CN109725151B - Human myoglobin detection test paper card and clinical application thereof - Google Patents
Human myoglobin detection test paper card and clinical application thereof Download PDFInfo
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Abstract
The invention relates to a novel human myoglobin fluorescence rapid detection test paper card and clinical application thereof, belonging to the field of immunochemistry. The invention prepares a plurality of antibodies, and performs pairing screening to obtain an antibody combination (M26 and M08) with sensitivity and specificity meeting the requirements; meanwhile, the preparation method is convenient for mass production and can meet the requirement of large-scale clinical application in the future. The antibody combination is debugged and optimized to obtain the fluorescent rapid detection test paper card of the human myoglobin, which has simple and convenient operation, sensitivity, specificity and related detection performance and can meet the requirement of human clinical sample detection. And various buffer solutions suitable for preparing the test paper card are obtained through targeted screening, the coefficient of variation CV of the prepared test card is less than 15%, the sensitivity is 0.88ng/L, and the method has the prospect of industrial application and clinical application value.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to two anti-human Myoglobin (MYO) antibodies, a preparation method thereof and application of the antibodies in human myoglobin detection.
Background
Myoglobin (MYO) is a skeletal and cardiac specific oxygen binding protein. The molecular weight is 16.7KDa, and the polypeptide chain is composed of a polypeptide chain containing 153 amino acid residues and a heme prosthetic group and is in a compact spherical shape.
Due to the small molecular weight of myoglobin, when myocytes are damaged, myoglobin is released into a circulatory system, rises within 1-2 hours, reaches a peak value within 12 hours, and gradually falls after 24 hours, so that the myoglobin is an important reference index for early diagnosis of diseases such as acute myocardial infarction and the like.
At present, the immunodetection method of myoglobin mainly comprises a colloidal gold immunochromatography method, an enzyme-linked immunoassay method, a chemiluminescence immunoassay method and the like. The colloidal gold immunochromatography method has the advantages of simple and convenient operation, rapidness, capability of single-part detection, convenience in storage, no need of special equipment and the like, but the use of the method is limited due to the defects of low sensitivity, difficulty in realizing quantification and the like. The enzyme-linked immunoassay has the characteristics of simple operation, long reagent validity period, no pollution, sensitivity higher than that of colloidal gold, good specificity, capability of measuring a result by an instrument and the like, but has the defects of narrow quantitative measurement range of the used marker enzyme and a substrate, narrow measurement range of the instrument and the like due to relatively low sensitivity, so that the application of the enzyme-linked immunoassay in the micro-immunoassay is limited. The chemiluminescence immunoassay method has the advantages of accuracy, high sensitivity, strong specificity and good precision, but the used instrument is expensive, the used reagent is imported reagent, and the chemiluminescence immunoassay method is difficult to develop in a common laboratory and is not suitable for emergency examination. Therefore, it is necessary to establish a detection method which has a detection time as short as possible, can perform bedside detection in addition to laboratory detection, and can quantitatively determine MYO, thereby providing accurate diagnosis basis for clinic.
In order to overcome the defects of the existing Myoglobin (MYO) detection technology, the invention provides a fluorescent quantitative test strip for rapidly detecting MYO and a preparation method and application thereof. According to the technical characteristics of immunofluorescence and MYO antigen-antibody system characteristics, the test strip provided by the invention is used for detecting MYO level, and has the characteristics of simplicity, rapidness, sensitivity, good specificity and the like, so that the test strip is suitable for application requirements of a detection center or a large hospital clinical department with high detection precision requirements.
Disclosure of Invention
The technical problem to be solved by the invention is to provide an antibody capable of effectively and specifically binding to human myoglobin. More specifically:
the first purpose of the invention is to provide two anti-human myoglobin antibodies.
The first anti-human myoglobin antibody (M26),
the heavy chain variable region comprises the following complementarity determining regions: the amino acid sequence is shown as the sequence SEQ ID NO:1, HCDR1 as set forth in sequence SEQ ID NO:2 and HCDR2 as shown in sequence SEQ ID NO: HCDR3 shown at 3;
the light chain variable region sequence comprises the following complementarity determining regions: the amino acid sequence is shown as the sequence SEQ ID NO: 4, LCDR1 shown as a sequence SEQ ID NO: 5 and LCDR2 as shown in sequence SEQ ID NO: LCDR3 shown in fig. 6.
Preferably, the amino acid sequence of the heavy chain variable region of the above antibody (M26) is represented by SEQ ID NO:7, and the amino acid sequence of the light chain variable region is represented by SEQ ID NO: 8.
