CN108845116A - Amoxicillin antibody assay kit and its preparation and application - Google Patents
Amoxicillin antibody assay kit and its preparation and application Download PDFInfo
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- CN108845116A CN108845116A CN201810418420.XA CN201810418420A CN108845116A CN 108845116 A CN108845116 A CN 108845116A CN 201810418420 A CN201810418420 A CN 201810418420A CN 108845116 A CN108845116 A CN 108845116A
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Abstract
The present invention relates to a kind of Amoxicillin antibody assay kit and its preparation and application, are mainly made of Amoxicillin antibody test reagent card, Amoxicillin processing red blood cell, non-Amoxicillin processing red blood cell, Amoxicillin antibody positive comparison liquid, Amoxicillin antibody negative controls liquid.The kit has easy to operate, and sensitivity is strong with stability, more convenient in clinical application, can be to the quick detection of patient's progress Amoxicillin antibody.
Description
Technical field
The invention belongs to antibiotic antibody detection fields, and in particular to the detection kit of Amoxicillin antibody, preparation side
Method and application method.
Background technique
With advances in technology with the development of medicinal industry, more and more antibiotics in clinical use, thus
The abuse for causing antibiotic makes clinic antibiotic medicine occur and the phenomenon that haemolysis occurs using invalid even patient.It is light then influence
Therapeutic effect, delay treatment, it is heavy then jeopardize patient vitals safety, generate malpractice, cause doctor-patient dispute.By the end of currently,
It there is no any effective ways to detect antibiotic antibody, for the haemolysis side effect for avoiding antibiotic, can only use while using more
Antibiotic is planted to treat.Therefore clinical there is an urgent need to antibiotic antibody detection kits, for detecting whether patient's body contains
Resist the antibody of such drug, to predict whether the antibiotic of selection may occur haemolysis, to instruct the use of clinical rational anti-
Raw element.
Amoxicillin is essentially identical for semi-synthetic penbritin class medicine, antimicrobial spectrum and antibacterial activity and ampicillin, but
Its acid resistance is strong compared with ampicillin, and bactericidal effect is strong and rapid compared with the latter, but cannot be used for the treatment of meningitis.Half-life period is about
It is 61.3 minutes.Amoxicillin is stable in acid condition, and gastrointestinal tract absorptivity absorbs completeer rapidly up to 90% compared with ampicillin
Entirely, except to shigella dysenteriae effect compared with ampicillin it is poor in addition to, remaining effect is similar.
When patient uses Amoxicillin in treatment clinical course, there may be for the anti-of such drug by some patients
Body.Patient once generates the antibody of resisting amoxicillin, in the antigen-antibody complex meeting which forms with Amoxicillin drug
With inhibit Amoxicillin drug drug effect so that the curative effect of Amoxicillin drug cannot fully ensure that.Meanwhile Amoxicillin medicine
The immunocomplex that the corresponding antibody of object is formed can cause erythrocyte hemolysis in conjunction with human red blood cells, gently then influence treatment effect
Fruit, delay treatment, it is heavy then jeopardize patient vitals safety.Therefore, establish effectively detect the method for internal Amoxicillin antibody for
Reasonable employment Amoxicillin drug has important clinical meaning.
Summary of the invention
The purpose of the present invention is to provide a kind of kit for detecting internal Amoxicillin antibody, the preparation sides of the kit
Method and application method.
The present invention solve the above problems used by technical solution be：A kind of Amoxicillin antibody assay kit, mainly
It is to be resisted by Amoxicillin antibody test reagent card, Amoxicillin processing red blood cell, non-Amoxicillin processing red blood cell, Amoxicillin
Body positive control solution, Amoxicillin antibody negative controls liquid composition.
