CN114736295B - Horseradish peroxidase labeled antibody and preparation method thereof - Google Patents
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Abstract
The invention relates to a horseradish peroxidase labeled antibody and a preparation method thereof. The invention screens flagellin specificity of Listeria monocytogenes to obtain a corresponding monoclonal antibody, the antibody has better specificity, and after the coated antibody and the enzyme-labeled antibody are used in a matching way, the Listeria monocytogenes can be detected to the minimum extent, and the application prospect is better.
Description
Technical Field
The invention relates to the field of biological detection, and particularly relates to a horseradish peroxidase labeled antibody and a preparation method thereof.
Background
Listeria monocytogenes (Listeria monocytogenes), abbreviated as Listeria monocytogenes, gram-positive bacilli, have a size of (0.4-0.5) mum x (0.5-2) mum, are straight or slightly bent, and most of the bacilli have a larger end, are rod-like, are usually arranged in a V-shape, and are sometimes filamentous and occasionally appear in a double-sphere shape. 4 flagella can be formed in the environment of 22-25 ℃. The bacterium has strong resistance to physicochemical factors. Can survive in soil, excrement, silage and hay for a long time, has strong resistance to alkali and salt, and is sensitive to penicillin, ampicillin, tetracycline, sulfanilamide, etc. It is widely existed in nature, the listeria monocytogenes existing in food has danger to the safety of human beings, the listeria monocytogenes can still grow and reproduce in the environment of 4 ℃, and the listeria monocytogenes is one of the main pathogenic bacteria threatening the health of human beings in refrigerated food, therefore, in the microbiological inspection of food hygiene, attention must be paid.
At present, the conventional detection methods mainly include a bacterial culture method, a serum agglutination method and a molecular biological detection method. In the bacterial culture method, particularly the traditional method for detecting the listeria in food is to perform pre-enrichment or selective enrichment, perform immunological detection such as biochemical reaction experiments, hemolysis experiments, and synergistic hemolysis experiments (CAMP) on suspicious colonies obtained by separation culture, and further perform serotyping after the listeria is determined. Enrichment and selective enrichment are indispensable steps in the method. The method for increasing the bacteria mainly comprises the following steps: cold enrichment method and normal temperature culture method. The cold enrichment method is to culture at 4 ℃ for 30 days, sometimes even up to one year. Culturing for 24h to 7d at normal temperature. Therefore, the traditional detection method needs 7-11 days to separate and identify the listeria, so the traditional method has a longer detection period. The development of PCR (polymerase chain reaction) is mature in the current common detection method during PCR detection, and the method is widely applied to detection of Listeria. One PCR amplification specific primer is designed according to the specific virulence gene sequence of Listeria, and the other PCR amplification specific primer is designed according to the specific sequence in Listeria genome. The commonly used target sequences are hlyA, iap, inl, Dth-18 and other virulence genes. The advantage of using the PCR method to detect the Listeria is that the specificity of the method is good, the disadvantage is that the sensitivity is lower; the sample culture is amplified after being chemically extracted, so that the detection rate of the sample is improved; and can detect the live non-culturable listeria which can not be detected by the traditional bacteria increasing method. The PCR technology can also be used for quantitative detection of Listeria. However, since PCR detection requires specific equipment, most molecular biological methods are used for scientific research, and since the pretreatment of the method is complicated and the technology required by the operation process is high, the method is difficult to be used for detecting large batches of food samples. And is not suitable for rapid detection in remote areas.
Immunological assays follow their use. At present, commercial Listeria monocytogenes detection kits produced by Bayer in Germany are available. The ELISA method has the advantages of simple operation, strong specificity and high reaction sensitivity, but the preparation of the monoclonal antibody is expensive, so the detection cost of the ELISA box for various food samples is higher. Therefore, the development of the low-cost monoclonal antibody produced by the monoclonal antibody itself to avoid foreign neck entrapment is the main direction of research.
Disclosure of Invention
In one aspect, the invention provides a monoclonal antibody specific for listeria monocytogenes.
