CN110468108A - Secrete hybridoma cell strain and its application of human ferritin light chain monoclonal antibody - Google Patents
Secrete hybridoma cell strain and its application of human ferritin light chain monoclonal antibody Download PDFInfo
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- CN110468108A CN110468108A CN201910738807.8A CN201910738807A CN110468108A CN 110468108 A CN110468108 A CN 110468108A CN 201910738807 A CN201910738807 A CN 201910738807A CN 110468108 A CN110468108 A CN 110468108A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/79—Transferrins, e.g. lactoferrins, ovotransferrins
Abstract
The invention discloses a strain of hybridoma strain 3F6, its deposit number is CCTCC NO:C2019173, its monoclonal antibody FTL-McAb1 that can secrete specific recognition human ferritin light chain, also disclose purposes of the monoclonal antibody FTL-McAb1 on detection people's ferritin, and the serum ferritin immunity detection reagent prepared by monoclonal antibody FTL-McAb1.
Description
Technical field
The present invention relates to field of immunodetection, concretely relating to one kind, can to secrete human ferritin light chain monoclonal anti-
The hybridoma cell strain of body, the monoclonal antibody secreted by the hybridoma cell strain, and in detection serum ferritin
Purposes, and the people's ferritin detection kit prepared by the monoclonal antibody.
Background technique
Hypoferric anemia (Iron deficiency anemia, IDA) is to cause to store up in vivo due to a variety of different
Iron deposit lacks, and influences anaemia caused by the generation of cytohaem, such anaemia red cell morphology becomes smaller, and central olistherozone expands
Greatly, it is poor to belong to microcytic hypochromic.About 50% is all the hypoferric anemia as caused by iron deficiency in the patient of anaemia.Iron deficiency
Property anaemia laboratory check mainly there are two purpose: first is that the iron deficiency stage locating for patient is determined, second is that clinically distinguishing
Hypoferric anemia (IDA) and anemia of chronic disease (ACD).
Studies have shown that the content of serum ferritin and body storage iron are proportional, the serum ferritins of lug/L
Corresponding to 8-10mg or per kilogram of body weight 120ug storage iron.A large amount of research confirms serum ferritin compared to other iron
Coherent detection is used to diagnose the superiority of diagnosis of iron deficiency anemia, show that serum ferritin is lacked as diagnosis from more parts of research
The AUC average value of the ROC curve of iron anaemia is 0.95 (95% confidence interval), and zinc protoporphyrin (zinc
Protoporphyrin, ZPP) be 0.77, mean corpuscular volume (MCV) (MCV) is 0.74, Transferrin turation
(transferrin saturation) is 0.74.As serum ferritin is more and more widely used and marks in clinical diagnosis
The foundation of standardization, serum ferritin have gradually replaced new goldstandard of the bone marrow iron stain as evaluation body storage iron state.No
It is storage iron in body only in this way, serum ferritin just has apparent variation at asiderosis phase (iron depletion, ID)
Lack sensitive marker.Clinically the detection of serum ferritin can screen out the early stage of iron deficiency, so as to adopt as soon as possible
The measures such as adjustment diet structure or supplement chalybeate are taken, the generation of hypoferric anemia is prevented.Serum ferritin level reduces, generally
Hypoferric anemia is represented, level increases the reason is that malignant tumour, inflammation, hepatopathy etc..Therefore, Concentration of Serum Ferritin is detected, it is right
Hypoferric anemia, the monitoring of malignant tumour, inflammation, hepatopathy it is significant.
Serum ferritin is mainly made of L subunit, contains only a small amount of H subunit, and the content of iron atom is also seldom.People's blood
Clear ferritin exists in blood with the form of nanoparticles of 24 aggressiveness, does not find unassembled subunit.
