CN110468108B - Hybridoma cell strain secreting human ferritin light chain monoclonal antibody and application thereof - Google Patents

Abstract

The invention discloses a hybridoma cell strain 3F6 with a preservation number of CCTCC NO: C2019173, which can secrete a monoclonal antibody FTL-McAb1 capable of specifically recognizing a human ferritin light chain, and also discloses application of the monoclonal antibody FTL-McAb1 in detection of human ferritin, and a human serum ferritin immunodetection kit prepared from the monoclonal antibody FTL-McAb 1.

Description

Hybridoma cell strain secreting human ferritin light chain monoclonal antibody and application thereof

Technical Field

The invention relates to the field of immunodetection, in particular to a hybridoma cell strain capable of secreting a human ferritin light chain monoclonal antibody, the monoclonal antibody secreted by the hybridoma cell strain, application of the monoclonal antibody in detection of human serum ferritin, and a human ferritin detection kit prepared from the monoclonal antibody.

Background

Iron Deficiency Anemia (IDA) is anemia caused by Iron deficiency stored in vivo due to various reasons and influencing the generation of cellular heme, and the red blood cells of the anemia become small in morphology, expand a central pale zone and belong to small cells with low chromogenicity. Iron deficiency anemia is caused by iron deficiency in approximately 50% of patients with anemia. Laboratory examinations of iron deficiency anemia have two main purposes: the method is characterized in that the iron deficiency stage of a patient is determined, and Iron Deficiency Anemia (IDA) and Anemia of Chronic Diseases (ACD) are clinically distinguished.

Studies have shown that the serum ferritin content is proportional to the body iron storage, lug/L corresponds to 8-10mg or 120ug per kg body weight. The superiority of serum ferritin over other iron-related assays for diagnosis of iron deficiency anemia was confirmed in a number of studies, from which it was obtained that the mean AUC of the ROC curve for diagnosis of iron deficiency anemia as serum ferritin was 0.95 (95% confidence interval), while zinc protoporphyrin (ZPP) was 0.77, mean red blood cell volume (MCV) was 0.74, and transferrin saturation (transferrin saturation) was 0.74. With the increasingly widespread use and standardization of serum ferritin in clinical diagnostics, serum ferritin has gradually replaced bone marrow iron staining as a new gold standard for assessing the iron storage status of the body. Moreover, serum ferritin has a clear change in Iron Deficiency (ID), which is a sensitive marker of iron deficiency in vivo. The detection of serum ferritin can screen the early stage of iron deficiency clinically, so that measures such as dietary structure adjustment or iron supplement can be taken as early as possible to prevent the occurrence of iron deficiency anemia. Human serum ferritin levels are reduced, typically representing iron deficiency anemia, and elevated levels are due to malignancy, inflammation, liver disease, and the like. Therefore, the detection of the serum ferritin content has important significance for monitoring iron-deficiency anemia, malignant tumors, inflammations and liver diseases.

Human serum ferritin is composed mainly of the L subunit, contains only a small amount of H subunit, and has a small iron atom content. Human serum ferritin is present in the blood as 24-mer nanoparticles, and no unassembled subunits are found.

The immunological detection has been more and more widely applied to the detection of human serum ferritin due to its advantages of high sensitivity and high speed. The most important limiting factor of the quality of the serum ferritin immunological detection kit is the quality of bioactive raw materials. In terms of methodology, the serum ferritin detection kit mostly adopts a double-antibody sandwich method, and the bioactive raw material is ferritin monoclonal antibody. The existing ferritin monoclonal antibody in the market has uneven quality level, which severely limits the quality of the kit.

In view of the above, it is necessary to develop a ferritin monoclonal antibody with high quality to meet the quality requirements of the kit.

Disclosure of Invention

The first purpose of the invention is to provide a hybridoma cell strain 3F6, which is preserved in China Center for Type culture collection (CCTCC for short), the preservation number of the hybridoma cell strain 3F6 is CCTCC NO: C2019173, and the hybridoma cell strain can secrete a monoclonal antibody FTL-McAb1 capable of specifically recognizing a human ferritin light chain.