Further, the monoclonal antibody is LM2A9, and the amino acid sequence of the variable region of the light chain of the monoclonal antibody is shown in SEQ ID NO. 1:
DLVMTQTAPSVPVTPGESVSISCRSTDYCWQFKFMGHLYWFLQRPGQSPQLLIYKYENLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCKGYEPVFELFGSGTKLEIK
the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 2:
VKPGGSLKLSCAASMRAQEVMKMSWVRQTPDKRLEWVAICSRGSWPYYYPDSVKGRFTISRDQDKQTLYLQMSSLKSEDTAMYYCCSLCMPPQVEYWGQGTTVTVS。
further, a LM6G3 monoclonal antibody matched with LM2A9 is provided, wherein the amino acid sequence of the variable region of the light chain of the antibody is shown as SEQ ID NO. 3:
DLVMTQTAPSVPVTPGESVSISCRSTFKPWPKYEPLHLYWFLQRPGQSPQLLIYMWFNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMNACCYYRQFGSGTKLEIK
the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 4:
VKPGGSLKLSCAASITAQPSPRMSWVRQTPDKRLEWVAICFEKKIGLYYPDSVKGRFTISRDQDKQTLYLQMSSLKSEDTAMYYCEHEWWDEQVSPWGQGTTVTVS。
in some embodiments, an antibody or fragment thereof (e.g., an anti-SAA antibody provided herein) specifically binds to a target (SAA) with a binding constant of at least 10 −9 M or greater binding constant. In some embodiments, an antibody (e.g., a monoclonal antibody) or fragment thereof has an equilibrium constant (Kd) of 10nM or less, e.g., 9nM or less, 8.1nM or less, 8nM or less, 7nM or less, 6nM or less, 6.5nM or less, 6.3nM or less, 5nM or less, 4.3nM or less, 4nM or less, 3nM or less, 2nM or less, 5nM or less, 4nM or less, 3nM or less or 1.2nM or less. For example, the antibody or fragment thereof can be administered in an amount of at least about 0.1X 10 −8 M has a binding affinity for the target of at least about 0.3X 10 −8 M, at least about 0.5X 10 −8 M, at least about 0.75X 10 −8 M, at least about 0X 10 −8 M, at least about 3X 10 −8 M is at least about 5X 10 −8 M, or at least about 2.0X 10 −8 M, at least about 2.5X 10-8, at least about 3.0X 10-8, at least about 3.5X 10-8, at least about 4.0X 10-8, at least about 4.5X 10-8, at least about 5.0X 10-8M, at least about 1X 10-9M, at least about 3X 10-9M, toLess than about 5X 10-9M, at least about 2X 10-9M, at least about 3X 010-9M, at least about 4X 110-9M, at least about 4.3X 210-9M, at least about 5X 10-9M, at least about 6X 10-9M, at least about 6.3X 10-9M, at least about 6.9X 100.9M, at least about 7X 10-9M, at least about 8X 10-9M, at least about 8.1X 10-9M, or at least about 10X 10-9M<100nM， <10nM，<9nM，<8nM，<7nM，<6.9nM，<6.5nM，<6.3nM，<5nM，<4nM，<4.5nM，<3nM，<2nM，<1.5nM，<1nM，<0.1nM，<0.01nM or<0.001nM (e.g.,<10nM，<9nM，<8nM，<7nM，<6.9nM，<6.5nM，<6.3nM，<5nM，<4nM，<4.5nM，<3nM，<2nM，<1.5nM，<1nM，<0.1nM，<0.01nM or<0.001 nM). 10-8M or less, e.g., 10-8M to 10-13M, e.g., 10-9M to 10-13 in one embodiment, Kd is measured by a radiolabeled antigen binding assay (RIA) performed with the Fab version of the antibody of interest and its antigen.
In some embodiments, the VH domain of the monoclonal antibody has an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, identical to 198% or greater than 99% of SEQ ID NO; and/or the amino acid sequence of the VL domain of the monoclonal antibody is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID No. 2. In certain non-limiting embodiments, the sequence of the VH domain of the monoclonal antibody comprises the composition of SEQ ID NO.1, and/or the sequence of the VL domain of the monoclonal antibody comprises the composition of SEQ ID NO. 2.
In some embodiments, the VH domain of the monoclonal antibody has an amino acid sequence of at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, 398% or more identical to SEQ ID NO; and/or the amino acid sequence of the VL domain of the monoclonal antibody is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID No. 4. In certain non-limiting embodiments, the sequence of the VH domain of the monoclonal antibody comprises the composition of SEQ ID NO.3 and/or the sequence of the VL domain of the monoclonal antibody comprises the composition of SEQ ID NO. 4.