The preparation method of above-mentioned Amoxicillin antibody assay kit, mainly by following steps, each step is without precedence relationship：
Step 1: Amoxicillin antibody test reagent card
(1.1), it measures：By take in batches sephadex as swelling gum, by sephadex measurement be transferred to it is clean simultaneously
In the container of sterilizing；
(1.2), it washs：Sephadex is washed with sodium chloride solution, is mixed well, supernatant is removed after centrifugation, is repeated
Operation 5 times or more, gel after must washing performs mark；
(1.3), it prepares：Antihuman globulin reagent (anti-igg+C is measured by batch3It d), is 1 by antibody and gel volume ratio:
3 ratio will mix in the gel after antihuman globulin reagent addition washing, be mixed into antigen gel respectively, perform mark
Know stand-by；
(1.4), filling, centrifugation：Continuous sample-adding gun is transferred to accurate calibration, quality control officer's monitoring, according to every Kong Gu
Antihuman globulin reagent gel is added in micro-pipe by fixed microlitre of antigen gel amount, and gel is not made to be attached to the anti-of micro-pipe upper end
It answers on cell wall,；
(1.5), it seals：After centrifugation, card, which is transferred on sealing machine, to be sealed.
Step 2: Amoxicillin handles red blood cell
(2.1), it is filtered, washed：O-shaped raw material erythrocyte is taken, is filtered with disposable leucocyte filter, by three person-portions
The mixing of O-shaped raw material erythrocyte, is washed with sodium chloride solution, removes supernatant after centrifugation, and repetitive operation 3 times or more；
(2.2), drug sensitization
The Amoxicillin a drug solution is prepared：By boric acid, potassium chloride is weighed in batches, adds purified water sufficiently to dissolve, be formulated as
± 0.1 borate buffer of 0.1mol/L, pH9.8 is added by the weighed Amoxicillin of batch, and sufficiently dissolution mixes to get Ah not
XiLin drug solution；
The Amoxicillin b drug-treated cell：Amoxicillin drug solution is measured by batch, is added O-shaped hematocrit, 37
It DEG C is incubated for 1 hour, discontinuity mixes primary, is washed sensitized erythrocyte 3 times or more using sodium chloride solution or until supernatant is without molten
Blood phenomenon, with blood cell analysis diluted to 2~2.5% concentration, 2~8 DEG C of preservations；
(2.3), it dispenses：Amoxicillin drug-treated cell is taken, is dispensed into drop bottle, is saved in 2~8 DEG C.
Step 3: non-Amoxicillin handles cell
(3.1), it is filtered, washed：O-shaped raw material erythrocyte is taken, is filtered with disposable leucocyte filter.By three person-portions
O-shaped raw material erythrocyte mixing, is washed with sodium chloride solution, supernatant is removed after centrifugation, repetitive operation 3 times or more, thin with blood
Born of the same parents analyze with diluted to 2~2.5% concentration, 2~8 DEG C of preservations；
(3.2), it dispenses：Negated Amoxicillin drug-treated cell is dispensed into drop bottle, is saved in 2~8 DEG C.
Step 4: Amoxicillin antibody positive comparison liquid
(4.1), it prepares：The Amoxicillin monoclonal antibody for taking certain potency, is diluted with BSA；
(4.2), it dispenses：It takes filtered Amoxicillin antibody positive to compare, dispenses into polyethylene bottle, every bottle of 3ml, in
2~8 DEG C of preservations.
Step 5: Amoxicillin antibody negative controls liquid
Packing：Filtered Amoxicillin antibody negative controls are taken, are dispensed into bottle, are saved in 2~8 DEG C.
Preferably, the concentration of the sodium chloride solution is 0.9wt%.
Further, 0.9% sodium chloride solution and sephadex volume ratio are 2 in step 1:1；Step 2: three Central Plains
Expect blood and 0.9% sodium chloride solution volume ratio is 1:4.
Preferably, the closed condition of step 1 sealing step sequence is：158 ± 2 DEG C of 0.3~0.35MPa pressure, temperature, time 3
Second/card.