The second purpose of the invention is to provide a monoclonal antibody FTL-McAb1 which is secreted by a hybridoma cell strain 3F6 and can specifically recognize a human ferritin light chain.

The third purpose of the invention is to provide the application of a monoclonal antibody FTL-McAb1 which specifically recognizes the light chain of human ferritin in a human ferritin detection kit.

The fourth purpose of the invention is to provide a human ferritin immunodetection kit prepared by a monoclonal antibody FTL-McAb1 which specifically recognizes a human ferritin light chain.

Preferably, the human ferritin immunoassay kit comprises a monoclonal antibody FTL-McAb1 which is secreted by hybridoma cell line 3F6 and specifically recognizes the light chain of human ferritin.

And (3) cell preservation:

the hybridoma cell strain 3F6 is obtained by self screening of the inventor, the preservation number is CCTCC NO: C2019173, the preservation date is 7 months and 19 days in 2019, the preservation unit is CCTCC, and the address is located in the eight-path Wuhan university No. 299 in the Wuchang area in Wuhan city, Hubei province.

Definition of terms:

the amino acid sequence of the human Ferritin light chain, namely Ferritin light chain (FTL for short), disclosed by the invention is published in UniProt (https:// www.uniprot.org/UniProt/P02792), and is specifically shown in SEQ ID NO:1, and the nucleotide sequence of the corresponding mRNA coding region is shown in SEQ ID NO: 2.

Monoclonal antibodies, abbreviated as monoclonal antibodies (McAb), are highly homogeneous antibodies raised against a particular epitope by a single B cell clone.

Human embryonic kidney cells (Human embryo kidney 293cells), HEK 293 for short, HEK-293,293 cells, can be used to express foreign proteins.

The human ferritin detection kit adopts an immunological detection method, utilizes antigen-antibody specific reaction to detect the content of human ferritin in a specimen, and is divided into an enzyme-linked immunosorbent assay, an immunochromatography assay, a chemiluminescence assay, an immunoturbidimetric immunoassay and the like according to a detection platform.

In order to achieve the above purpose, according to the general steps of preparing monoclonal antibodies, human ferritin is firstly needed to be obtained as an immunogen, the inventor finds that natural human ferritin is adopted as the immunogen, although the immune titer is high, the antigen quantity is too small, the price is too expensive, and the human ferritin expressed by a prokaryotic expression system such as escherichia coli is probably because the human ferritin conformation expressed by a prokaryotic expression system is different from the natural ferritin, the immune titer is lower, so the immunogen is the first choice of the human ferritin expressed by a eukaryotic system, and the embodiment of the invention adopts the self-developed HEK-293 recombinant expression FTL as the immunogen. However, ferritin derived from other routes, methods and expression systems is also useful as an immunogen and is not limited to HEK-293 recombinant expression.

Extracting RNA from human tissues (such as blood, liver and spleen), carrying out reverse transcription to obtain cDNA of FTL, using the cDNA as a template, obtaining FTL gene fragments by using a PCR method, adding a base A to two 3' ends of the fragments by using a PCR enzyme, connecting the fragments to a T vector, selecting positive clones, carrying out DNA sequence determination, thus obtaining the FTL gene, or carrying enzyme cutting sites on two ends of an RT-PCR primer, directly carrying out enzyme cutting on a PCR product, connecting the PCR product to a vector subjected to the same enzyme cutting, selecting the positive clones, and carrying out sequencing. It is also feasible to use a whole-gene synthesis method to send the FTL gene to a commercial gene synthesis company for synthesis. The obtained FTL gene is connected to an expression vector by adopting an enzyme digestion or non-enzyme digestion gene cloning method.