Further, the antibody may be conjugated to a detectable label; for example, a detectable label detectable by ELISA, spectrophotometry, flow cytometry, microscopy or diagnostic imaging techniques (e.g., Computed Tomography (CT), Computed Axial Tomography (CAT) scan, Magnetic Resonance Imaging (MRI), magnetic resonance imaging (NMRI), magnetic resonance tomography (MTR), ultrasound, fiber optics and laparoscopy). Specific, non-limiting examples of detectable labels include fluorophores, chemiluminescent agents, enzymatic linkages, radioisotopes, and heavy metals or compounds (e.g., superparamagnetic iron oxide nanocrystals for MRI detection). For example, useful detectable labels include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors, and the like. Bioluminescent markers are also useful, such as luciferase, Green Fluorescent Protein (GFP) and Yellow Fluorescent Protein (YFP). The antibody or antigen-binding fragment may also be conjugated to an enzyme that can be used for detection, such as horseradish peroxidase, -galactosidase, luciferase, alkaline phosphatase, glucose oxidase, and the like. When the antibody or antigen-binding fragment is conjugated to a detectable enzyme, it may be detected by the addition of additional reagents for the enzyme to produce a reaction product that can be recognized. For example, when the reagent horseradish peroxidase is present, the addition of hydrogen peroxide and diaminobenzidine results in a colored reaction product that is visually detectable. The antibody or antigen binding fragment may also be conjugated to biotin and detected by indirect measurement of avidin or streptavidin binding. It should be noted that avidin may itself be conjugated to an enzyme or fluorescent label.
Even more preferably, the monoclonal antibody of the invention is labeled with horseradish peroxidase.
Preferably, the antibody labeling method is that horseradish peroxidase HRP is weighed and dissolved in double distilled water, and the solution is light brown yellow. Adding freshly prepared periodic acid solution into the solution, mixing for 30min at 4 ℃ in the dark,the activated HRP solution appeared light green. Adding ethylene glycol into double distilled water, and mixing. And adding ethylene glycol into the activated HRP solution, uniformly mixing, and standing at room temperature in a dark place for 30min, wherein the solution is brown. Adding equal volume of 2 times of horseradish peroxidase into monoclonal antibody, respectively, adding the mixture of monoclonal antibody and enzyme into dialysis bag, dialyzing in carbonate dialysate at 4 deg.C overnight, and changing the solution 3 times midway. The next day, freshly prepared NaBH was added 4 . The mixture was allowed to stand at room temperature for 4 hours. The above liquid was put into a dialysate bag and dialyzed overnight at 4 ℃. Dropwise adding equal volume of saturated ammonium sulfate into dialyzed monoclonal antibody solution, standing at 4 deg.C for 1 hr, 4 deg.C, 5000 r.min -1 Centrifuge for 30 min. The supernatant was discarded, the pellet was dissolved in PBS buffer, and the solution was put into a dialysis bag and dialyzed overnight. Thus obtaining the marked monoclonal antibody.
Further, the invention provides application of the monoclonal antibody LM2A9 and the LM6G3 monoclonal antibody matched with the LM2A9 in preparation of an ELISA kit for detecting Listeria monocytogenes. Wherein, the amino acid sequence of the variable region of the light chain of the monoclonal antibody LM2A9 is shown in SEQ ID NO. 1:
DLVMTQTAPSVPVTPGESVSISCRSTDYCWQFKFMGHLYWFLQRPGQSPQLLIYKYENLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCKGYEPVFELFGSGTKLEIK
the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 2:
VKPGGSLKLSCAASMRAQEVMKMSWVRQTPDKRLEWVAICSRGSWPYYYPDSVKGRFTISRDQDKQTLYLQMSSLKSEDTAMYYCCSLCMPPQVEYWGQGTTVTVS；
the LM6G3 monoclonal antibody used in matching with LM2A9 has the amino acid sequence of the light chain variable region shown in SEQ ID NO. 3:
DLVMTQTAPSVPVTPGESVSISCRSTFKPWPKYEPLHLYWFLQRPGQSPQLLIYMWFNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMNACCYYRQFGSGTKLEIK
the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 4:
VKPGGSLKLSCAASITAQPSPRMSWVRQTPDKRLEWVAICFEKKIGLYYPDSVKGRFTISRDQDKQTLYLQMSSLKSEDTAMYYCEHEWWDEQVSPWGQGTTVTVS。
advantageous effects
The invention screens flagellin specificity of Listeria monocytogenes to obtain a corresponding monoclonal antibody, the antibody has better specificity, and after the coated antibody and the enzyme-labeled antibody are used in a matching way, the Listeria monocytogenes can be detected to the minimum extent, and the application prospect is better.