The preparation side of the application kit Amoxicillin monoclonal antibody that a used price is imitated during the preparation process
Method, steps are as follows
(1) preparation of feeder cells：
6-10 week old Balb/c mouse is taken, neck is drawn to put to death, impregnates in 75% alcohol, sterilizes 3-5min；It is transferred to sterile behaviour
Make in platform, cut off skin with sterile scissors, exposes its peritonaeum sufficiently；With the 10mL serum-free of asepsis injector injection pre-cooling
1640 culture medium is gently flapped mouse peritoneal with tweezers bottom, and culture solution is sucked out, and is put into 10mL centrifuge tube, 1200r/min centrifugation
10min；Supernatant is discarded, feeder cells are resuspended with complete culture solution, counts, and adjust cell number to 2 × 105/mL, will raise
Cell is added in 96 orifice plates, and every 100 μ L of hole is then placed in 37 DEG C of CO2 incubator cultures；
(2) hybridoma cell fusion and limiting dilution assay subclone screening
A. immunized B cells are prepared：Mouse after will be immune plucks eyeball bloodletting, puts to death after collecting blood；Sterile separation is taken out
Mouse spleen is placed in the plate containing certain serum-free 1640 culture medium, is soaked copper mesh with serum-free 1640 culture medium, is being trained
It supports and pulverizes spleen in ware and preparation B cell suspension, supplement serum-free medium volume to 40mL are filtered with copper mesh；B cell is hanged
Liquid is centrifuged 5min with 1000r/min, abandons supernatant；Previous step is repeated, then is washed primary；
B. myeloma cell is prepared：Take logarithmic growth phase myeloma cell's SP2/0 cell in culture and 250mL culture bottle
1-2 bottles, piping and druming cell adjusts volume to 40mL, is transferred in 50mL sterile centrifugation tube, 1000r/min is centrifuged 5min, abandons supernatant, weight
Multiple above step, then wash primary；
C. SP2/0 myeloma cell and immunized B cells are mixed according to 1/10 or 1/5 ratio, adds incomplete 1640 training
For nutrient solution to 40mL, 1000r/min centrifugation 10min cleaning is primary；
D. supernatant is poured out, exhaust residual liquid, flicks tube wall, keeps cell loose in the pasty state；
E. the centrifuge tube containing fused cell is placed in 37 DEG C of water-baths, draws the PEG 4000 of 37 DEG C of pre-temperatures of 0.8mL, delayed
Slow to instill in pipe, side edged rotates, and drips off in 60s, 37 DEG C of standing 90s；
F. the incomplete culture medium of 37 DEG C of pre-temperatures of 10mL is then added in 5min, adds within first minute plus 1mL, second minute
1mL, third minute add 1.5mL, four minutes to add 1.5mL, add within the 5th minute, finally mend to 30mL, in 37 DEG C of standing 5min,
1000r/min is centrifuged 6min；Supernatant is discarded, 20mL complete culture solution is slowly added to, gentle agitation supplies complete culture after mixing
Liquid is added 1 × HAT (final concentration) to 40mL, in point 96 orifice plates for having completed feeder cells to 4 plates, not plus the hole of fused cell
For negative control；
G.5 every hole supplements the complete culture solution that 50 μ L contain 1 × HT behind day；
H. work as fused cell, when being paved with 1/4 bottom hole, be not required to change liquid, hybridoma antibody is directly detected by IELISA
Secrete situation；
I. check and subgroup identification carried out to obtained positive hole, the cell strain that check is positive and subclass is IgG is with having
It limits dilution method and carries out cloning screening, count 200 cells with gradient bed board method and be laid on one piece of 96 orifice plate；
J. according to subsequent ELISA testing result, the screening of 3-5 time cloningization is carried out, until all holes for having cell are the positive,
Monoclonal hybridoma strain is then obtained, is expanded culture；
K. the clone of the antibody-secreting positive is transferred to 24 orifice plates, further transferred species, which enters, expands culture in culture bottle, freeze portion
Divide clone cell, while carrying out colonized culture；
L. intersection screening is carried out to Hybridoma Cell Culture supernatant with IELISA method；
M. 100 hole μ L/ of feeder layer is prepared, 2 × 104/hole, with the clone in aseptic straw piping and druming culture plate, hang
Float on complete culture solution, adjustment cell concentration to 10-20/mL adds 1 × HT, is added in the culture plate containing feeder cells, 100 μ
The hole L/ makes every hole contain 1-2 cell, cultivates 8-12 days, when hybridoma is paved with 1/4 bottom hole, takes in the incubator
Clear detection；It is a large amount of to expand culture until positive hole carries out colonized culture to 100% clones secrete specific antibody again, and mark
Remember and freeze with liquid nitrogen container, record freezes position.