Expression vectors and expression systems are compatible, and expression systems known to those skilled in the art, including prokaryotic expression systems, including E.coli expression systems, and eukaryotic expression systems, including yeast expression systems, insect cell expression systems, and mammalian expression systems, can be used to express the FTL protein. And (3) introducing the expression vector connected with the FTL gene into a host cell of a corresponding expression system, wherein the corresponding introduction technology of a prokaryotic expression system is transformation, and the corresponding introduction technology of a eukaryotic expression system is transfection. And (3) adding corresponding antibiotics for selective screening during transformation or transfection, selecting recombinant daughter cells carrying FTL genes, carrying out expression identification, selecting cell strains with large expression quantity of FTL, and carrying out amplification culture. The cultured cells are collected by centrifugation (in the case of intracellular expression) or the supernatant (in the case of secretory expression) is collected and purified to obtain the recombinant FTL protein.

Immunizing a mouse with natural or recombinant FTL protein for several times, measuring the serum titer of the mouse by ELISA, selecting the high-value mouse to take splenocytes to fuse with myeloma cells, screening positive monoclonal cell strains, performing amplification culture, and injecting the positive monoclonal cell strains into the abdominal cavity of the mouse to produce ascites, or producing a culture by an in vitro culture method. And collecting ascites or culture rich in the antibody, and purifying to obtain the FTL monoclonal antibody. These monoclonal antibodies were sub-typed.

The FTL monoclonal antibody is marked with HRP, and is matched with the unmarked monoclonal antibody ELISA to detect FTL with different concentrations, so as to investigate performance indexes such as sensitivity, specificity, linear range and the like, select the strain with the best performance, and carry out further performance verification. Finally, a monoclonal antibody FTL-McAb1 is selected and secreted by a hybridoma cell strain 3F 6. Because ferritin is 24 subunits, the invention can be realized by only using one monoclonal antibody for coating and labeling at the same time in order to simplify raw materials and a kit preparation process.

The monoclonal antibody FTL-McAb1 is used for coating and marking, the content of human serum ferritin in an immunodetection specimen is detected, and a detection platform comprising an enzyme-linked immunosorbent assay, an immunochromatography, an immunoturbidimetry, a chemiluminescence method and the like has a good effect.

Has the advantages that:

the monoclonal antibody FTL-McAb1 secreted by the hybridoma cell strain 3F6 is used for detecting the content of human serum ferritin in a specimen, has the advantages of high sensitivity, good specificity, wide linear range and good universality of different detection platforms, and can replace the FTL monoclonal antibody in the existing kit.

Drawings

FIG. 1: SDS-PAGE electrophoresis of purified recombinant expression FTL.

FIG. 2: SDS-PAGE of FTL monoclonal antibody FTL-McAb1 after purification.

FIG. 3: and (3) clinical correlation of FTL immunochromatography detection results and Yapei reagents.

FIG. 4: clinical relevance between FTL chemiluminescence detection results and Yapei reagent.

FIG. 5: and (3) the detection result of the FTL immunoturbidimetry is clinically related to the yapei reagent.

Detailed Description

Example 1: FTL antigen preparation

Firstly, the mRNA coding region (the sequence is shown as SEQ ID NO: 2) of the FTL is taken as a template, and a pair of primers (the sequences are respectively shown as SEQ ID NO:3 and SEQ ID NO:4) are designed.

Taking fresh human liver cancer tissue, extracting RNA by a Trizol one-step method of Saimer Feishale, carrying out reverse transcription on the RNA into cDNA by using an AMV reverse transcription kit, and carrying out PCR amplification by using the primers by using the cDNA as a template.

And (3) carrying out agarose gel electrophoresis on the PCR product, cutting off a target band after the molecular weight is confirmed to be correct, and recovering by using an AXYGEN gel recovery kit to obtain the FTL gene fragment.

The 3' end of the FTL gene fragment obtained by amplification is provided with a base A, the base A is connected to a pMD-18T vector of TaKaRa, positive clone is identified by PCR and sequenced, 100 percent of homology with SEQ ID NO. 2 is obtained by sequence comparison, the gene is proved to be successfully obtained, and the pMD-18T-FTL clone is obtained.