Immunology detection is got on detection serum ferritin due to its high sensitivity, fireballing advantage
To be more widely applied.The main restraining factors of serum ferritin immunological detecting kit quality are exactly bioactivity former material
The quality of material.For from methodology, serum ferritin detection kit mostly uses greatly double antibody sandwich method, and bioactivity is former
Material is exactly ferritin monoclonal antibody.And the existing ferritin monoclonal antibody quality level in market is irregular, it is serious to limit
The quality of kit is made.
In consideration of it, it is necessary to develop the ferritin monoclonal antibody of high quality, to meet the quality requirement of kit.
Summary of the invention
The first object of the present invention is to provide a kind of hybridoma cell strain 3F6, is preserved in China typical culture collection
The heart (China Center for Type CultureCollection, abbreviation CCTCC), hybridoma cell strain 3F6 deposit number
It is CCTCC NO:C2019173, the monoclonal antibody FTL-McAb1 of specific recognition human ferritin light chain can be secreted.
The second object of the present invention is to provide a kind of specific recognition people ferritin as secreted by hybridoma cell strain 3F6
The monoclonal antibody FTL-McAb1 of light chain.
The third object of the present invention is to provide the monoclonal antibody FTL-McAb1 of specific recognition human ferritin light chain in people
Purposes in ferritin detection kit.
The fourth object of the present invention is to provide the monoclonal antibody FTL-McAb1 preparation of specific recognition human ferritin light chain
People's ferritin immunity detection reagent.
Preferably, people's ferritin immunity detection reagent, contains the spy as secreted by hybridoma cell strain 3F6
The monoclonal antibody FTL-McAb1 of opposite sex identification human ferritin light chain.
Cyropreservation:
A strain of hybridoma strain 3F6 of the invention is that the present inventor voluntarily screens and obtains, deposit number
For CCTCC NO:C2019173, the deposit date is 19 days, depositary institution CCTCC July in 2019, address was located at Hubei Province's force
No. 299 Wuhan Universitys of the Wuchang District Han Shi Bayi Road are in the school.
Term definition:
Signified human ferritin light chain of the invention, i.e. Ferritin light chain (abbreviation FTL), amino acid sequence
It is published in UniProt (https: //www.uniprot.org/uniprot/P02792), specifically such as SEQ ID NO:1 institute
Show, corresponding mRNA coding region nucleotide sequence is as shown in SEQ ID NO:2.
Monoclonal antibody (monoclonal antibody), abbreviation monoclonal antibody (McAb) are to be cloned to generate by single B cell
Height is uniform, antibody only for a certain specific antigen epitope.
Human embryonic kidney cells (Human embryonic kidney 293cells), abbreviation HEK 293, HEK-293,293 are thin
Born of the same parents can be used to express foreign protein.
People's ferritin detection kit, uses immunological detection method, using Ag-Ab specific reaction, is used to
The content for detecting people's ferritin in sample, is divided into enzyme-linked immunization according to detection platform, immunochromatographic method, chemoluminescence method, exempts from
Epidemic disease turbidimetry etc..
To achieve the goals above, according to the general step of preparation monoclonal antibody, it is necessary first to obtain people's ferritin work
For immunogene, the inventors discovered that, although high as immunogene immunizing potency using natural human ferritin, amount of antigen is too
It is few, it is too expensive, and use people's ferritin of the prokaryotic expressions such as Escherichia coli, it may be possible to because of the people of prokaryotic expression
Ferritin conformation is variant with native ferritin, and immunizing potency is relatively low, therefore people's ferritin of immunogene first choice eukaryotic expression,
Used in the embodiment of the present invention is exactly that the HEK-293 voluntarily developed recombinantly expresses FTL as immunogene.But other approach, side
The ferritin that method and expression system obtain also is available as immunogene, it is not limited to HEK-293 recombinant expression.
RNA is extracted from tissue (such as blood, liver, spleen), reverse transcription obtains the cDNA of FTL, then is with cDNA
Template obtains FTL genetic fragment with PCR method, and PCR enzyme adds base " A " at segment two 3 ' ends, then the segment is connected to T
On carrier, picks out positive colony and carry out determined dna sequence, thus obtain FTL gene, it can also be at RT-PCR primer both ends
Take restriction enzyme site, PCR product direct enzyme cutting is connected on the carrier after same digestion, selects positive colony and be sequenced.