A second anti-human myoglobin antibody (M08),
the heavy chain variable region comprises the following complementarity determining regions: the amino acid sequence is shown as the sequence SEQ ID NO: 9, HCDR1 as shown in sequence SEQ ID NO: 10 and HCDR2 as shown in sequence SEQ ID NO: HCDR3 shown in fig. 11;
the light chain variable region sequence comprises the following complementarity determining regions: the amino acid sequence is shown as the sequence SEQ ID NO: 12, LCDR1 as shown in sequence SEQ ID NO: 13 and LCDR2 as shown in sequence SEQ ID NO: LCDR3 shown at 14.
Preferably, the amino acid sequence of the heavy chain variable region of the above antibody (M08) is represented by SEQ ID NO:15, and the amino acid sequence of the antibody light chain variable region is represented by SEQ ID NO: 16.
The second purpose of the invention is to provide two single-chain antibodies, wherein the amino acid sequence of the single-chain antibody (M26) is shown as SEQ ID NO: 17; the amino acid sequence of the single-chain antibody (M08) is shown in SEQ ID NO: 18.
The third objective of the invention is to provide two nucleotide sequences for encoding the single-chain antibody, wherein the nucleotide sequence for encoding the single-chain antibody (M26) is shown as SEQ ID NO:19, and the nucleotide sequence for encoding the single-chain antibody (M08) is shown as SEQ ID NO: 20.
The fourth purpose of the invention is to provide an expression vector containing the nucleotide sequence.
The fifth object of the present invention is to provide a recombinant host cell containing the above expression vector. The host cell may be Escherichia coli, yeast or mammalian cell, preferably Escherichia coli.
It is a sixth object of the present invention to provide a method for producing the above single-chain antibody, comprising:
1) culturing the recombinant host bacteria under proper conditions to express the antibody;
2) then purifying and collecting the antibody from the host bacteria.
The seventh purpose of the invention is to provide the application of the anti-human myoglobin antibody in detecting the content of human myoglobin.
An eighth object of the present invention is to provide a set of antibody pairs that can pair and detect human myoglobin, combinations M26 and M08; the antibody has high detection sensitivity and good specificity to the combination.
The ninth purpose of the invention is to provide a fluorescent quantitative detection test paper card for detecting human myoglobin by using the anti-human myoglobin antibody, which comprises a sample pad, a reaction membrane, a water absorption pad and a fluorescent combination pad; the fluorescent binding pad is sprayed with an antibody M08 marked by fluorescent microspheres, the reaction membrane is provided with a detection band and a quality control band, and the position of the detection band is coated with an antibody M26.
Preferably, the sealing liquid used for the test paper card is: 1% -10% BSA, 50mM pH8.0 boric acid buffer.
The preferable fluorescent antibody diluent for the test paper card is: 0-10% BSA, 0-10% trehalose, 0-0.025% Tween 20, 50mM pH8.0 boric acid buffer.
The preferable coated antibody diluent for the test paper card is as follows: 0-3% methanol, 0-0.025% Tween-20, 0.05mM PBS pH7.2.
The sample diluent used for the test paper card is preferably: 0-0.5% BSA, 0.05% antipyrine, 0-0.025% Tween-20, 0.05% proclin300, 0.05mM pH7.2-8.0 in boric acid buffer.
The invention prepares a plurality of antibodies, and performs pairing screening to obtain an antibody combination (M26 and M08) with sensitivity and specificity meeting the requirements; meanwhile, the preparation method is convenient for mass production and can meet the requirement of large-scale clinical application in the future. The antibody combination is debugged and optimized to obtain the fluorescent quantitative detection test paper card which is simple and convenient to operate, and has sensitivity, specificity and related detection performance meeting the requirements of human clinical sample detection.
Drawings
FIG. 1 shows the specific detection effect of antibodies M08 and M26 (Western Blot),
wherein FIG. 1a is a Western Blot image of antibody M08; FIG. 1b is a Western Blot image of antibody M26. Lane 1 is standard protein (Marker); lane 2 is human MYO.
FIG. 2 is a schematic view of a fluorescent quantitative detection test paper card. 1 is a sample pad, 2 is a reaction film, 3 is a water absorption pad, 4 is a quality control line (C line), 5 is a detection line (T line), 6 is a fluorescent bonding pad, and 7 is a PVC sheet.
FIG. 3 shows a fitting curve of the detection range of the fluorescent quantitative detection test paper card. Wherein the abscissa is protein concentration (ng/mL); the ordinate is the detection value; r2Is 0.998.
FIG. 4 shows a linear range fitting curve of the fluorescence quantitative detection test paper card. Wherein the abscissa is the theoretical concentration (ng/mL); the ordinate is the detection value; r2Is 0.995.
FIG. 5 shows the correlation between the fluorescence quantitative detection test paper card of the present invention and the detection result of the chemiluminescence method. Wherein the abscissa is the value determined by intense chemiluminescence (ng/mL); the ordinate is the detection value; r2Was 0.982.