Extracting pMD-18T-FTL plasmid by using an AXYGEN plasmid extraction kit, cutting an FTL gene fragment by using BamHI and EcoRI enzymes of NEB, connecting the FTL gene fragment to a plasmid vector pcDNA3.1(+) which is cut by the same enzyme, transforming a DH5 α strain by using a connecting substance, selecting a monoclonal which is positive by PCR identification for sequencing verification, and proving that the pcDNA3.1(+) -FTL plasmid is successfully constructed.

HEK-293 cells were pre-cultured to logarithmic growth phase, inoculated with 24 wells of 80-90% density, transfected with the PEI/pcDNA3.1(+) -FTL transfection reagent/plasmid mixture, and gene-transfected cells were screened with antibiotic G418.

And (3) carrying out passage on the positive cells, detecting the supernatant by an ELISA method, carrying out high expression screening, and successfully obtaining a cell strain pcDNA3.1(+) -FTL (HEK-293)3D2 capable of stably and highly expressing the FTL, wherein the concentration of the FTL in the supernatant reaches 10 mg/L.

3D2 cells are cultured by multiple times of bottle-rotating amplification, 5L of cell supernatant is collected and purified by S200 and DEAE, and finally 40mg of recombinant FTL protein is obtained, the purity reaches more than 95%, and more than 80% of target protein exists in a 24-polymer form.

TABLE 1 ELISA test results for FTL monoclonal antibodies

Example 2 FTL antibody preparation

FTL recombinant antigen prepared in example 1 was dialyzed against 20mM PBS ph7.4 and diluted to 1mg/ml with dialysate, with adjuvant according to 1: mixing at a ratio of 1, and emulsifying.

The emulsified FTL antigen is used for immunizing Balb/c mice with the age of 8 weeks, each mouse is immunized with 100ug of the antigen, Freund's complete adjuvant is used for primary immunization, and Freund's incomplete adjuvant is used for boosting immunization.

After three immunization injections, tail vein blood is collected, the titer is measured, and the cells with the titer exceeding 100 ten thousand are selected for cell fusion.

72 hours before fusion, 30-50ug FTL antigen was injected intraperitoneally.

The mice to be fused were sacrificed, the spleens were removed, ground in a 200 mesh screen while washing with RPMI1640 medium, the cell suspension was transferred to a 50ml centrifuge tube and washed 4 times with RPMI1640 medium, each time centrifuged at 15000rpm for 5 min.

Myeloma cells were washed 4 times with RPMI1640 medium, centrifuged 5min at 15000rpm each, and finally washed 1 time with splenocytes.

Centrifuging the mixed spleen cells and myeloma cells at 1500rpm for 5min, removing supernatant, flicking the bottom of the tube, and flicking the cells; slowly adding 1ml of PEG1450 preheated to 37 ℃ in advance, uniformly mixing while adding, and finishing adding within 60 s; then adding 40ml of RPMI1640 culture medium pre-warmed to 37 ℃ in advance to terminate the fusion reaction, and centrifuging at 1500rpm for 5 min; the cells were flicked up, transferred to RPMI1640 HAT cell culture medium, mixed well and plated.

After plating, changing liquid regularly and detecting supernatant, selecting high-value positive holes for subcloning, after 3 times of subcloning, finally selecting 18 hybridoma monoclonal cells, wherein the supernatant has higher response (OD value is more than 1.0) to human ferritin light chain expressed by HEK-293 and natural liver ferritin.

Respectively injecting 18 hybridoma cells into a mouse, collecting generated ascites, centrifuging to remove precipitates, primarily purifying the supernatant by using saturated ammonium sulfate, finely purifying by Protein A column affinity chromatography, and dialyzing into a PBS buffer solution to finally obtain 18 monoclonal antibodies, wherein the concentration of the monoclonal antibodies is over 5.0mg/ml, and the purity of the monoclonal antibodies is over 95%.