Using full genome synthetic method, gives FTL gene to commercialization gene chemical synthesis company and synthesize and feasible.It obtains
FTL gene be connected on expression vector using the gene clone method of digestion or non-digestion.
Expression vector and expression system be it is corresponding, expression system well known to those skilled in the art has protokaryon table
Up to system, including escherichia expression system, there is an eukaryotic expression system, including yeast expression system, insect cell expression system,
Mammalian expression systems may serve to expression FTL albumen.The expression vector for being connected to FTL gene is imported into corresponding expression
In the host cell of system, wherein the corresponding leading-in technique of prokaryotic expression system is conversion, the corresponding importing of eukaryotic expression system
Technology is transfection.Conversion or transfection need to be added corresponding antibiotic and carry out selective screening, select and carry FTL gene
Daughter cell is recombinated, expression identification is carried out, selects the big cell strain of FTL expression quantity, expand culture.The thin of culture is collected by centrifugation
Born of the same parents' (when intracellular expression), or supernatant (when secreting, expressing) is collected, by purifying, obtain recombination FTL albumen.
For several times with natural or recombination FTL protein immunization mouse, ELISA measures mice serum potency, and the high person of selective value takes
Splenocyte is merged with myeloma cell, screens positive monoclonal cell strain, and mouse abdomen will be injected into after its amplification cultivation
Chamber produces ascites, and extracorporeal culture-ing productive culture object can also be used.The ascites or culture for being rich in antibody are collected, process is pure
Change, obtains FTL monoclonal antibody.These monoclonal antibodies are subjected to subtype identification.
FTL labeling of monoclonal antibody HRP is matched to detect various concentration with unlabelled monoclonal antibody ELISA
FTL selects best one plant of performance to investigate the performance indicators such as its sensitivity, specificity, the range of linearity, carries out further
Performance verification.One plant of monoclonal antibody FTL-McAb1 finally is had selected, as secreted by hybridoma cell strain 3F6.Because of iron egg
White is 24 subunits, in order to simplify raw materials and reagents box preparation process, so only being used to be coated with and mark simultaneously with one plant of monoclonal antibody
Also it is able to achieve goal of the invention.
It is used to be coated with and mark with monoclonal antibody FTL-McAb1, the serum ferritin content in immune detection sample,
Detection platform includes that enzyme-linked immunization, immunochromatographic method, immunoturbidimetry, chemoluminescence method etc. all have better effects.
The utility model has the advantages that
Monoclonal antibody FTL-McAb1 secreted by hybridoma cell strain 3F6 of the invention, for detecting the people in sample
Concentration of Serum Ferritin has high sensitivity, and specificity is good, and the range of linearity is wide, the good advantage of different detection platform versatilities, energy
Enough substitute the FTL monoclonal antibody in available reagent box.
Detailed description of the invention
Fig. 1: recombinant expression FTL SDS-PAGE electrophoresis after purification.
The SDS-PAGE electrophoresis of Fig. 2: FTL monoclonal antibody FTL-McAb1 after purification.
Fig. 3: FTL immunochromatography testing result and Abbott Laboratories' reagent clinical correlation.
Fig. 4: FTL chemiluminescence detection result and Abbott Laboratories' reagent clinical correlation.
Fig. 5: FTL immunoturbidimetry testing result and Abbott Laboratories' reagent clinical correlation.
Specific embodiment
The preparation of embodiment 1:FTL antigen
First with the code area mRNA of FTL (sequence is as shown in SEQ ID NO:2) for template, pair of primers (sequence is designed
Respectively such as SEQ ID NO:3 and SEQ ID NO:4).
Fresh human liver cancer tissue is taken, RNA is extracted with silent your the Trizol one-step method of winged generation of match, with AMV reverse transcription reagent box handle
RNA reverse transcription is cDNA, then using cDNA as template, carries out PCR amplification with above-mentioned primer.