Detailed Description
Definition of
"antibody", also known as immunoglobulin, is a large Y-shaped protein secreted by B lymphocytes, and is an immunoglobulin molecule capable of specifically binding to a target antigen, such as a protein, a sugar, a polynucleotide, a lipid, a polypeptide, a small molecule compound, etc., through complementary sites (antigen-binding sites) at the two bifurcated tips of the Y.
"Single chain antibody" (scFv) refers to the variable region of the heavy chain (V) of an antibodyH) And light chain variable region (V)L) A single-chain fusion protein is formed by connecting 15-20 amino acid short peptides (linkers), and the linkers used for connection are usually rich in glycine and serine, so that the stability and flexibility of a single-chain antibody are facilitated. The connection mode can be VLIs connected to VHC-terminal, or vice versa. Despite the removal of the constant region and the introduction of the linker, the single-chain antibody retains the specificity of the antibody to the antigen, and has the characteristics of small molecular weight, strong penetration, weak antigenicity and the like.
Complementary-determining regions (CDRs), also called hypervariable regions. Patterned at the amino acid end of the antibody monomer is the most critical region for binding of the target antigen to the antibody, and in immune network theory, the complementarity determining regions of each antibody are also called idiotypes or genotypes.
Example 1 preparation of anti-human MYO hybridoma cell line
1. Animal immunization
BALB/c female mice (purchased from Kyowa Kavens laboratory animals Co., Ltd.) were immunized with recombinant human MYO (E.coli recombinant expression, manufactured by this company) following the general immunization protocol. For specific immunization, see "guidelines for antibody preparation and use". And tracking the serum titer of the immune mice by adopting an indirect ELISA method, selecting the immune mice with the highest serum titer, and performing fusion experiments on the spleen cells and myeloma cells of the mice.
2. Cell fusion
(1) Preparation of spleen cells
Immunized mice, eyeballs are picked and blood is taken, after cervical vertebra is cut off, the immunized mice are placed in 75% (v/v) alcohol for soaking for 10 minutes, spleens of the immunized mice are taken out from a sterile operating platform, the spleens are placed in a cell screen, cells are fully ground, the cells are screened, the spleen is centrifugally washed for a plurality of times by using sterile 1640 culture medium (purchased from Gibco company), and then the cells are resuspended to prepare single cell suspension, and the single cell suspension is counted for standby.
(2) Preparation of feeder cells
Taking one female BALB/c mouse 8-10 weeks old, picking an eyeball to obtain negative serum, and immersing the negative serum in 75% (v/v) alcohol for 10 minutes after the cervical vertebra is cut off; the abdominal skin was aseptically peeled, the peritoneum was exposed, and about 10mL of 1640HT medium (purchased from SIGMA) was injected into the abdominal cavity of the mouse with a syringe, and the abdomen was gently massaged and air-blown several times. Sucking the culture medium containing the macrophages and injecting the culture medium into 20% 1640HAT culture medium for later use;
taking one female BALB/c mouse with the age of 2-3 weeks, and immersing the mouse in 75% (v/v) alcohol for 10 minutes after the mouse dies after cervical vertebra breakage; aseptically placing thymus into a cell screen, grinding, sieving to obtain thymocytes, and placing the thymocytes into the 20% 1640HAT culture medium containing macrophages for later use.
(3) Cell fusion
Mouse myeloma cell line SP2/0 was selected at the logarithmic growth phase and collected and counted. Get about 108The above spleen cells were combined with 2X 107Each of the above SP2/0 cell lines was mixed in a fusion tube, centrifuged at 1000rpm for 10 minutes, and the supernatant was discarded (discarded as clean as possible), and the fusion tube was gently rubbed back and forth on the palm of the hand to loosen the pellet. 1mL of preheated PEG1450 (polyethylene glycol 1450, available from SIGMA) was added slowly and quickly over 60 seconds, 30mL of 1640HT medium was added and stopped, centrifuged at 1000rpm for 10 minutes, the supernatant was removed, the precipitate was loosened by gentle rubbing, and added to 20% of 1640HAT medium obtained in step 2.
Mixing the HAT culture medium, subpackaging at 200 μ L/well into 96-well cell culture plate, standing at 37 deg.C and 5% CO2Cultured in a cell culture box. After one week, 20% 1640HAT medium was replaced with 10% 1640HT medium, and after 3 days, the supernatant was examined.
3. Screening of anti-human MYO specific hybridoma strain
(1) Preparation of the test plate: diluting recombinant human MYO (Escherichia coli system expression, prepared by the company) to 1 mu g/mL by using CB coating solution, coating a 96-hole ELISA plate, coating 100 mu L/hole, coating overnight at 2-8 ℃, washing and patting dry at one time; PBST buffer containing 2% bovine serum albumin was blocked (200 ul/well) for 2 hours at 37 ℃; patting dry for later use.