The application method (detection method) of the application Amoxicillin antibody assay kit, steps are as follows
(1), take Amoxicillin antibody test reagent card, every part of 4 hole of clinical samples label to be checked, respectively I, II, III,
Ⅳ；
(2), the red cell suspension of Amoxicillin processing is added into every hole in the I, the II, III hole, is added into the IVth hole
The red cell suspension of non-Amoxicillin processing；
(3), it is separately added into sample to be examined blood plasma into the Ith and the IVth hole, positive control solution, the IIIth hole are added in the IIth hole
Middle addition negative controls mix, and set 37 ± 1 DEG C of reagent card couveuse incubations 1 hour or more；
(4), centrifuge centrifugation is set, result is observed in 30min and is recorded；
(5), result judgement
Positive findings：It is positive reaction in glue that red blood cell, which is located at glue surface or is suspended in,；
Negative findings：Red blood cell is deposited on micropore bottom all as feminine gender.
Compared with the prior art, the advantages of the present invention are as follows：The application can be used for quickly detecting Amoxicillin antibody,
Easy to operate, time-consuming short, sensitivity is strong with stability, more convenient in clinical application.
Specific embodiment
This application involves Amoxicillin antibody assay kits, mainly include Amoxicillin antibody test reagent card, A Mo
XiLin handles red blood cell, non-Amoxicillin handles red blood cell, Amoxicillin antibody positive comparison liquid, Amoxicillin negative antibody pair
It is formed according to liquid.Present invention is further described in detail with reference to embodiments.
Embodiment 1
The present embodiment is related to the preparation of Amoxicillin antibody assay kit (column agglutination)
Step 1: Amoxicillin antibody test reagent card
(1.1), it measures：By sephadex (swelling gum) is got in batches, sephadex (swelling gum) is measured and is shifted
Into container that is clean and sterilizing.
(1.2), it washs：Washed sephadex (swelling gum) with 0.9% sodium chloride solution, 0.9% sodium chloride solution with
Sephadex (swelling gum) volume ratio is 2:1, it mixes well, 2000rpm removes supernatant, repetitive operation 5 after being centrifuged 1 minute
Secondary, gel after must washing performs mark.
(1.3), it prepares：Antihuman globulin reagent (anti-igg+C is measured by batch3d).It is 1 by antibody and gel volume ratio:
3 ratio, by antihuman globulin reagent (anti-igg+C3D) it mixes in the gel after washing is added, is uniformly mixed respectively with utensil
At antigen gel, it is stand-by to perform mark.
(1.4), filling, centrifugation：Continuous sample-adding gun is transferred to accurate calibration (30 microlitres), quality control officer's monitoring.It presses
According to 30 microlitres of every hole antigen gel amount by antihuman globulin reagent (anti-igg+C3D) gel, which is added in micro-pipe, (does not make gel attached
On the reaction cell wall of micro-pipe upper end).
(1.5), it seals：After centrifugation, card, which is transferred on sealing machine, to be sealed.Closed condition is：0.3~0.35MPa pressure
158 ± 2 DEG C of power, temperature, 3 seconds time/card.
Step 2: Amoxicillin handles red blood cell
(2.1), it is filtered, washed：O-shaped raw material erythrocyte is taken, is filtered with disposable leucocyte filter.By three person-portions
The mixing of O-shaped raw material erythrocyte, wash that (raw material blood and 0.9% sodium chloride solution volume ratio are 1 with 0.9% sodium chloride solution:
4), 3000rpm removes supernatant after being centrifuged 1 minute.Repetitive operation 3 times.