Subtype identification was performed on 18 monoclonal antibodies, 12 of which were IgG and 6 of which were IgM.

EXAMPLE 3 monoclonal antibody pairing

And (3) marking 12 IgG monoclonal antibodies by a sodium periodate method, dissolving 5mg HRP in 0.5ml of water, adding 0.5ml of fresh 0.06M NaIO4 aqueous solution, uniformly mixing, and standing at 4 ℃ for 30 min.

After being taken out, 0.5ml of 0.16M ethylene glycol aqueous solution was added, and after standing at room temperature for 30min, 1ml of an aqueous solution containing 5mg of the purified antibody was added, followed by mixing and filling into a dialysis bag, and dialyzing against 0.05M carbonate buffer solution of pH9.51 overnight.

After the mixture was taken out, 0.2ml of NaBH4 solution (5mg/ml) was added, and the mixture was placed in a refrigerator for 2 hours.

Adding equal volume of saturated ammonium sulfate, standing in refrigerator for 30min, centrifuging, resuspending the precipitate with 2.5ml of 20mM PBS, adding equal volume of glycerol, mixing, and storing.

The coated antibody is diluted to 0.5-4ug/ml with 20mM PB7.4, coated on a 96-well enzyme label plate at 100ul per well, and coated at 37 ℃ for 2 h.

And (3) taking out the enzyme label plate, discarding the coating solution, washing for 1 time by using PBST, adding 200ul of sealing solution into each hole, and sealing for 2 hours at 37 ℃.

Taking out the enzyme label plate, discarding the confining liquid, adding the diluted ferritin sample into the plate, reacting at 37 ℃ for 30min with each hole being 100 ul.

Taking out the enzyme label plate, discarding the sample, washing with PBST for 5 times, adding 100ul enzyme-labeled secondary antibody into each hole, and reacting at 37 ℃ for 30 min.

And (3) taking out the enzyme label plate, discarding the enzyme-labeled secondary antibody, washing with PBST for 5 times, adding 50ul of tetramethyl benzidine and 50ul of hydrogen peroxide into each hole, and reacting at 37 ℃ for 15 min.

And (4) taking out the ELISA plate, adding 2M sulfuric acid into each hole to terminate the reaction, and putting the ELISA plate into an ELISA reader for reading.

A monoclonal antibody with the best performance is selected to be respectively coated and marked, namely FTL-McAb1, and the FTL-McAb1 is secreted by a hybridoma cell strain 3F 6.

Example 4 immunochromatography assay

(1) Preparing colloidal gold: adding 100ml of ultrapure water into an erlenmeyer flask, heating to boiling on a magnetic heating stirrer, adding 1ml of 1% chloroauric acid (Sigma-Aldrich company, product number: 16961-25-4) solution, immediately adding 1ml of 1% trisodium citrate (Sigma-Aldrich company, product number: 6132-04-3) aqueous solution after boiling, continuing boiling for 10 minutes, and naturally cooling.

(2) Labeling with colloidal gold: 10ml of the above colloidal gold was put into a beaker, and 0.1M K was added while stirring₂CO₃Adjusting the pH value to 7.0, and continuing stirring for 5 minutes; adding a certain amount of monoclonal antibody FTL-McAb1, and continuing stirring for 15-30 minutes; adding 0.1ml of 10% BSA, and continuing stirring for 15-30 minutes; 8000g was centrifuged for 59 minutes, the supernatant was discarded, and the precipitate was resuspended in colloidal gold diluent (20mM PB, 250mM NaCl, 2% BSA, 1% Sucrose, 0.01% Proclin300), to a constant volume of 1ml, and stored at 4 ℃ after being thoroughly mixed.

(3) Preparing a gold label pad: diluting the gold-labeled compound by 10 times of colloidal gold diluent, soaking glass fiber (Watman company), drying at 37 ℃ for 2 hours to obtain a gold-labeled pad, drying, sealing and storing for later use.