PCR product carries out agarose gel electrophoresis and purpose band is cut, with AXYGEN glue after confirmation molecular weight is correct
QIAquick Gel Extraction Kit is recycled to arrive FTL genetic fragment.
Obtained FTL genetic fragment is expanded, 3 ' ends all have a base " A ", are connected to TaKaRa's
On pMD-18T carrier, the positive clone of PCR identification is sequenced, by sequence alignment and SEQ ID NO:2 have 100% it is same
Source property, it was demonstrated that gene obtains successfully, obtains pMD-18T-FTL clone.
PMD-18T-FTL plasmid is extracted with AXYGEN plasmid extraction kit to be cut with BamHI the and EcoRI enzyme of NEB
FTL genetic fragment is connected on the plasmid vector pcDNA3.1 (+) by same digestion, and attachment converts DH5 α bacterial strain, is chosen
Choosing carries out sequence verification by the positive monoclonal of PCR identification, it was demonstrated that the success of pcDNA3.1 (+)-FTL plasmid construction.
Culture HEK-293 cell is inoculated with 24 holes and cultivates to 80-90% density, transfected with it to logarithmic growth phase in advance
Transfection reagent/plasmid mixture of PEI/pcDNA3.1 (+)-FTL has transfected the cell of gene with antibiotic G418 screening.
Positive cell is passed on, carries out high expression screening with ELISA method detection supernatant, successfully obtaining one plant can be steady
Fixed height expresses cell strain pcDNA3.1 (+)-FTL (HEK-293) 3D2 of FTL, and FTL concentration reaches 10mg/L in supernatant.
By multiple rolling bottle amplification cultivation 3D2 cell, it is collected into the cell conditioned medium of 5L, is purified with S200 and DEAE, most
40mg recombination FTL albumen is obtained eventually, and purity reaches 95% or more, and 80% object above albumen to be existed with 24 dimer forms.
1 FTL monoclonal antibody ELISA testing result of table
2 FTL Antibody preparation of embodiment
FTL recombinant antigen 20mM PBS pH7.4 dialysis prepared by embodiment 1, and 1mg/ is diluted to dialyzate
Ml is mixed with adjuvant according to 1:1 ratio, emulsification.
The immune 8 week old Balb/c mouse of FTL antigen after emulsification, every mouse immune 100ug, initial immunity Freund are complete
Full adjuvant, booster immunization incomplete Freund's adjuvant.
After immune three needles, tail vein is adopted, measures potency, selecting potency is more than 1,000,000 progress cell fusions.
Fusion impact in first 72 hours is immune, to intraperitoneal injection 30-50ug FTL antigen.
Mouse to be fused is put to death, spleen is taken, mills in 200 mesh screens, while being washed with RPMI1640 culture medium, it will be thin
Born of the same parents' suspension is transferred in 50ml centrifuge tube, is washed 4 times with RPMI1640 culture medium, and each 15000rpm is centrifuged 5min.
Myeloma cell is washed 4 times with RPMI1640 culture medium, each 15000rpm is centrifuged 5min, last 1 time thin with spleen
Born of the same parents wash together.
Mixed splenocyte and myeloma cell 1500rpm are centrifuged 5min, discards supernatant, flicks tube bottom, by cell
It bounces；It is slowly added to the PEG1450 that 1ml shifts to an earlier date pre-temperature to 37 degree, side edged mixes, and adds in 60s；40ml is added immediately to shift to an earlier date
1640 culture medium of RPMI of pre-temperature to 37 degree terminates fusion reaction, and 1500rpm is centrifuged 5min；Cell is bounced, RPMI is transferred to
In 1640 HAT cell culture mediums, bed board after mixing.
Liquid and detection supernatant are periodically changed after bed board, high level positive hole is selected and is subcloned, after 3 subclones, most
Select 18 plants of hybridoma monoclonal cells eventually, supernatant to the HEK-293 human ferritin light chain expressed and native liver ferritin all
There is higher reaction (value > 1.0 OD).