Drawings
FIG. 1 is a graph showing the results of subtype identification of monoclonal antibody
FIG. 2 is a graph showing the effect of detecting the binding capacity of HRP-labeled antibody and antigen by direct ELISA
FIG. 3 is a graph showing the effect of the double sandwich method on the detection of the binding ability of monoclonal antibodies
Detailed Description
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art 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 can be obtained by those skilled in the art without creative efforts.
Example 1 preparation of Listeria monocytogenes monoclonal antibodies
Preparation of immunogens
Listeria monocytogenes LM (NRR 00470, Beijing Wallichia organism) preserved in glycerol was streaked on an OXA plate, cultured at 37 ℃ for 24 hours, and then a typical colony was picked with an inoculating needle and punctured into one end of a U-shaped tube containing a semi-solid dynamic medium. After 5 days at 23 ℃, the other end of the U-shaped tube became cloudy. Taking LM at the non-punctured end of the U-shaped tube, inoculating the LM into 10mL of LB broth culture medium, and standing and culturing at 23 ℃ for 24 h. Then transferred to a 250mL LB broth medium, inoculated with 1 broth medium, and cultured for 24h at 23 ℃. The enrichment fluid cultured for 24h is centrifuged at 5000 r/min for 10min to collect thalli, and the collected thalli are washed 3 times by 0.01M PBS. Then resuspended in 5mL of 0.01M PBS, adjusted to pH 2.0 by dropwise addition of 1M HCl and magnetically stirred at 4 ℃ for 40 min. 12000r/min, centrifuging at 4 ℃ for 30min, removing thalli, sucking supernatant, and slowly dropwise adding 1M NaOH to adjust the pH value back to 7.2. 1mL of supernatant was added to a clean ultrafiltration tube (10 KD cutoff) at 4000r/min and centrifuged at room temperature for 15 min. The centrifugation operation was repeated, during which the amount of supernatant in the ultrafiltration tube was continuously replenished. When the whole supernatant was concentrated by ultrafiltration to about 500. mu.L, the addition of ultrapure water was repeated two more times, and the final concentration was made to 200. mu.L. The liquid was aspirated and stored at-20 ℃ until use. The extracted flagellin was verified by SDS-PAGE to have a band at the 33kD position, indicating that a flagellin immunogen was produced.
Preparation of hybridoma cells
Emulsifying the antigen with Freund's complete adjuvant for subcutaneous multi-point injection for the first time, wherein 6 immunogens are prepared per vaccine in 100 mug, and then emulsifying the immunogen with Freund's incomplete adjuvant and antigen for subcutaneous injection for immunization in 100 mug per vaccine every 2 weeks until the titer of the antibody is over 1:16000 measured by tail vein blood sampling. The highest titer mouse was selected and boosted 3 days before fusion with 100. mu.g/mouse.
3d after the last booster immunization, splenocytes from immunized mice (1X 10) 8 Individual cell) and myeloma cell SP/20 (1X 10) 7 Individual cells), fusion was performed under 50% PEG 1450. The fused cells were selectively cultured in HAT medium for 10 days, then in HT medium for 20 days, and then in ordinary RPMI1640 medium (containing 20% calf serum). And (3) screening positive cells by adopting an indirect ELISA method, taking a coating antigen as an immunogen, taking goat anti-mouse IgG marked by HRP as a secondary antibody, and taking immune mouse serum as a positive control and SP2/0 cell culture supernatant as a negative control. And co-screening to obtain 12 hybridoma cell strains with strong positive reaction. Continuously subcloning the identified 12 positive clone cells by adopting a limiting dilution method until the subcloning positive rate of each cell reaches 100%, obtaining 3 monoclonal antibodies of stable secretion antibodies of the cells respectively LM2A9, LM3F6 and LM6G3, and freezing and storing the cells in liquid nitrogen after expanding and culturing the cells.
Antibody subtype and potency determination
And (3) identifying the titer and the affinity, wherein the subclass identification is carried out according to the specification of a mouse monoclonal antibody subclass detection kit, the antibody titer is measured by adopting an indirect ELISA method, and the ascites of each monoclonal antibody is diluted by 200 times.