(2) Screening of positive clones: adding 100 μ L/well of cell culture supernatant to be detected into the detection plate, performing action at 37 deg.C for 30 min, washing, drying, adding 100 μ L/well HRP-labeled goat anti-mouse IgG, performing action at 37 deg.C for 30 min, washing, drying, adding 100 μ L/well TMB color development solution, performing light-shielding development at 37 deg.C for 15min, adding 50 μ L of 2M H per well2SO4The reaction was stopped and the value read at OD 450. Positive well determination principle: OD450 value/negative control value is not less than 2.1. Selecting positive clone strains to carry out cell cloning screening. After three to four rounds of cloning screening, the positive rate of the monoclonal cell strain is determined to be a stable cell strain with 100 percent of positive rate, and the cell strain is determined. Hybridoma cell strains M08 and M26 both have higher titer, and then the hybridoma cell strains are further subjected to antibody variable region sequence sequencing analysis.
Example 2 determination of variable region sequences of antibodies of hybridoma cell lines
The sequences of the variable regions of the antibodies of the hybridoma cell lines M08 and M26 were determined.
Extraction of RNA: total RNA extraction was performed on the hybridoma cell lines M08 and M26 and reverse transcription was immediately performed with reference to the instructions of a cell total RNA extraction kit (purchased from Roche Co.);
reverse transcription of RNA into DNA: performing reverse transcription on the total RNA extracted in the previous step by referring to Thermo Scientific reversed First strand cDNA Synthesis Kit (purchased from Thermo company), preparing cDNA, and freezing and storing at-20 ℃ for later use;
c. PCR amplification and recovery of variable region sequences: performing PCR amplification on variable region sequences of a heavy chain and a light chain by using cDNA obtained in the previous step as a template and a universal primer of a mouse IgG subtype monoclonal antibody variable region sequence as a primer, and recovering a PCR product by using a DNA gel recovery kit (purchased from TIANGEN company);
d. cloning and sequencing of variable region sequences: according to the specification of cloning vector pMD18-T kit (purchased from Takara), the heavy chain and light chain variable region genes were ligated with pMD18-T vector, respectively, to transform E.coli DH 5. alpha. positive clones were picked up and submitted to Nanjing Kingsry Biotech Ltd for sequencing.
The amino acid sequence of the heavy chain variable region of the antibody of the hybridoma cell strain M26 obtained by sequencing is shown as SEQ ID NO 7, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO: shown in fig. 8. The Vbase2 database analyzes the above sequences, and the amino acid sequences of the complementarity determining regions of the heavy chain variable region are: as shown in sequence SEQ ID NO:1, HCDR1 as set forth in sequence SEQ ID NO:2 and HCDR2 as shown in sequence SEQ ID NO: HCDR3 shown at 3; the amino acid sequence of each complementarity determining region of the light chain variable region is: as shown in sequence SEQ ID NO: 4, LCDR1 shown as a sequence SEQ ID NO: 5 and LCDR2 as shown in sequence SEQ ID NO: LCDR3 shown in fig. 6.
The amino acid sequence of the heavy chain variable region of the antibody of the hybridoma cell strain M08 obtained by sequencing is shown as SEQ ID NO:15, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO: shown at 16. The Vbase2 database analyzes the above sequences, and the amino acid sequences of the complementarity determining regions of the heavy chain variable region are: as shown in sequence SEQ ID NO: 9, HCDR1 as shown in sequence SEQ ID NO: 10 and HCDR2 as shown in sequence SEQ ID NO: HCDR3 shown in fig. 11; the amino acid sequence of each complementarity determining region of the light chain variable region is: as shown in sequence SEQ ID NO: 12, LCDR1 as shown in sequence SEQ ID NO: 13 and LCDR2 as shown in sequence SEQ ID NO: LCDR3 shown at 14.
Example 3 recombinant expression and purification of Single chain antibodies
According to the sequencing results of example 2, linker peptides (GGGGS) were added between the heavy and light chain variable regions of M26 and M08 antibodies, respectively3And synthesizing the whole gene, and constructing and recombining the expression vector of the single-chain antibody. The recombinant expression of the single-chain antibody is specifically as follows:
a) construction of expression plasmids for single-chain antibodies M26 and M08
The nucleotide sequence of the single-chain antibody M26 is shown as SEQ ID NO. 19, the amino acid sequence is shown as SEQ ID NO. 17, the nucleotide sequence of the single-chain antibody M08 is shown as SEQ ID NO. 20, and the amino acid sequence is shown as SEQ ID NO. 18. Introducing NcoI enzyme cutting sites into the upstream of single-chain antibody M26 and M08 genes, introducing histidine tags and XhoI enzyme cutting sites into the downstream of the single-chain antibody M26 and M08 genes, carrying out whole-gene synthesis, and constructing into a pUC57 plasmid (provided by Nanjing King Shirui Biotech Co., Ltd.), so as to obtain a long-term storage plasmid, wherein the plasmids are respectively marked as pUC57-M26-scFv and pUC 57-M08-scFv. PCR amplification was performed.