18 strain of hybridoma are injected into mouse respectively, collect the ascites of generation, sediment, supernatant saturation are removed in centrifugation
Pure at the beginning of ammonium sulfate, Protein A column affinity chromatography is consummate, then carries out dialysis PBS buffer solution, has finally obtained 18 plants of monoclonal antibodies, concentration
All in 5.0mg/ml or more, purity is all 95% or more.
Subtype identification is carried out to 18 plants of monoclonal antibodies, wherein 12 plants are IgG, in addition 6 plants are IgM.
The pairing of 3 monoclonal antibody of embodiment
12 plants of IgG monoclonal antibodies are marked using Over-voltage protection, 5mg HRP is taken to be dissolved in 0.5ml water, are added fresh
0.06M NaIO4 aqueous solution 0.5ml is mixed, 4 degree of placement 30min.
0.16M glycol water 0.5ml is added after taking-up, after being placed at room temperature for 30min, the antibody purification containing 5mg is added
It 1 milliliter of aqueous solution, mixes and fills bag filter to 0.05M, the carbonate buffer solution dialysed overnight of pH9.51.
NaBH4 solution (5mg/ml) 0.2ml is added after taking-up, sets refrigerator 2h.
Isometric saturated ammonium sulfate is added, after refrigerator places 30min, centrifugation, precipitating is resuspended with 2.5ml 20mM PBS, is added
Isometric glycerol mixes, and saves.
Coated antibody is diluted to 0.5-4ug/ml with 20mM PB7.4, every hole adds 100ul to be coated in 96 hole elisa Plates, and 37
Degree coating 2h.
ELISA Plate is taken out, coating buffer is discarded, is washed 1 time with PBST, 200ul confining liquid, 37 degree of closing 2h are added in every hole.
ELISA Plate is taken out, confining liquid is discarded, the ferritin sample diluted is added in plate, every hole 100ul, 37 degree of reactions
30min。
ELISA Plate is taken out, sample is discarded, is washed 5 times with PBST, ELIAS secondary antibody 100ul, 37 degree of reaction 30min is added in every hole.
ELISA Plate is taken out, ELIAS secondary antibody is discarded, is washed 5 times with PBST, tetramethyl benzidine is added in every hole and hydrogen peroxide is each
50ul, 37 degree of reaction 15min.
ELISA Plate is taken out, every hole is added 2M sulfuric acid and terminates reaction, and ELISA Plate is put into readings in microplate reader.
Selected one plant of optimal monoclonal antibody of performance is coated with and marks respectively, i.e. FTL-McAb1, they are by hybridoma cell strain
Secreted by 3F6.
4 immunochromatographyassay assay of embodiment
(1) prepared by colloidal gold: 100ml ultrapure water being added in conical flask, is heated to boiling in magnetic heating stirrer, add
Enter 1% gold chloride of 1ml (Sigma-Aldrich company, article No.: 16961-25-4) solution, 1ml 1% is added immediately after boiling
Trisodium citrate (Sigma-Aldrich company, article No.: 6132-04-3) aqueous solution continues boiling 10 minutes, then naturally cold
But.
(2) colloid gold label: taking the above-mentioned colloidal gold of 10ml to be put into beaker, and 0.1M K is added in stirring2CO3Adjust pH to
7.0, continue stirring 5 minutes；A certain amount of monoclonal antibody FTL-McAb1 is added, continues stirring 15-30 minutes；0.1ml 10% is added
BSA continues stirring 15-30 minutes；8000g be centrifuged 59 minutes, abandon supernatant, by precipitating with colloidal gold dilution (20mM PB,
250mM NaCl, 2%BSA, 1%Sucrose, 0.01%Proclin300) it is resuspended, it is settled to 1ml, 4 degree are put after mixing well
It saves.