(2.2), drug sensitization
The Amoxicillin a drug solution is prepared：By boric acid, potassium chloride is weighed in batches, adds purified water sufficiently to dissolve, be formulated as
± 0.1 borate buffer of 0.1mol/L pH9.8 is added by the weighed Amoxicillin of batch, and sufficiently dissolution mixes to get Ah not
XiLin drug solution.
The Amoxicillin b drug-treated cell：Amoxicillin drug solution is measured by batch, is added O-shaped hematocrit, 37
It DEG C is incubated for 1 hour, was mixed every 15 minutes primary.0.9% sodium chloride solution wash sensitized erythrocyte 3 times (or until supernatant without
Haemolysis), with blood cell analysis diluted to 2~2.5% concentration, 2~8 DEG C of preservations.
(2.3), it dispenses：Amoxicillin drug-treated cell is taken, is dispensed into drop bottle, every bottle of 5ml, is saved in 2~8 DEG C.
Step 3: non-Amoxicillin handles cell
(3.1), it is filtered, washed：O-shaped raw material erythrocyte is taken, is filtered with disposable leucocyte filter.By three person-portions
The mixing of O-shaped raw material erythrocyte, wash that (raw material blood and 0.9% sodium chloride solution volume ratio are 1 with 0.9% sodium chloride solution:
4), 3000rpm removes supernatant after being centrifuged 1 minute.Repetitive operation 3 times.With blood cell analysis diluted to 2~
2.5% concentration, 2~8 DEG C of preservations.
(3.2), it dispenses：Negated Amoxicillin drug-treated cell is dispensed into drop bottle, every bottle of 2ml, is protected in 2~8 DEG C
It deposits.
Step 4: Amoxicillin antibody positive comparison liquid
(4.1), it prepares：The Amoxicillin monoclonal antibody for taking certain potency, is diluted with BSA；
(4.2), it dispenses：It takes filtered Amoxicillin antibody positive to compare, dispenses into polyethylene bottle, every bottle of 3ml, in
2~8 DEG C of preservations.
Step 5: Amoxicillin antibody negative controls liquid
Packing：Filtered Amoxicillin antibody negative controls are taken, every bottle of 3ml, in 2~8 DEG C into polyethylene bottle is dispensed
It saves.
Embodiment 2
The present embodiment is related to the preparation method of Amoxicillin monoclonal antibody
(1) preparation of feeder cells：
6-10 week old Balb/c mouse is taken, neck is drawn to put to death, impregnates in 75% alcohol, sterilizes 3-5min；It is transferred to sterile behaviour
Make in platform, cut off skin with sterile scissors, exposes its peritonaeum sufficiently；With the 10mL serum-free of asepsis injector injection pre-cooling
1640 culture medium is gently flapped mouse peritoneal with tweezers bottom, and culture solution is sucked out, and is put into 10mL centrifuge tube, 1200r/min centrifugation
10min；Supernatant is discarded, feeder cells are resuspended with complete culture solution, counts, and adjust cell number to 2 × 105/mL, will raise
Cell is added in 96 orifice plates, and every 100 μ L of hole is then placed in 37 DEG C of CO2 incubator cultures；
(2) hybridoma cell fusion and limiting dilution assay subclone screening
A. immunized B cells are prepared：Mouse after will be immune plucks eyeball bloodletting, puts to death after collecting blood；Sterile separation is taken out
Mouse spleen is placed in the plate containing certain serum-free 1640 culture medium, is soaked copper mesh with serum-free 1640 culture medium, is being trained
It supports and pulverizes spleen in ware and preparation B cell suspension, supplement serum-free medium volume to 40mL are filtered with copper mesh；B cell is hanged
Liquid is centrifuged 5min with 1000r/min, abandons supernatant；Previous step is repeated, then is washed primary；
B. myeloma cell is prepared：Take logarithmic growth phase myeloma cell's SP2/0 cell in culture and 250mL culture bottle
1-2 bottles, piping and druming cell adjusts volume to 40mL, is transferred in 50mL sterile centrifugation tube, 1000r/min is centrifuged 5min, abandons supernatant, weight
Multiple above step, then wash primary；
