevated ALT levels; certain medications, industrial toxins, or environmental pollutants can also cause elevated ALT levels, suggesting liver damage.

[0003] Clinical methods for detecting alanine aminotransferase (ALT) are primarily based on its catalytic activity. These include continuous monitoring methods, which determine activity by monitoring the formation rate of a specific product (such as pyruvate) in the ALT catalytic reaction; endpoint methods, which measure the final concentration of the product; and enzyme-coupled methods, which indirectly determine ALT activity by coupling the ALT-catalyzed reaction with another enzyme reaction and measuring changes in the product or substrate of the second enzyme reaction. Essentially, all these methods detect the catalytic activity of ALT, rather than the amount of ALT.

[0004] The problem is that ALT activity is affected by many factors, including time, temperature, pH, protease, metal cations, oxidants, anti-ALT antibodies, chemical inhibitors, freeze-thaw cycles, and light exposure. These factors can lead to inaccurate ALT activity test data, especially when samples are improperly stored or stored for a long time, requiring further determination of ALT concentration.

[0005] Mass spectrometry can be used to measure ALT concentration very accurately, but this method requires sophisticated equipment, is costly, and takes a long time to complete, so it is usually used for research rather than routine clinical testing. Summary of the Invention

[0006] The purpose of this invention is to solve the above-mentioned problems in ALT detection and to provide a rapid, convenient, and low-cost ALT reagent and detection method.

[0007] In a first aspect, the present invention provides an alanine aminotransferase (ALT) detection reagent, comprising a capture antibody, a detection antibody, and a buffer solution. The capture antibody is a nanobody AC1 that specifically binds to ALT, and the amino acid sequence of the nanobody AC1 is SEQ ID NO.1. The detection antibody is a nanobody AT1 that specifically binds to ALT, and the amino acid sequence of the nanobody AT1 is SEQ ID NO.2. When ALT comes into contact with the antibody, it can be captured by the nanobody. For ease of purification, nanobodies AC1 and AT1 also have a His-tag, and for ease of detection, nanobody AT1 also has an HA-tag.

[0008] Secondly, this invention provides a method for preparing an alanine aminotransferase (ALT) detection reagent. The reagent comprises two anti-ALT nanobodies, which are selected from an immunological library obtained through immunization of camels using a T7 phage display platform. ALT-binding nanobody conjugates are then obtained through ELISA screening. Subsequently, the conjugates are sequenced, an expression vector and recombinant bacteria are constructed, and the nanobodies are obtained after induction and purification. The binding affinity of the nanobodies to ALT is measured, and the nanobodies with the strongest affinity are selected as the capture antibody AC1. Another nanobodies binding to different ALT binding sites are identified using the SPR method and selected as the detection antibody AT1. The detection antibody AT1 carries a HA-tag and can be specifically recognized by an anti-HA antibody (secondary antibody). The enzyme-labeled secondary antibody can be any commercially available HRP-labeled anti-HA-tag antibody.

[0009] Thirdly, the present invention provides a method for detecting ALT using the aforementioned alanine aminotransferase (ALT) detection reagent. This detection method is not for diagnostic purposes. The nanobody AC1 is first biotinylated and then coated onto an avidin ELISA plate with biotin. The process involves sample addition, incubation, plate washing, addition of capture antibody AT1, plate washing, addition of colorimetric antibody, plate washing, color development, termination, and reading. The concentration of ALT in the sample is calculated using a standard curve.

[0010] Beneficial effects: The ALT detection reagent of the present invention includes a capture antibody AC1 and a detection antibody AT1 that can specifically recognize ALT. It is a nanobody that can specifically bind to ALT. AC1 is biotinylated and immobilized on an avidin ELISA plate to capture ALT in serum. The detection antibody AT1 has an HA-tag, which binds ALT at the head and HA antibody at the tail. The concentration of ALT can be obtained at a very low cost by ELISA, without considering changes in ALT activity. It is a more accurate measurement indicator and complements ALT activity data to meet the needs of ALT detection. Attached Figure Description

[0011] Figure 1 This is a kinetic sensing image of the dissociation of the nanobodies AC1 and AT1 prepared in Example 4 with ALT.