(3) prepared by gold-labelled pad: will impregnate glass fibre after 10 times of the colloidal gold dilution dilutions of above-mentioned gold mark compound
(Watman company), 37 degree are dried 2 hours, that is, gold-labelled pad is made, and drying is sealed spare.
(4) nitrocellulose filter (NC) film is coated with: diluting monoclonal antibody with detection line dilution (10mM PBS+2% sucrose)
Detection line working solution is made in FTL-McAb1 to 1mg/ml, is made pair with same diluted sheep anti mouse monoclonal antibody to 0.5mg/ml
According to line working solution, with point film instrument by both working solutions draw to nitrocellulose filter (Millipore company, article No.:
HF135002 on corresponding position), 37 degree drying 8 hours.
(5) by the auxiliary materials group such as above-mentioned gold-labelled pad, the nitrocellulose filter being coated with and blotting paper, polyester sheet, sample pad
Dress up ferritin gold mark detection kit.
(6) detection method: add 100ul sample to be tested (such as: serum) at sample pad, be placed at room temperature for after 5 minutes, sentence
Calmly as a result, criterion is as follows,
1. only a band occurs in quality control region, occur in test section without band, for negative (-)；
2. there is the appearance of two band, wherein one is located at quality control region, another is located at test section, indicates positive (+)；
3. quality control region does not occur band, show that incorrect operating process or detection block damage of having gone bad.In the case,
Specification should be read over again, and is retested with new test strips.
(7) contrasting detection: according to above-mentioned colloidal gold strip preparation process, by ferritin monoclonal antibody of the invention
FTL-McAb1 is coated with, and ferritin monoclonal antibody FTL-McAb1 according to the present invention is marked, is then matched
Test strips are assembled into, the clinical samples after detecting 71 parts of Abbott Laboratories (Abbott) company's ferritin detection detection kit detections, knot
Fruit shows that test strips detection sensitivity can achieve 2ng/ml, range of linearity 5-500ng/ml, with Abbott Laboratories' testing result correlation
R=0.97 (Fig. 3: FTL immunochromatography testing result and Abbott Laboratories' reagent clinical correlation).
It can be seen that the immuno-chromatographic test paper strip of ferritin monoclonal antibody preparation according to the present invention is had excellent performance.
The detection of 5 chemoluminescence method of embodiment
(1) magnetic bead coated antibody: taking 2mg PM3-008 magnetic bead into 1.5ml centrifuge tube, with 500ul MEST (10mM
MES, pH6.0,0.05%Tween 20) washing 2 times, abandon supernatant.EDC (5mg/ml) and 200ul that 200ul is newly prepared is added
NHS (5mg/ml) is mixed, 37 degree of activation 30min, abandons supernatant.Be added 500ul BST (5mM boric acid, 0.05%
Tween 20, pH9.0), it mixes, abandons supernatant, then washed 2 times with 500ul BST.100ug monoclonal antibody FTL-McAb1 is added to magnetic
Pearl, rolling mix, and abandon supernatant, then washed 2 times with 1ml BST, save liquid with 5ml magnetic bead and are resuspended.
(2) acridinium ester label antibody: take monoclonal antibody FTL-McAb1,200ul that 600ul 0.1M PBS is added
(pH8.0) in, the AE (being dissolved in methylformamide) of 150ul 0.5mM is added, mixes, room temperature is protected from light 20min.Add bad ammonia
Acid solution (PBS that 8mg lysine is dissolved in 200ul pH8.0) places 15min, to the PBS dialysed overnight of 10mM pH6.5, next day
It is purified with sephadex G 50.
(3) chemoluminescence method detects: into the microwell plate close addition antibody of 50ul marked by magnetic bead, 25ul antigen or
Sample to be measured, 50ul analysis buffer, are incubated at room temperature 15min, and cleaning solution 200ul is carefully washed 3 times every time, blotted.Every hole adds
Enter with analysis buffer according to 1:100 times of diluted acridinium ester label antibody 50ul, reacts at room temperature 10min, cleaning solution 200ul is every
It secondary careful washing 5 times, blots.200ul cleaning solution is added in every hole, is blown with pipettor even, is transferred in 2ml luminous tube, magnetic frame
On blot supernatant.100ul preexciting liquid A liquid is added, upper machine testing adds 100ul exciting liquid B liquid, detects luminous value.