After a conventional PCR procedure, agarose gel electrophoresis analysis revealed that the two product sizes were consistent with the expected sizes. After recovery and purification of the gene products obtained by PCR, they were digested simultaneously with NcoI (# R0193S, available from New England BioLabs) and XhoI (# R0146S, available from New England BioLabs) and ligated to pET28a (69864, available from Merck) plasmid using T4 ligase, transformed into DH5a competent cells (CB101, available from Beijing Tiangen Biochemical technology Co., Ltd.), and cultured overnight at 37 ℃ on LB plates containing kanamycin (0408, available from Amresco). The positive clone bacteria are screened for sequencing the next day, the sequence is compared and is completely consistent with the expected sequence, and the expression plasmids of the single-chain antibody M26 are obtained and are marked as pET28a-M26-scFv and pET28 a-M08-scFv.
b) Construction, screening and expression of single-chain antibody M26 and M08 recombinant escherichia coli engineering strains
The method comprises the following specific steps:
the plasmid pET28a-M26-scFv and pET28a-M08-scFv sequenced correctly in step a of example 3 were transformed into an E.coli BL21(DE3) competent strain (CB105, from Beijing Tiangen Biochemical technology Co., Ltd.) and cultured overnight at 37 ℃ on kanamycin plates. Positive colonies were picked the next day, inoculated into LB medium containing 50. mu.g/mL kanamycin, and cultured overnight at 37 ℃. 50 μ L of the overnight culture was inoculated into 5mL of LB induction medium containing 50 μ g/mL of kanamycin, and cultured with shaking at 37 ℃. When OD600 is 1.0, 1mmol/L of IPTG (available from Amresco) is used for induction expression. Meanwhile, the culture solution of Escherichia coli without IPTG addition is used as a negative control. Collecting bacterial liquid at 12000rpm after 4h for 3min, washing the precipitate with precooled PBS, adding 5 xSDS gel sample adding buffer solution, heating at 100 ℃ for 10min, centrifuging at 12000rpm at room temperature for 1min at high speed, and taking supernatant. The E.coli culture broth without IPTG addition was also treated in this procedure.
c) Purification of Single chain antibodies M26 and M08
Single-chain antibodies M26 and M08 were purified using a histidine-tagged affinity column, selected as HisTrap HP, pre-packed column, as follows:
inoculating single-chain antibodies M26 and M08 recombinant escherichia coli engineering strain glycerol tubes into LB liquid culture medium containing 50 mu g/ml kanamycin according to the inoculation amount of 1%, culturing at 37 ℃ and 220rpm until OD600 is approximately equal to 1.0, adding 1mM IPTG into the culture medium for induction expression, and after the induction expression is performed for 4 hours, centrifuging and collecting thalli. Resuspend with precooled PBS, centrifuge at 12000rpm at 4 ℃ for 15 min; and repeating the steps once. The supernatant was aspirated off, the pellet weight was weighed, and 5mL of binding buffer was added per gram (wet weight of pellet): 300mM NaCl, 20mM NaH2PO4, 10mM Imidazole, pH 7.5. After mixing well, 5. mu.L of 100mmol/L PMSF and 5. mu.L of 100mg/mL lysozyme were added per gram of the cells (wet weight of the cells), and incubated on ice for 20 min. Placing the sample on ice, crushing thallus with probe-type ultrasonic instrument, performing ultrasonic treatment 120 times with 5s interval each time for 5s, circulating three times, waiting for 2min between circulating to cool the sample each time,wait for the sample to cool. Centrifuge at 12000rpm for 15min at 4 ℃ and filter through a 0.45 μm filter.
HisTrap HP affinity column purification: the cell lysates of the single-chain antibodies M26 and M08 obtained by the above pretreatment were affinity-purified using a fully automated intelligent protein purification system (AKTA avant150, available from GE healthcare Co.), and the column was HisTrap HP (17-5248-02, available from GE healthcare Co.). The binding buffer was 300mM NaCl, 20mM NaH2PO410mM Imidazole, pH7.5, and elution buffer 300mM NaCl, 20mM NaH2PO4500mM Imidazole, pH 7.5. Linear elution was performed during elution and the individual elution peaks were collected.