[0012] Figure 2 This is the linear standard curve for detecting ALT using the ELISA method of this invention;

[0013] Figure 3 This is the four-parameter logistic curve for detecting ALT using the ELISA method of this invention. Detailed Implementation

[0014] The following examples illustrate specific embodiments of the present invention. However, the embodiments of the present invention are not limited to these examples, and any selections and modifications can be made within the scope of the technical effects to be achieved by the present invention. The technical terms and abbreviations used in this invention have their conventional meanings known to those skilled in the art; unless otherwise specified, all materials in the following examples were obtained through commercial channels.

[0015] Example 1: Construction of an anti-ALT nanobody library.

[0016] The phage display library used in this invention is an immune library based on T7 phage, and the establishment steps are as follows:

[0017] (1) Recombinant human ALT was purchased from Shanghai Zeye Biotechnology Co., Ltd. 1 mL of 100 μg / mL recombinant human ALT was mixed with an equal volume of Freund's complete adjuvant and used to immunize one Bactrian camel (number: 10078). Immunization was repeated every 1-2 weeks for a total of 7 immunizations. After four immunizations, jugular vein blood was collected from the camel, peripheral blood lymphocytes were isolated, and total RNA was extracted (PuerLink™ RNA Mini Kit, Life Technologies: 12183018A).

[0018] (2) Total RNA was reverse transcribed into cDNA and the VHH gene was amplified by two rounds of nested PCR;

[0019] The first round of PCR used cDNA as a template, with UP primer1 and DOWN primer1 as upstream and downstream primers, respectively. After amplification, a band of 650-750 bp was recovered. This band was then used as the template for the second round of PCR, with UP primer2 and DOWN primer2 as upstream and downstream primers, respectively. A PCR product of 450-500 bp was recovered.

[0020] UP primer1: 5'-GATGGTGGTCCTGGCTGCTCT-3' (SEQ ID NO.3);

[0021] DOWN primer1: 5'-GGTACGTGCTGTTGAACTGTTAG-3' (SEQ ID NO.4);

[0022] UP primer2: 5'-TACGGTAGTCGAATTCCGCCCAGGTGCAGCTC-3' (SEQ ID NO.5);

[0023] DOWN primer2: 5'-AGCGACTAAGCTTTGAGGAGACGGGACAC-3' (SEQ ID NO. 6).

[0024] (3) The PCR product was digested with EcoRI and HindIII and subjected to agarose gel electrophoresis. The 400-500 bp gene band was recovered, which is the VHH gene fragment.

[0025] (4) The T7 vector (T7Serelect® 10-3 Cloning Kit, MeterckMetillipore Novagen®: 70550-3) and the VHH gene fragment were ligated using T4 ligase;

[0026] (5) The ligation product is mixed with the packaging protein to form a complete T7 phage. The mixture is then amplified to obtain the original phage library.

[0027] (6) The titer of the original library was found to be 9.82 × 10⁻⁶. 9 pfu / mL, diversity 1.9×10 6 .

[0028] Example 2: Screening of anti-ALT nanobodies

[0029] (1) Bacterial culture

[0030] Inoculate with a volume of 1%. E. coli BLT5403 cells were cultured in Carb-LB liquid medium on a shaker at 37°C and 170 rpm until the OD600 reached 0.5–1.0. Cell density was estimated using an empirical formula for E. coli cell density. :

[0031] ;

[0032] (2) Phage infection and titer determination

[0033] The multiplicity of infection was set at 0.001~0.01, and bacteriophages and host bacteria were collected according to this standard. E. coliBLT5403 was cultured in a mixed culture. The culture conditions were 37℃ and 170 rpm. The bacterial cell lysis was observed at any time. Once lysis was complete, the culture was stopped immediately, and the cells were centrifuged (10000×g, 15 min) and the precipitate was discarded. The supernatant was recovered and the phage titer was determined.