(4) 144 parts of clinical samples that detection Abbott Laboratories (Abbott) kit detected are matched with monoclonal antibody of the present invention, from calibration
As can be seen that being coated with magnetic bead with monoclonal antibody FTL-McAb1 in curve, acridinium ester, this plant of monoclonal are marked with FTL-McAb1
Antibody establish serum ferritin chemiluminescence detection reagent sensitivity be 0.2ng/ml, range of linearity 1-1000ng/ml,
With Abbott Laboratories testing result correlation R=0.96 (Fig. 4: FTL chemiluminescence detection result and Abbott Laboratories' reagent clinical correlation).
The detection of 6 immunoturbidimetry of embodiment
(1) microspherulite diameter 307nm, concentration 100mg/ml (10%), carboxyl density the activation of polystyrene microsphere: are selected
59, microballoon is diluted 20 times with 50mM pH6.0MES-HCl, NHS is added and adds EDC later, wherein EDC:COOH=2:1,
NHS:COOH=3.4:1 is activated 20 minutes.
(2) it prepares coupling antibody: with the ratio of every pipe 75ug antibody/1mg microballoon, being diluted with 50mM pH6.0MES anti-
Body FTL-McAb1.
(3) microballoon coupled antibody: the microballoon 16000rpm/min after the completion of activating is centrifuged 15 minutes, supernatant is abandoned, with dilution
The MES of antibody redissolves microballoon (ultrasonic 75s)；After redissolution, in antibody and ultrasonic (75s), microballoon: antibody=250 is added in microballoon:
500.Every pipe conjugate is placed on overturning instrument, room temperature overturns 2h.
(4) it terminates: 5ul/ml ethanol amine is added and terminates 30 minutes.
(5) close: centrifugation terminate after conjugate (16000rpm/min, 15min), abandon supernatant, with R2 (confining liquid,
20mM pH8.0Gly-Tris, 3% trehalose, 2%BSA, 0.1%Tween-20,0.05%proclin) it redissolves, 37 degree of baking ovens
One night, the second machine testing in the sky.
(6) testing result: the clinical samples that 32 parts of Abbott Laboratories (Abbott) kits of detection were surveyed, sensitivity 5ng/ml, line
Property range 10-2000ng/ml, with Abbott Laboratories testing result correlation R=0.97 (Fig. 5: FTL immunoturbidimetry testing result with it is refined
Train reagent clinical correlation).
In summary: specific recognition people ferritin secreted by hybridoma cell strain 3F6 is obtained by embodiment 1,2,3
The monoclonal antibody FTL-McAb1 of light chain is detected, the human serum iron in sample by the different detection platform of embodiment 4,5,6
Protein content all has high sensitivity, and specificity is good, and the range of linearity is wide, and the good advantage of different detection platform versatilities can be replaced
For the FTL monoclonal antibody in available reagent box.