Purity is identified by SDS-PAGE electrophoresis, single-chain antibodies M26 and M08 collection tubes with higher purity meeting the requirements are combined, buffer solution is changed into PBS solution, ultrafiltration concentration (1mg/ml) is carried out, and filtration sterilization is carried out and the mixture is stored at the temperature of minus 20 ℃ for standby.
One skilled in the art will recognize that the recombinant protein may be unlabeled, may have additional labels, or may incorporate additional forms of linker peptides. Protein L affinity filler can be used for purification whether tagged or tagged in a different form.
Example 4 evaluation of antibody Performance
1. Western blot identification of antibodies M08 and M26
a. Polyacrylamide gel electrophoresis: preparing 12% separation gel and 5% concentration gel, loading standard protein and recombinant MYO (Escherichia coli system expression, manufactured by this company) respectively, and performing electrophoresis at constant pressure for 1 hr;
b. film transfer: the membrane was rotated for 1 hour under constant current (35 mA/membrane) to transfer the proteins on the polyacrylamide gel to a nitrocellulose membrane. Staining SDS-PAGE gel subjected to membrane transfer by Coomassie brilliant blue G250, and observing the residual condition of protein;
c. and (3) sealing: TBST buffer containing 5% skimmed milk was blocked (blocking solution) overnight at 4 ℃; washing with a washing solution (TBST, for details, TBST buffer of TaKaRa) once for 10 minutes after blocking;
d. antigen-antibody reaction: diluting a confining liquid (according to a volume ratio of 1: 1000), adding horseradish peroxidase labeled M08(M08-HRP, 1mg/mL, labeled by a classic sodium periodate method in the company, the same below) and horseradish peroxidase labeled M26(M26-HRP, 1mg/mL, labeled by a classic sodium periodate method in the company, the same below) into the two cellulose nitrate membranes respectively, and reacting at room temperature for 1 hour; TBST washes 5 times for 10 minutes each;
e. and (3) color development and photographing: sucking up residual liquid on the nitrocellulose membrane, adding a mixed solution (purchased from Thermo company) of 2mL of a stable peroxidase solution (1mL) and a luminol/enhancer solution (1mL) into the nitrocellulose membrane, uniformly wetting the surface of the nitrocellulose membrane, carrying out a reaction at room temperature in a dark place for one minute, and then photographing in a gel imaging system (purchased from GE company) (see figure 1) to obtain a result.
As seen in the detection results, the antibodies M08 and M26 both have good specificity, and can specifically detect human MYO.
2. Evaluation of single-chain antibodies M26 and M08 on time-resolved fluorescence detection platform
M26 antibody was diluted to 1mg/ml with 0.05mol/L PBS pH7.2 and streaked onto nitrocellulose; diluting the M08 antibody marked by the time-resolved fluorescent microspheres by 10 times by using 0.05mol/L boric acid buffer solution with pH8.0, and spraying the diluted M08 antibody on a bonding pad; the film was applied, cut into strips and mounted as shown in FIG. 2 (see example 5 for details of preparation). The detection card is used for respectively detecting MYO antigen (escherichia coli recombinant expression, prepared by the company) with the concentration content of 200 and 50ng/ml and PBS (0.05 mol/L) with the pH value of 7.2, and the detection results are as follows:
from the results, the double-antibody sandwich detection system composed of M26 and M08 can be applied to a time-resolved fluorescence detection platform and can detect MYO antigen with a certain concentration.
Example 5 preparation of time-resolved fluoroimmunoassay card for Myoglobin
The detection method adopts the principle of double-antibody sandwich immunochromatography.
1. Solution preparation
0.05mol/L pH8.0 boric acid buffer preparation: taking 0.1mol/L of H3BO370ml of Na with a concentration of 0.025mol/L2B4O7·10H2Adjusting pH to 8.0 with O, diluting to 100ml, standing at 4 deg.C for use, and prolonging effective period for 3 months.
1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC, available from SIGMA) solution preparation: 1.5g EDC is added with 100ml deionized water to prepare a water solution which is placed at 4 ℃ for standby, and the effective period is 3 months.
Preparation of a sealing liquid: containing 10% BSA (the percentages are mass volume ratio), 0.05mol/L pH8.0 boric acid buffer, using 0.22um filter membrane filtration, placed at 4 degrees C for standby, the effective period of 7 days.
Preparing a fluorescent antibody diluent: containing 1% BSA, 10% trehalose, 0.025% Tween-20, 0.05mol/L pH8.0 boric acid buffer, filtering with 0.22um filter membrane, standing at 4 deg.C for use, and validity period of 7 days.
Preparation of coating antibody diluent: PBS containing 3% methanol, 0.025% Tween-20, 0.05mol/L pH7.2, filtering with 0.22um filter membrane, standing at 4 deg.C for use, and validity period of 7 days.