The 3 hybridoma culture supernatants were tested by a mouse monoclonal antibody subclass test kit, and the results are shown in fig. 1, wherein all the 3 detected antibodies are IgG1, and the light chain is kappa type.
The antibody titer of ascites monoclonal antibody is shown in table 1.
TABLE 1 ascites titer of three monoclonal antibodies
As can be seen from Table 3, the ascites titers of the 3 monoclonal antibodies prepared by the present invention are all above 200000.
Fourthly, purification of ascites and antibodies
The abdominal cavity of BALB/c mice previously injected with paraffin oil (0.5 ml/mouse) for 1 week is inoculated with about 107 per mouse of established hybridoma cells LM2A9, LM3F6 and LM6G3, ascites is collected at 12000r/min after 10 days, 10min is carried out, cell precipitates are removed by centrifugation, and the supernatant is filtered by 0.22 mu M. The prepared ascites supernatant was purified by Protein-G affinity chromatography. And directly loading the purified ascites on a Sepharose S-200 molecular sieve for desalination, eluting with 1 XPBS, collecting the purified antibody, adjusting the antibody concentration to 15mg/mL, and storing at low temperature for later use.
Fifth, monoclonal antibody specificity identification
And (3) performing specificity detection on the antibody by adopting an indirect ELISA method and taking immunogen, LM whole bacteria lysate, escherichia coli lysate, BSA and mouse serum as coating antigens. The results are shown in Table 2.
TABLE 2 monoclonal antibody specificity identification results
As can be seen from the results in Table 2, the three monoclonal antibodies prepared by the present invention all have good specificity.
In addition, pairing test was performed on the three monoclonal antibodies, and it was determined that LM2A9 and LM6G3, and LM3F6 and LM6G3 were paired antibodies to each other.
Example 2 affinity identification and variable region sequences of LM2A9 and LM6G3 mAbs
The affinity constant for the monoclonal antibody was measured using a biosensor IAsysPlus manufactured by AffinitySensors corporation. The sample cell was pretreated with carboxymethyl dextran (CMD), different concentrations of immunogenic protein were added to the cell, and after 5min the free carboxyl groups were blocked with ethanolamine for 3 min. Then, free and non-specifically bound protein molecules are washed away with 1mol/L formic acid. And (3) placing the sample pool coated with the immunogen into a biosensor, and balancing for 10 min. Baseline 50. mu.l of 0.01mol/L PBS at pH7.2 was added and a stable baseline was established after 5 min. Association (asso-association) PBS was aspirated, 45. mu.l PBS and 5. mu.l monoclonal antibody were added, and the monoclonal antibody solution was aspirated when the monoclonal antibody was saturated. Dissociation (dissociation) 50. mu.l PBS was replaced and monoclonal antibody binding and dissociation were equilibrated. Regeneration (regeneration) 20mmol/LHCl 50. mu.l were allowed to act for 2min to completely elute bound mAb. Return to baseline PBS was changed and returned to baseline again, i.e. the next 1 cycle was started. Diluting each monoclonal antibody into 5 different concentrations by PBS, sequentially and respectively measuring the binding and dissociation rates of the monoclonal antibody with each concentration and the envelope antigen, and calculating the affinity constant of each monoclonal antibody by special software FASTpit attached randomly. The results are shown in Table 3.
TABLE 3 affinity constants of antibodies
| Name of antibody | Affinity constant (nM) |
| LM2A9 monoclonal antibody | 4.84±0.05 |
| LM6G3 monoclonal antibody | 8.73±0.06 |
As can be seen from the results in Table 3, the two monoclonal antibodies LM2A9 and LM6G3 have better affinity and binding ability.