[0034] A two-layer culture medium was prepared. The lower layer was Carb-LB solid medium, and the upper layer was Carb-LB solid medium mixed with bacteriophage and bacterial suspension. The mixing ratio of the upper layer medium was: 100 μL of bacteriophage at different dilutions, 300 μL of host bacterial suspension, and 4 mL of undiluted Carb-LB solid medium. After thorough mixing, the mixture was quickly poured into the petri dishes containing the lower layer medium and spread evenly. Each sample was prepared in triplicate, with a sample without added bacteriophage serving as a blank control. After the upper layer medium solidified, the plates were inverted and incubated at 37°C. The appearance of phage plaques was closely monitored, and incubation was stopped when the plaques reached a suitable size. Plaques with 40–400 plaques were selected for counting, and the phage titer (number of plaques per milliliter of sample, pfu / mL) was calculated based on the dilution factor.

[0035] Perform phage enrichment using the following steps:

[0036] Antigen coating. The antigen concentration is 5 μg / mL, 100 μL is added to each well, and the mixture is incubated at 37℃ for 1 h, followed by incubation at 4℃ for 8 h.

[0037] Blocking. After coating with antigen, the 96-well plate was washed three times with 1×TBS, and then 300 μL of skim milk powder was added to each well for blocking. The blocking conditions were 37°C for 1 h and then 4°C for 8 h.

[0038] Add phage. After blocking, wash the 96-well plate 5 times with 1×TBST solution, calculate the volume of phage added, and make up the volume with sterile LB medium if the volume is less than 100 μL; add the phage to the 96-well plate after premixing the blocking agent for 1 h, and place it on a horizontal shaker at room temperature for 1 h.

[0039] Elute the phage. After incubation, discard the supernatant. Wash the 96-well plate five times each with 1×TBST and 1×TBS solutions. Add 100 μL of T7 elution buffer to each well and incubate at 37°C and shake at 100 rpm for 30 min to complete phage elution. Aspirate the eluent into sterile LB medium, determine the phage titer as described above, and calculate the recovery rate.

[0040] ;

[0041] Enrichment of bacteriophages. The elution buffer was amplified, and the titer of the amplified bacteriophages was measured. The amplification product from the previous elution buffer was used for the next round of screening. After two rounds of enrichment, the enrichment degree was calculated. This process was repeated until the enrichment degree approached 1.0, at which point enrichment was stopped, and the next experiment was performed.

[0042] ;

[0043] (3) Screening of nanobodies.

[0044] First, dilute the antigen (ALT) to 10 μg / mL with TBS, and add 100 μL to a 96-well plate. Incubate at 4°C for 12 h. Aspirate the antigen dilution from the wells, wash the plate three times with TBS, blot dry, and add 300 μL / well of 1% protein-free blocking buffer (purchased from Sangon Biotech Co., Ltd.). Incubate at room temperature for 2 h (alternate between 1% protein-free blocking buffer and 1% BSA during screening). Aspirate the blocking agent from the wells, wash the plate six times with TBST, blot dry, and add 100 μL / well of amplified phage. Incubate at room temperature for 30 min. Wash the plate 10 times with TBST, add T7 elution buffer (1% SDS) to elute the phage, incubate at room temperature for 30 min, and amplify the elution buffer for the next round of screening.

[0045] Example 3: Construction of genetically engineered bacteria

[0046] (1) After four rounds of screening, the screening eluent was amplified on solid, plaques were picked, and PCR amplification was performed using plaque amplification solution as template and UP primer3 and DOWN primer3 as upstream and downstream primers.

[0047] UP primer3: 5'-TTGATTAACATATGGCCCAGGTGCAGCTCGT-3' (SEQ ID NO.7);

[0048] DOWN primer3: 5'-TTAAGGAACTCGAGCACGGTGACCAGCCTC-3' (SEQ ID NO. 8).

[0049] (2) A portion of the PCR products were sequenced externally to obtain the nanobody sequence information.

[0050] (3) The other part of the PCR product was double-digested with NdeI and XhoI, and the digested products were recovered. At the same time, the digested products and vector were recovered by the same method. The digested products and vector were ligated with T4 ligase, and the ligation product was transformed into Escherichia coli to obtain genetically engineered bacteria expressing ALT-specific nanobodies.