Sample diluent: contains 0.5% BSA, 0.025% Tween-20, 0.05% antipyrine, 0.05% proclin300, 0.05mol/L boric acid buffer solution with pH of 8.0, and is stored at 2-8 deg.C for 1 year.
2. Preparation of myoglobin fluorescence detection card
1) Time-resolved fluorescent microsphere labeling
The M08 antibody labeling method is as follows (taking 500ul reaction system as an example): add 400ul boric acid buffer solution into 2ml centrifuge tube, add 100ul concentration 1% no-load fluorescent microsphere (purchased from Thermo corporation) with particle size of 200nm, mix well by vortex oscillation. Then, 10ul of EDC solution was added thereto, and the mixture was shaken at room temperature for 15 min. Centrifuging at 14000rpm at 10 deg.C for 10min, removing supernatant, dissolving precipitate with 0.5ml boric acid buffer, ultrasonic dispersing at 100W for 1min (ultrasonic 3s interval of 3 s).
Adding 75ul of M08 antibody with the concentration of 1mg/ml into the activated microspheres, and carrying out constant-temperature shaking reaction at 25 ℃ at 250r/min for 2 h; 58ul of confining liquid is added, and the reaction is carried out for 4 hours with constant temperature shaking. Centrifuging at 14000rpm and 10 deg.C for 15min, washing for 2 times, removing supernatant, dissolving precipitate with 0.5ml boric acid buffer solution, dissolving precipitate with fluorescent antibody diluent after final centrifugation, ultrasonically dispersing, and storing at 4 deg.C.
2) Spray coating of fluorescent bonding pads
The M08 antibody fluorescent binding pad 6 spot method was as follows: the M08 fluorescent antibody prepared above was diluted 10-fold with a fluorescent antibody diluent and sprayed onto the entire piece of conjugate pad (glass fiber 300mm in length and 12mm in width).
3) Coating of nitrocellulose membranes
The nitrocellulose membrane coating method is as follows: 0.5ml of M26 antibody with the concentration of 4mg/ml is added into a 5ml graduated centrifuge tube, and coated with the antibody diluent to 1ml, and coated on the nitrocellulose membrane 2 at the position of T line 5. 0.5ml of anti-His antibody with the concentration of 4mg/ml is added into a centrifuge tube, and coated with the diluted coating antibody solution to 1ml, and coated on the position of the C line 4 of the nitrocellulose membrane 2.
4) Film sticking, slitting and clamping
The detection card comprises a sample pad 1, a fluorescence combination pad 6, a nitrocellulose membrane (reaction membrane) 2 coated with an M26 antibody, and a water absorption pad 3, wherein the water absorption pad 3 is sequentially arranged from left to right, the nitrocellulose membrane is slightly contacted with the water absorption pad, a T line 5 of the nitrocellulose membrane is arranged on the left, a C line 4 of the nitrocellulose membrane is arranged on the right (as shown in figure 2), cutting is carried out according to the size of the card shell, the nitrocellulose membrane is arranged in the card shell, and the detection card is prepared.
5) Kit assembly
Taking an aluminum foil bag and a drying agent; opening a heat sealing machine and preheating; packing the detection card and the drying agent into an aluminum foil bag; sealing the aluminum foil bag by using a heat sealing machine; and (6) sticking a label.
3. Application method of myoglobin fluorescence detection card
1) Diluting a sample to be tested: adding 1mL of sample diluent into a clean centrifuge tube, accurately sucking 10 mu L of serum/plasma sample, adding into the centrifuge tube, and oscillating and fully mixing.
2) Sample adding and interpretation: and sucking 50 mu L of diluted sample by using a pipette gun, slowly adding the diluted sample into the sample adding hole, starting timing, and quantitatively judging the result by using a fluorescence immunochromatography instrument within 10-15 minutes. If the time exceeds 15 minutes, the result is invalid.
4. Evaluation of detection effect of myoglobin fluorescence detection card
1) Precision: the M26 (coated) -M08 (labeled) test cards were tested for 10 replicates each of 20, 50, 200ng/ml MYO reference using the test card. The experimental result shows that the coefficient of variation CV of the three concentration detection results is less than 15%.
| Concentration point (ng/ml) | 20 | 50 | 200 |
| CV | 11.4％ | 10.5％ | 8.5％ |
2) Detection range: m26 (coated) -M08 (labeled) detection cards were used to detect different concentrations of MYO antigen, 0, 50, 105, 205, 415ng/mL, and the fitted curve and detection range were 0-415ng/mL (see FIG. 3).
3) Linear range: preparing 5 series concentration samples (0, 50, 100, 200 and 400ng/ml) from the high-value sample and the sample diluent according to a certain proportion, detecting by using an M26 (coating) -M08 (marking) detection card, detecting each sample for 3 times, performing regression statistics on the result and the theoretical concentration, and judging whether the concentration is linear in the concentration range. The linear range was 0-400ng/mL (see FIG. 4).