C. SP2/0 myeloma cell and immunized B cells are mixed according to 1/10 or 1/5 ratio, adds incomplete 1640 training
For nutrient solution to 40mL, 1000r/min centrifugation 10min cleaning is primary；
D. supernatant is poured out, exhaust residual liquid, flicks tube wall, keeps cell loose in the pasty state；
E. the centrifuge tube containing fused cell is placed in 37 DEG C of water-baths, draws the PEG 4000 of 37 DEG C of pre-temperatures of 0.8mL, delayed
Slow to instill in pipe, side edged rotates, and drips off in 60s, 37 DEG C of standing 90s；
F. the incomplete culture medium of 37 DEG C of pre-temperatures of 10mL is then added in 5min, adds within first minute plus 1mL, second minute
1mL, third minute add 1.5mL, four minutes to add 1.5mL, add within the 5th minute, finally mend to 30mL, in 37 DEG C of standing 5min,
1000r/min is centrifuged 6min；Supernatant is discarded, 20mL complete culture solution is slowly added to, gentle agitation supplies complete culture after mixing
Liquid is added 1 × HAT (final concentration) to 40mL, in point 96 orifice plates for having completed feeder cells to 4 plates, not plus the hole of fused cell
For negative control；
G.5 every hole supplements the complete culture solution that 50 μ L contain 1 × HT behind day；
H. work as fused cell, when being paved with 1/4 bottom hole, be not required to change liquid, hybridoma antibody is directly detected by IELISA
Secrete situation；
I. check and subgroup identification carried out to obtained positive hole, the cell strain that check is positive and subclass is IgG is with having
It limits dilution method and carries out cloning screening, count 200 cells with gradient bed board method and be laid on one piece of 96 orifice plate；
J. according to subsequent ELISA testing result, the screening of 3-5 time cloningization is carried out, until all holes for having cell are the positive,
Monoclonal hybridoma strain is then obtained, is expanded culture；
K. the clone of the antibody-secreting positive is transferred to 24 orifice plates, further transferred species, which enters, expands culture in culture bottle, freeze portion
Divide clone cell, while carrying out colonized culture；
L. intersection screening is carried out to Hybridoma Cell Culture supernatant with IELISA method；
M. 100 hole μ L/ of feeder layer is prepared, 2 × 104/hole, with the clone in aseptic straw piping and druming culture plate, hang
Float on complete culture solution, adjustment cell concentration to 10-20/mL adds 1 × HT, is added in the culture plate containing feeder cells, 100 μ
The hole L/ makes every hole contain 1-2 cell, cultivates 8-12 days, when hybridoma is paved with 1/4 bottom hole, takes in the incubator
Clear detection；It is a large amount of to expand culture until positive hole carries out colonized culture to 100% clones secrete specific antibody again, and mark
Remember and freeze with liquid nitrogen container, record freezes position.
Embodiment 3
The present embodiment is related to the use of Amoxicillin antibody assay kit
1, Amoxicillin antibody test reagent card, every part of 4 hole of clinical samples label (respectively I, II, III, IV) to be checked are taken.
2, each 25 μ l of red cell suspension of Amoxicillin processing is added into every hole in the I, the II, III hole, into the IVth hole
The 25 μ l of red cell suspension of non-Amoxicillin processing is added.
3, it is separately added into 50 μ l of sample to be examined blood plasma into the Ith and the IVth hole, 50 μ l of positive control solution is added in the IIth hole,
50 μ l of negative controls is added in IIIth hole.It mixes, sets 37 ± 1 DEG C of reagent card couveuse and be incubated for 1 hour.
4, LB-3000 medical centrifuge centrifugation 5min (900rpm × 2min, 1500rpm × 3min), observation in 30min are set
As a result it and records.
5, result judgement
Positive findings：It is positive reaction in glue that red blood cell, which is located at glue surface or is suspended in,.
Negative findings：Red blood cell is deposited on micropore bottom all as feminine gender.