Sequence table
<120>hybridoma cell strain and its application of human ferritin light chain monoclonal antibody are secreted
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 175
<212> PRT
<213> Homo sapiens
<400> 1
Met Ser Ser Gln Ile Arg Gln Asn Tyr Ser Thr Asp Val Glu Ala Ala
1 5 10 15
Val Asn Ser Leu Val Asn Leu Tyr Leu Gln Ala Ser Tyr Thr Tyr Leu
20 25 30
Ser Leu Gly Phe Tyr Phe Asp Arg Asp Asp Val Ala Leu Glu Gly Val
35 40 45
Ser His Phe Phe Arg Glu Leu Ala Glu Glu Lys Arg Glu Gly Tyr Glu
50 55 60
Arg Leu Leu Lys Met Gln Asn Gln Arg Gly Gly Arg Ala Leu Phe Gln
65 70 75 80
Asp Ile Lys Lys Pro Ala Glu Asp Glu Trp Gly Lys Thr Pro Asp Ala
85 90 95
Met Lys Ala Ala Met Ala Leu Glu Lys Lys Leu Asn Gln Ala Leu Leu
100 105 110
Asp Leu His Ala Leu Gly Ser Ala Arg Thr Asp Pro His Leu Cys Asp
115 120 125
Phe Leu Glu Thr His Phe Leu Asp Glu Glu Val Lys Leu Ile Lys Lys
130 135 140
Met Gly Asp His Leu Thr Asn Leu His Arg Leu Gly Gly Pro Glu Ala
145 150 155 160
Gly Leu Gly Glu Tyr Leu Phe Glu Arg Leu Thr Leu Lys His Asp
165 170 175
<210> 2
<211> 421
<212> RNA
<213> Nucleotide sequence of coding region
<400> 2
agagccccag acgcagaaac caccgacggg aggcagccgc aacagccggc aagaccgcag 60
gccccacacc acccccgggc cacgaccgcg agagggccgg aaggcggagc caccccgcga 120
aggccgagga gaagcgcgag ggcacgagcg cccgaagagc aaaaccagcg ggcggccgcg 180
cccccaggac acaagaagcc agcgaagaga gggggaaaac cccagacgcc agaaagcgcc 240
aggcccggag aaaaagcgaa ccaggcccgg accagcccgg gcgcccgcac ggacccccac 300
cggacccgga gaccacccag agaggaagga agcacaagaa gaggggacca ccgaccaacc 360
ccacaggcgg gggcccggag gcgggcgggc gagacccgaa aggccaccca agcacgacga 420
a 421
<210> 3
<211> 24
<212> DNA
<213> amplified polymorphic
<400> 3
ggatccatga gctcccagat tcgt 24
<210> 4
<211> 27
<212> DNA
<213> amplified polymorphic
<400> 4
gaattcttag tcgtgcttga gagtgag 27
Claims (5)
1. the hybridoma cell strain 3F6 of one plant of secretion human ferritin light chain monoclonal antibody, which is characterized in that it is preserved in
CCTCC, deposit number are CCTCC NO:C2019173.
2. specific recognition human ferritin light chain secreted by a kind of hybridoma cell strain 3F6 as described in claims 1
Monoclonal antibody FTL-McAb1.
3. a kind of monoclonal antibody FTL-McAb1 of specific recognition human ferritin light chain described in claims 2 is being detected
Application in serum ferritin.
4. a kind of monoclonal antibody FTL-McAb1 system with specific recognition human ferritin light chain described in claims 2
Standby serum ferritin immunity detection reagent.
5. serum ferritin immunity detection reagent according to claim 4, which is characterized in that contain claim
Monoclonal antibody FTL-McAb1 described in 2.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114573668A (en) * | 2020-11-30 | 2022-06-03 | 中国人民解放军军事科学院军事医学研究院 | Japanese encephalitis virus-like particle and preparation method thereof |
Citations (1)
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| CN105821004A (en) * | 2016-04-22 | 2016-08-03 | 成都正能生物技术有限责任公司 | Ferritin light chain monoclonal antibody and application thereof |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105821004A (en) * | 2016-04-22 | 2016-08-03 | 成都正能生物技术有限责任公司 | Ferritin light chain monoclonal antibody and application thereof |
Non-Patent Citations (2)
| Title |
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| PENGFEI WANG等: "Nanoscale magnetic imaging of ferritins in a single cell", 《SCIENCE ADVANCES》 * |
| 金蕾 等: "铁蛋白轻链的原核表达、纯化及其多克隆抗体的制备", 《第二军医大学学报》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114573668A (en) * | 2020-11-30 | 2022-06-03 | 中国人民解放军军事科学院军事医学研究院 | Japanese encephalitis virus-like particle and preparation method thereof |
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