[0051] Example 4: Nanobody Preparation and Activity Detection

[0052] Preparation of ALT nanobodies:

[0053] (1) The basic culture medium for nanobodies is TB medium. The inoculum is 1% and cultured at 37°C for 4 h. The inducing agent galactoside (IPTG) (final concentration 0.25 mM, the same below) is added for overnight induction.

[0054] (2) After induction, the cells containing nanobodies were centrifuged at 4000 rpm for 20 min to obtain the bacterial cells.

[0055] (3) Add lysis buffer (10mM imidazole, 500mM NaCl, pH7.4 0.02M PB) to the obtained bacteria at a ratio of 1:10, and use a 700bar high-pressure homogenizer to disrupt the cells;

[0056] (4) Centrifuge at 4℃ and 10000 rpm for 20 min and collect the supernatant;

[0057] (5) The supernatant was filtered through a 0.45 μm filter and then purified by separation of nanobodies using an affinity chromatography column (GE Healthcare, US). The packing material of the affinity chromatography column was Ni Sepharose High Performance.

[0058] (6) The nanobody purified by affinity chromatography was subjected to SDS-PAGE electrophoresis to determine its purity. The protein solution with high purity was selected and the protein concentration was determined by BCA method.

[0059] Nanobody activity detection:

[0060] The binding affinity of nanobodies to ALT was analyzed using SPR technology. ALT was amino-coupled to a CM5 sensor chip at a density of 500–800 RU. Seven different concentrations of nanobodies were injected at a flow rate of 45 μL / min in all experiments. Chip regeneration conditions were glycine-HCl pH 1.5. The kinetic parameters Ka, Kd, ​​and KD were calculated using binding curves obtained at different nanobodies concentrations. Simultaneously, nanobodies with different epitopes were identified using manual mode, ultimately selecting nanobodies AC1 (SEQ ID NO.1) and AT1 (SEQ ID NO.2). Figure 1 The curves in the middle, from top to bottom, are the response curves of the nanobody at concentrations of 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, and 1.5625 nM. The kinetic parameters shown in Table 1 were calculated by fitting the equations.

[0061] Table 1 Kinetic parameters of anti-ALT nanobodies

[0062] Antibody ka(1 / Ms) kd(1 / s) kD(M) AC1 <![CDATA[2.63×10 4 ]]> <![CDATA[1.60×10 -5 ]]> <![CDATA[6.09×10 -10 <!-- 4 -->]]> AT1 <![CDATA[2.18×10 4 ]]> <![CDATA[3.82×10 -4 ]]> <![CDATA[1.75×10 -8 ]]>

[0063] Example 5: Reagent Kit Preparation

[0064] The detection kit comprises the following components: an ELISA plate coated with capture antibody AC1, detection antibody reagent AT1, enzyme-labeled secondary antibody (anti-HA antibody), chromogenic solution, and stop solution. The enzyme-labeled secondary antibody used in this invention is HRPAnti-HA tag antibody (ab128131) purchased from Abcam.

[0065] Specific operation methods:

[0066] (1) Biotinylation of capture antibody: The present invention uses Thermo Scientific EZ-Link NHS-Biotin reagent. The biotinylation process is as follows: AC1 is diluted to 2 mg / mL, 3 µL of biotin reagent is added to 1 mL of protein solution, and the reaction is carried out at room temperature for 30 min.

[0067] (2) Coating capture antibody: Dilute AC1 to 10 µg / mL (high concentration, supersaturated coating), add 300 µL / well to avidin microplate, and react overnight at 4°C.

[0068] (3) Blocking: Remove excess liquid, wash the plate 3 times with PBS, and then block with 0.5% BSA and biotin.

[0069] (4) Washing: Wash the plate 5 times with 0.05% PBST.

[0070] (5) Adding samples: Add the test sample and ALT standards of different concentration gradients respectively, and react at 37℃ for 1 h.

[0071] (6) Washing: Wash the plate 5 times with 0.05% PBST.