(4) Nitrocellulose (NC) membrane coating: the monoclonal antibody FTL-McAb1 was diluted to 1mg/ml with a test line diluent (10mM PBS + 2% sucrose) to prepare a test line working solution, the goat anti-mouse monoclonal antibody was diluted to 0.5mg/ml with the same diluent to prepare a control line working solution, the two working solutions were drawn to the corresponding positions on a nitrocellulose membrane (Millipore, cat # HF135002) with a spotting instrument, and dried at 37 ℃ for 8 hours.

(5) And (3) assembling the gold-labeled pad, the coated nitrocellulose membrane, absorbent paper, a polyester plate, a sample pad and other auxiliary materials into the ferritin gold-labeled detection kit.

(6) The detection method comprises the following steps: adding 100ul of sample (such as serum) to the sample pad, standing at room temperature for 5min, determining the result according to the following criteria,

① one band appeared in the control zone only, and no band appeared in the test zone, which was negative (-);

② two bands appear, one in the control region and the other in the test region, indicating a positive (+);

③ No bands appear in the quality control zone, indicating an incorrect procedure or a test card has been tampered with.

(7) And (3) comparison and detection: according to the preparation process of the colloidal gold test strip, the ferritin monoclonal antibody FTL-McAb1 is coated, the ferritin monoclonal antibody FTL-McAb1 related to the invention is marked, then the test strip is assembled in a matching way, and a clinical specimen detected by 71 Abbott ferritin detection kit is detected, wherein the result shows that the test strip detection sensitivity can reach 2ng/ml, the linear range is 5-500ng/ml, and the correlation R with the Abbott detection result is 0.97 (figure 3, the FTL immunochromatographic detection result is clinically correlated with Abbott reagents).

Therefore, the immunochromatographic test strip prepared by the ferritin monoclonal antibody provided by the invention has excellent performance.

Example 5 detection by chemiluminescence method

(1) Magnetic bead coated antibody: 2mg of PM3-008 magnetic beads were placed in a 1.5ml centrifuge tube, washed 2 times with 500ul MEST (10mM MES, pH6.0, 0.05% Tween 20), and the supernatant was discarded. 200ul of freshly prepared EDC (5mg/ml) and 200ul of NHS (5mg/ml) were added, mixed well, activated at 37 ℃ for 30min, and the supernatant was discarded. 500ul of BST (5mM boric acid, 0.05% Tween 20, pH9.0) was added, mixed well, the supernatant discarded, and washed 2 times with 500ul of BST. 100ug of monoclonal antibody FTL-McAb1 was added to the beads, the mixture was stirred and mixed, the supernatant was discarded, the mixture was washed with 1ml of BST for 2 times, and the mixture was resuspended in 5ml of a magnetic bead storage medium.

(2) Acridinium ester labeled antibody: monoclonal antibody FTL-McAb1, 200ul was added to 600ul 0.1M PBS (pH8.0), 150ul 0.5mM AE (dissolved in methyl formamide) was added, mixed well, and reacted at room temperature with exclusion of light for 20 min. Lysine solution (8mg lysine in 200ul PBS pH8.0) was added and left for 15min, and then dialyzed against 10mM PBS pH6.5 overnight, followed by purification on Sephadex G50 the next day.

(3) And (3) detecting by a chemiluminescence method: 50ul of magnetic bead labeled antibody, 25ul of antigen or specimen to be detected and 50ul of analysis buffer solution are added into the sealed microporous plate, the plate is incubated for 15min at room temperature, 200ul of washing solution is carefully washed for 3 times each time, and the plate is sucked dry. Each well was added with assay buffer at a rate of 1: the acridinium ester labeled antibody diluted 100 times is reacted at room temperature for 10min in 50ul, and the washing solution is carefully washed 5 times each time in 200ul and then sucked dry. 200ul of washing solution was added to each well, blown up evenly with a pipette, transferred to a 2ml luminotron, and the supernatant was blotted dry on a magnetic rack. Adding 100ul of the pre-excitation liquid A, detecting on a machine, adding 100ul of the excitation liquid B, and detecting the luminescence value.