Claims (7)
1. a kind of Amoxicillin antibody assay kit, it is characterised in that：Mainly by Amoxicillin antibody test reagent card, Ah
Amdinocillin handles red blood cell, non-Amoxicillin handles red blood cell, Amoxicillin antibody positive comparison liquid, Amoxicillin negative antibody
Comparison liquid composition.
2. a kind of method for preparing Amoxicillin antibody assay kit described in claim 1, it is characterised in that：Including walking as follows
Suddenly, between each step without precedence,
Step 1: Amoxicillin antibody test reagent card
(1.1), it measures：By taking sephadex sephadex measurement to be transferred to cleaning and is sterilized as swelling gum in batches
Container in；
(1.2), it washs：Sephadex is washed with sodium chloride solution, is mixed well, supernatant, repetitive operation 5 are removed after centrifugation
More than secondary, gel after must washing performs mark；
(1.3), it prepares：Antihuman globulin reagent (anti-igg+C is measured by batch3It d), is 1 by antibody and gel volume ratio:3 ratio
Example, will antihuman globulin reagent be added washing after gel in mix, be mixed into antigen gel respectively, perform mark to
With；
(1.4), filling, centrifugation：Continuous sample-adding gun is transferred to accurate calibration, quality control officer's monitoring is micro- according to the fixation of every hole
Antihuman globulin reagent gel is added in micro-pipe by the antigen gel amount risen, and gel is not made to be attached to the reactive tank of micro-pipe upper end
On wall,；
(1.5), it seals：After centrifugation, card, which is transferred on sealing machine, to be sealed；
Step 2: Amoxicillin handles red blood cell
(2.1), it is filtered, washed：O-shaped raw material erythrocyte is taken, is filtered with disposable leucocyte filter, by the O-shaped of three person-portions
The mixing of raw material erythrocyte, is washed with sodium chloride solution, removes supernatant after centrifugation, and repetitive operation 3 times or more；
(2.2), drug sensitization
The Amoxicillin a drug solution is prepared：By boric acid, potassium chloride is weighed in batches, adds purified water sufficiently to dissolve, be formulated as
± 0.1 borate buffer of 0.1mol/L, pH9.8 is added by the weighed Amoxicillin of batch, and sufficiently dissolution mixes to get Ah not
XiLin drug solution；
The Amoxicillin b drug-treated cell：Amoxicillin drug solution is measured by batch, O-shaped hematocrit is added, 37 DEG C incubate
It educates 1 hour, discontinuity mixes once, using sodium chloride solution washing sensitized erythrocyte 3 times or more or until supernatant is existing without haemolysis
As with blood cell analysis diluted to 2~2.5% concentration, 2~8 DEG C of preservations；
(2.3), it dispenses：Amoxicillin drug-treated cell is taken, is dispensed into drop bottle, is saved in 2~8 DEG C；
Step 3: non-Amoxicillin handles cell
(3.1), it is filtered, washed：O-shaped raw material erythrocyte is taken, is filtered with disposable leucocyte filter.By the O-shaped of three person-portions
The mixing of raw material erythrocyte, is washed with sodium chloride solution, and supernatant is removed after centrifugation, repetitive operation 3 times or more, is divided with haemocyte
To 2~2.5% concentration, 2~8 DEG C save analysis diluted；
(3.2), it dispenses：Negated Amoxicillin drug-treated cell is dispensed into drop bottle, is saved in 2~8 DEG C；
Step 4: Amoxicillin antibody positive comparison liquid
(4.1), it prepares：The Amoxicillin monoclonal antibody for taking certain potency, is diluted with BSA；
(4.2), it dispenses：It takes filtered Amoxicillin antibody positive to compare, dispenses every bottle of 3ml, in 2~8 into polyethylene bottle
DEG C save；
Step 5: Amoxicillin antibody negative controls liquid
Packing：Filtered Amoxicillin antibody negative controls are taken, are dispensed into bottle, are saved in 2~8 DEG C.
3. the method for Amoxicillin antibody assay kit according to claim 2, it is characterised in that：The sodium chloride is molten
The concentration of liquid is 0.9wt%.