4) Accuracy-recovery: the detection results of the MYO antigens added in amounts of 50ng/ml, 100 ng/ml and 200ng/ml in the detection card of M26 (coating) -M08 (labeling) are shown in the table below.
| Concentration point (ng/ml) | 50 | 100 | 200 |
| Recovery rate | 103％ | 97％ | 102％ |
5. Accuracy-methodological alignment:
the results show that the MYO detection card of M26 (coating) -M08 (marking) has better performance, and a commercial Myoglobin (MYO) quantitative detection kit (chemiluminescence method) is selected as a comparison product for verification. 30 clinical patient specimens were selected, numbered in the order of 1 to 30, and the experiments were performed simultaneously with the control product and the MYO fluorescence detection card of M26 (coated) -M08 (labeled) to be evaluated, measured in the sample order of 1, 2, 3. Correlation coefficient R of detection results of comparison and product to be evaluated2The result obtained by the two methods is better correlated (as shown in fig. 5).
6. Formula screening:
in addition to the above-mentioned best preparation example 1, the applicant tried various preparation schemes, for example, the following 3 sets of test cards were prepared and applied as follows:
sequence listing
<110> Jiangsu Zhonghong bioengineering institute of drug creation Limited
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Claims (8)
1. The human myoglobin fluorescence quantitative determination test paper card comprises a sample pad, a reaction membrane, a water absorption pad and a fluorescence combination pad; the fluorescent binding pad is sprayed with a fluorescent microsphere-labeled anti-human myoglobin-labeled antibody, the reaction membrane is provided with a detection zone and a quality control zone, and the position of the detection zone is coated with the anti-human myoglobin-coated antibody;
the coating antibody is an anti-human myoglobin antibody, comprising:
the heavy chain variable region comprises the following complementarity determining regions: the amino acid sequence is shown as the sequence SEQ ID NO:1, HCDR1 as set forth in sequence SEQ ID NO:2 and HCDR2 as shown in sequence SEQ ID NO: HCDR3 shown at 3;
and the light chain variable region comprises the following complementarity determining regions: the amino acid sequence is shown as the sequence SEQ ID NO: 4, LCDR1 shown as a sequence SEQ ID NO: 5 and LCDR2 as shown in sequence SEQ ID NO: LCDR3 shown at 6;
the labeled antibody is an anti-human myoglobin antibody, comprising:
the heavy chain variable region comprises the following complementarity determining regions: the amino acid sequence is shown as the sequence SEQ ID NO: 9, HCDR1 as shown in sequence SEQ ID NO: 10 and HCDR2 as shown in sequence SEQ ID NO: HCDR3 shown in fig. 11;
and the light chain variable region sequence comprises the following complementarity determining regions: the amino acid sequence is shown as the sequence SEQ ID NO: 12, LCDR1 as shown in sequence SEQ ID NO: 13 and LCDR2 as shown in sequence SEQ ID NO: LCDR3 shown at 14.
2. The human myoglobin fluorescence quantitative detection test paper card as claimed in claim 1, characterized in that the amino acid sequence of the heavy chain variable region of the coating antibody is shown as SEQ ID NO. 7, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 8.
3. The human myoglobin fluorescence quantitative detection test paper card as claimed in claim 2, characterized in that said coating antibody is a single chain antibody, and the amino acid sequence thereof is shown in SEQ ID NO. 17.
4. The human myoglobin fluorescence quantitative detection test paper card as claimed in claim 1, characterized in that the amino acid sequence of the heavy chain variable region of the labeled antibody is shown as SEQ ID NO. 15, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 16.
5. The human myoglobin fluorescence quantitative detection test paper card of claim 4, wherein the labeled antibody is a single chain antibody, and the amino acid sequence of the labeled antibody is shown in SEQ ID NO. 18.
6. The human myoglobin fluorescence quantitative detection test paper card according to any one of claims 1 to 5, characterized in that the blocking solution used in preparing the detection test paper card is 50mM pH8.0 PB buffer solution containing 1% to 10% BSA.
7. The human myoglobin fluorescence quantitative detection test paper card according to any one of claims 1 to 5, characterized in that the fluorescence antibody diluent used in preparing the detection test paper card is 50mM pH8.0 PB buffer solution containing 0% to 10% BSA, 0% to 10% trehalose, 0% to 0.025% Tween 20.
8. The human myoglobin fluorescence quantitative detection test paper card according to any one of claims 1-5, characterized in that the coated antibody diluent used in preparing the detection test paper card is 0-3% methanol, 0-0.025% tween-20, 0.05mM PBS pH 7.2.
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