(4) 144 clinical specimens detected by the Abbott kit for monoclonal antibody pairing detection of the invention are characterized in that a monoclonal antibody FTL-McAb1 is used to coat magnetic beads, FTL-McAb1 is used to mark acridine ester, the sensitivity of the human serum ferritin chemiluminescence detection reagent established by the monoclonal antibody is 0.2ng/ml, the linear range is 1-1000ng/ml, and the correlation with the Abbott detection result is R & ltg & gt 0.96 (figure 4: the correlation between the FTL chemiluminescence detection result and the Abbott reagent is clinical).

Example 6 immunoturbidimetric assay

(1) Activating the polystyrene microspheres: the microspheres were selected to have a particle size of 307nm, a concentration of 100mg/ml (10%), a carboxyl density of 59, and were diluted 20-fold with 50mM pH6.0MES-HCl, and addition of NHS followed by EDC, where EDC: COOH ═ 2:1, NHS: COOH 3.4:1, activation for 20 min.

(2) Preparing an antibody for coupling: antibody FTL-McAb1 was diluted with 50mM MES, pH6.0, at a rate of 75ug antibody/1 mg of microspheres per tube.

(3) Coupling the microspheres with the antibody: centrifuging the activated microspheres at 16000rpm/min for 15min, discarding the supernatant, and re-dissolving the microspheres with diluted antibody MES (ultrasonic 75 s); after reconstitution, the microspheres were added to the antibody and sonicated (75s), microspheres: antibody 250: 500. Each tube of conjugate was placed on a tumbling apparatus and tumbled for 2h at room temperature.

(4) And (4) terminating: addition of 5ul/ml ethanolamine was stopped for 30 minutes.

(5) And (3) sealing: the terminated conjugate was centrifuged (16000rpm/min,15min), the supernatant was discarded, and the resultant was reconstituted with R2 (blocking solution, 20mM pH8.0Gly-Tris, 3% trehalose, 2% BSA, 0.1% Tween-20, 0.05% proclin), oven at 37 ℃ overnight, and then subjected to on-machine detection on the next day.

(6) And (3) detection results: and (3) detecting 32 clinical samples detected by an Abbott kit, wherein the sensitivity is 5ng/ml, the linear range is 10-2000ng/ml, and the correlation R with the detection result of Abbott is 0.97 (figure 5: the detection result of FTL immunoturbidimetry is clinically correlated with Abbott reagent).

In summary, the following steps: the monoclonal antibody FTL-McAb1 which is secreted by the hybridoma cell strain 3F6 and specifically recognizes the light chain of human ferritin is obtained in the embodiments 1, 2 and 3, and the detection of different detection platforms in the embodiments 4, 5 and 6 shows that the content of human ferritin in a specimen has the advantages of high sensitivity, good specificity, wide linear range and good universality of different detection platforms, and can replace the FTL monoclonal antibody in the existing kit.

Sequence listing

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Claims (5)

1. A hybridoma cell strain 3F6 secreting human ferritin light chain monoclonal antibody is characterized in that the hybridoma cell strain is preserved in CCTCC, and the preservation number is CCTCC NO: C2019173.

2. A monoclonal antibody FTL-McAb1 secreted from the hybridoma cell line 3F6 of claim 1 and specifically recognizing a light chain of human ferritin.

3. Use of the monoclonal antibody FTL-McAb1 specifically recognizing human ferritin light chain according to claim 2 in the preparation of a kit for detecting human serum ferritin.

4. A human serum ferritin immunoassay kit prepared using the monoclonal antibody FTL-McAb1 specifically recognizing human ferritin light chain of claim 2.

5. The human serum ferritin immunoassay kit of claim 4 comprising the monoclonal antibody FTL-McAb1 of claim 2.

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