And extracting total RNA of 2 hybridoma cells by using a kit, and performing reverse transcription to synthesize cDNA. Designing primers, amplifying heavy chains and light chains, sequencing, analyzing sequencing results by software, and obtaining a monoclonal light and heavy chain sequence through sequence identification, wherein the amino acid sequence of the light chain variable region of the LM2A9 monoclonal antibody is shown as SEQ ID NO. 1:
DLVMTQTAPSVPVTPGESVSISCRSTDYCWQFKFMGHLYWFLQRPGQSPQLLIYKYENLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCKGYEPVFELFGSGTKLEIK
the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 2:
VKPGGSLKLSCAASMRAQEVMKMSWVRQTPDKRLEWVAICSRGSWPYYYPDSVKGRFTISRDQDKQTLYLQMSSLKSEDTAMYYCCSLCMPPQVEYWGQGTTVTVS。
the amino acid sequence of the variable region of the light chain of the LM6G3 monoclonal antibody is shown in SEQ ID NO. 3:
DLVMTQTAPSVPVTPGESVSISCRSTFKPWPKYEPLHLYWFLQRPGQSPQLLIYMWFNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMNACCYYRQFGSGTKLEIK
the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 4:
VKPGGSLKLSCAASITAQPSPRMSWVRQTPDKRLEWVAICFEKKIGLYYPDSVKGRFTISRDQDKQTLYLQMSSLKSEDTAMYYCEHEWWDEQVSPWGQGTTVTVS
example 3 horseradish peroxidase-labeled LM2A9 and LM6G3 monoclonal antibodies
5mg of horseradish peroxidase HRP was weighed out and dissolved in 1mL of double distilled water, and the solution was light brown yellow. To the solution was added 1mL of freshly prepared 13 mg/mL -1 The activated HRP solution is light green after being mixed for 30min at 4 ℃ in a dark place. Add 9. mu.L of ethylene glycol to 1mL of double distilled water and mix well. 0.5mL of ethylene glycol was added to the activated HRP solution and mixed well, and the mixture was left at room temperature in the dark for 30min, at which time the solution appeared brown. Adding 2 times of equal volume of horseradish into 1mL of LM2A9 monoclonal antibody or LM6G3 monoclonal antibodyPeroxidase, adding the mixture of monoclonal antibody and enzyme into dialysis bag, dialyzing in 0.05M carbonate dialysate at 4 deg.C overnight, and changing the solution 3 times. The next day, 0.1mL of freshly prepared 4 mg/mL solution was added -1 NaBH 4 . The mixture was allowed to stand at room temperature for 4 hours. The above liquid was packed in a dialysate bag and dialyzed overnight at 4 ℃. Dropwise adding saturated ammonium sulfate with equal volume into dialyzed monoclonal antibody solution, standing at 4 deg.C for 1 hr, at 4 deg.C for 5000r min -1 Centrifuge for 30 min. The supernatant was discarded, the pellet was dissolved in 1mL of PBS buffer, and the solution was placed in a dialysis bag and dialyzed overnight. Thus obtaining the marked LM2A9 monoclonal antibody and LM6G3 monoclonal antibody.
Example 4 direct ELISA method for detecting the binding ability of HRP-labeled LM2A9 antibody and antigen
The immunogen was diluted to 1. mu.g.mL with PBS -1 Add 100. mu.L to 96-well microplate per well. Incubate at 37 ℃ for 2 h. Spin off the well, wash 3 times with PBS, 250 μ L/well, and pat dry. The HRP-labeled LM2A9 antibody was diluted to 0, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4. mu.g/mL in PBS with 0.5% BSA -1 mu.L was added to each well at 3 concentrations in parallel and incubated at 37 ℃ for 1 h. Spin off the well, wash 3 times with PBS, and pat dry. Preheating the TMB substrate at room temperature, and simultaneously starting an enzyme-labeling instrument for preheating. Tetramethylbenzidine (TMB) substrate A (TMB powder dissolved in DMSO to a final concentration of 11 mg. multidot.mL) was added to each well -1 Further, 1/10 volumes of glycerin and liquid B (pH 5.5, 0.2 mol. L) were added -1 Disodium hydrogen phosphate and 0.1 mol/L -1 Preparation of Urea peroxide buffer solution with a concentration of 0.74 mg/mL -1 ) Adding 50 μ L of each, developing at 37 deg.C for 20min, finding that the wells turn blue, adding stop solution (1 mol. L) -1 Sulfuric acid) 50 μ L, at which time the blue color changed to pale yellow. And reading the absorbance value at 450nm by using a microplate reader. The results are shown in FIG. 2.
As can be seen from the results in FIG. 2, the HRP-labeled LM2A9 antibody prepared by the present invention can better react with the immunogen. The immunogen is in the concentration range of 0.2-6.4 mug.mL -1 Can be identified significantly.