[0072] (7) Detection of antibody binding: Dilute AT1 to 2 µg / mL with 1×PBS, add 100 µL / well to the enzyme label well, and bind at 37℃ for 1 h.

[0073] (8) Washing: Wash the plate 5 times with 0.05% PBST.

[0074] Add secondary antibody: Dilute anti-HRP / Anti-HA antibody 1:2000 with 1×PBS, and add 100 µL / well to the enzyme-labeled wells. Incubate at 37℃ for 1 h.

[0075] (9) Washing: Wash the plate 5 times with 0.05% PBST.

[0076] (10) Color development: Add 100 µL of TMB working solution to each well and react at 37°C in the dark for 6 min. After the color development reaction is complete, add 50 µL of 2 M H2SO4 to terminate the reaction.

[0077] (11) Reading: Read OD using an ELISA reader 630 and OD 450 Absorbance value at the location.

[0078] Test results as follows Figure 2 and Figure 3 As shown, the ALT concentration in the sample can be calculated based on the absorbance value and the standard curve. The detection limit is 0.5 pg / mL, and the optimal detection range of the linear standard curve is 0.1–1.0 ng / mL. The formula is: y = 0.6186x - 0.0122, R² = 0.9926. The detection range is 0–3.0 µg / mL when fitted with a four-parameter Logistic curve. The formula is: y = A2 + (A1-A2) / (1 + (x / x0)^p), A1=0.09447, A2=2.99289, x0=903.15466, p=2.63714, R² = 0.9975.

[0079] Both R 2 All values ​​are greater than 0.99, indicating high goodness of fit and high reliability. This demonstrates that the ELISA detection method based on the nanobody of this invention has strong reliability and high sensitivity.

[0080] In summary, the detection reagent prepared by this invention is a nanobody composition, which has high specificity, high sensitivity, stable structure, and low cost. The alanine aminotransferase (ALT) detection kit prepared using the nanobody of this invention also has the advantages of high sensitivity, low cost, and good storage resistance. The ALT detection method based on the above kit using the ELISA method yields accurate, reliable, and highly sensitive results, especially in accurately measuring the specific ALT content. It complements the currently used ALT activity detection methods and makes up for the shortcomings of existing methods.

[0081] Obviously, those skilled in the art can make various modifications and variations to this application without departing from the spirit and scope of this application. Therefore, if such modifications and variations fall within the scope of the claims of this application and their equivalents, this application also intends to include such modifications and variations.

Claims

1. A reagent for detecting alanine aminotransferase (ALT), characterized in that, The detection reagent comprises a capture antibody and a detection antibody, wherein the capture antibody is a nanobody AC1 specifically binding to glutamate-oxaloacetate transaminase, and the amino acid sequence of the nanobody AC1 is SEQ ID NO. 1; and the detection antibody is a nanobody AT1 specifically binding to glutamate-oxaloacetate transaminase, and the amino acid sequence of the nanobody AT1 is SEQ ID NO.

2. 2.A nucleic acid encoding the nanobody AC1 of the glutamate-oxaloacetate transaminase detection reagent according to claim 1. 3.A nucleic acid encoding the nanobody AT1 of the glutamate-oxaloacetate transaminase detection reagent according to claim 1. 4.A recombinant vector or a recombinant cell containing the nucleic acid according to claim 2 or 3.

5. A glutamate pyruvate transaminase test kit, characterized by, The kit comprises the capture antibody AC1 and the detection antibody AT1 according to claim 1, and further comprises a biotinylation reagent, an HRP-labeled anti-HA-tag antibody and an enzyme-labeled plate.

6. The use of the glutamic pyruvic transaminase test kit as claimed in claim 5, which is a non-disease diagnostic purpose use, characterized by, The nanobody AC1 is first biotinylated, and then coated on an avidin ELISA plate through biotin, and then subjected to sample addition, incubation, plate washing, addition of the detection antibody AT1, plate washing, addition of an HRP-labeled anti-HA-tag antibody, plate washing, color development, termination and reading, and the concentration of glutamate-oxaloacetate transaminase in the sample is calculated through a standard curve.

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