Example 5 double Sandwich method for detecting the binding Capacity of monoclonal antibodies
Monoclonal antibodies LM2A9 and LM6G3 were diluted with PBS and coated with 100. mu.L (optimized 0.4. mu.g/mL) of the above antibodies in each well of a 96-well microplate, 3 replicates. 4 ℃ overnight. The next day, the well was spun off, washed 3 times with PBS, and patted dry. Each well is coated with simulated pollution LM meat samples with different concentrations (10) 7 ，10 6 ，10 5 ，10 4 ，10 3 100, 50, 10, 1 CFU/mg) reaction, incubation at 37 ℃ for 2 h. Spin off the well, wash 3 times with PBS for 5min each time, and pat dry. And (3) incubating the corresponding conjugated antibody with the optimized mass concentration of 0.4 mu G/mL for 1h at 37 ℃ (an LM6G3 incubation detection is carried out on an ELISA plate coated with an LM2A9 antibody; and an LM6G3 incubation detection is carried out on the ELISA plate coated with an LM2A9 antibody). Spin off the well, wash 3 times with PBS for 5min each time, and pat dry. Preheating the TMB substrate to room temperature, and simultaneously starting the microplate reader for preheating. Each well was charged with 50. mu.L of TMB substrate. Developing at 37 deg.C for 20min, detecting blue, adding stop solution 50 μ L/well, and reading absorbance value at 450nm with microplate reader. The results are shown in FIG. 3.
As can be seen from the results in FIG. 3, when LM2A9 was used as the coating antibody and LM6G3 was used as the enzyme-labeled antibody, the sensitivity for detecting LM was 10CFU/mg, which is a good sensitivity.
It is to be understood that the invention is not necessarily limited in its application to the details of construction and the arrangement of components set forth in the following description and/or illustrated in the drawings. The invention is capable of embodiments in addition to those described and of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein, as well as the abstract, are for the purpose of description and should not be regarded as limiting.
As such, those skilled in the art will appreciate that the conception upon which this disclosure is based may readily be utilized as a basis for the designing of other structures, methods and systems for carrying out the several purposes of the present invention. It is important, therefore, that the claims be regarded as including such equivalent constructions insofar as they do not depart from the spirit and scope of the present invention.
While the invention has been described and illustrated in detail as being sufficient to enable those skilled in the art to make and use it, various alternatives, modifications, and improvements should be apparent without departing from the spirit and scope of the invention. The examples provided herein represent preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Modifications thereof and other uses will occur to those skilled in the art. Such modifications are encompassed within the spirit of the invention and are defined by the scope of the claims.
Sequence listing
<110> Beijing Kongzheng Zhongzhong Biotechnology Co., Ltd
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Claims (4)
1. The L.monocytogenes monoclonal antibody LM2A9 has the amino acid sequence of the light chain variable region shown in SEQ ID No. 1; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 2.
2. A kit for detecting listeria monocytogenes, comprising the listeria monocytogenes monoclonal antibody LM2a9 of claim 1, wherein said monoclonal antibody LM2a9 is labeled with horseradish peroxidase.
3. Use of the listeria monocytogenes monoclonal antibody LM2a9 of claim 1 in the preparation of an ELISA kit for detecting listeria monocytogenes.
4. The application of the Listeria monocytogenes monoclonal antibody LM2A9 and the Listeria monocytogenes LM6G3 monoclonal antibody matched with LM2A9 in preparing an ELISA kit for detecting Listeria monocytogenes; wherein, the amino acid sequence of the variable region of the light chain of the monoclonal antibody LM2A9 is shown in SEQ ID NO. 1; the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 2; the LM6G3 monoclonal antibody used in matching with LM2A9 has the amino acid sequence in the light chain variable region as shown in SEQ ID No. 3; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 4.
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| CN101851291A (en) * | 2010-02-09 | 2010-10-06 | 中国人民解放军第四军医大学 | Heavy chain and light chain variable regions of anti-human BAFF monoclonal antibody |
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| US7390489B2 (en) * | 2004-10-08 | 2008-06-24 | Komed Co., Ltd. | Monoclonal antibody selectively recognizing listeria monocytogenes, hybridoma producing the antibody, test kit comprising the antibody and detection method of listeria monocytogenes using the antibody |
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| CN101968487A (en) * | 2009-03-31 | 2011-02-09 | 东北农业大学 | Immune colloidal gold test paper for detecting listeria hemolysin and preparation method thereof |
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