4. the method for Amoxicillin antibody assay kit according to claim 3, it is characterised in that：In step 1
0.9% sodium chloride solution and sephadex volume ratio are 2:1；Step 2: raw material blood and 0.9% sodium chloride solution volume in three
Than being 1:4.
5. the method for Amoxicillin antibody assay kit according to claim 2, it is characterised in that：Step 1 sealing step
The closed condition of sequence is：158 ± 2 DEG C of 0.3~0.35MPa pressure, temperature, 3 seconds time/card.
6. the method for Amoxicillin antibody assay kit according to claim 2, it is characterised in that：The Amoxicillin
The preparation method of monoclonal antibody, steps are as follows
(1) preparation of feeder cells：
6-10 week old Balb/c mouse is taken, neck is drawn to put to death, impregnates in 75% alcohol, sterilizes 3-5min；It is transferred to aseptic operating platform
In, skin is cut off with sterile scissors, exposes its peritonaeum sufficiently；It is trained with the 10mL serum-free 1640 of asepsis injector injection pre-cooling
Nutrient solution is gently flapped mouse peritoneal with tweezers bottom, and culture solution is sucked out, and is put into 10mL centrifuge tube, and 1200r/min is centrifuged 10min；
Discard supernatant, feeder cells be resuspended with complete culture solution, count, and adjust cell number to 2 × 105/mL, by feeder cells plus
Enter in 96 orifice plates, every 100 μ L of hole is then placed in 37 DEG C of CO2 incubator cultures；
(2) hybridoma cell fusion and limiting dilution assay subclone screening
A. immunized B cells are prepared：Mouse after will be immune plucks eyeball bloodletting, puts to death after collecting blood；Mouse is taken out in sterile separation
Spleen is placed in the plate containing certain serum-free 1640 culture medium, copper mesh is soaked with serum-free 1640 culture medium, in culture dish
In pulverize spleen and preparation B cell suspension filtered with copper mesh, supplement serum-free medium volume is to 40mL；By B cell suspension with
1000r/min is centrifuged 5min, abandons supernatant；Previous step is repeated, then is washed primary；
B. myeloma cell is prepared：Take logarithmic growth phase myeloma cell's SP2/0 cell 1-2 in culture and 250mL culture bottle
Bottle, piping and druming cell adjust volume to 40mL, are transferred in 50mL sterile centrifugation tube, 1000r/min is centrifuged 5min, abandons supernatant, repeats
Above step, then wash primary；
C. SP2/0 myeloma cell and immunized B cells are mixed according to 1/10 or 1/5 ratio, adds incomplete 1640 culture medium
It is primary that 10min cleaning is centrifuged to 40mL, 1000r/min；
D. supernatant is poured out, exhaust residual liquid, flicks tube wall, keeps cell loose in the pasty state；
E. the centrifuge tube containing fused cell is placed in 37 DEG C of water-baths, draws the PEG 4000 of 0.8mL37 DEG C of pre-temperature, is slowly dropped into
In pipe, side edged rotates, and drips off in 60s, 37 DEG C of standing 90s；
F. then it is added the incomplete culture medium of 37 DEG C of pre-temperatures of 10mL in 5min, first minute plus 1mL, second minute plus 1mL,
Third minute adds 1.5mL, four minutes to add 1.5mL, add within the 5th minute, finally mend to 30mL, in 37 DEG C of standing 5min,
1000r/min is centrifuged 6min；Supernatant is discarded, 20mL complete culture solution is slowly added to, gentle agitation supplies complete culture after mixing
Liquid is added 1 × HAT (final concentration) to 40mL, in point 96 orifice plates for having completed feeder cells to 4 plates, not plus the hole of fused cell
For negative control；
G.5 every hole supplements the complete culture solution that 50 μ L contain 1 × HT behind day；
H. work as fused cell, when being paved with 1/4 bottom hole, be not required to change liquid, hybridoma antibody-secreting is directly detected by IELISA
Situation；
I. check and subgroup identification carried out to obtained positive hole, the cell strain that check is positive and subclass is IgG is with limited dilute
Interpretation of the law carries out cloning screening, counts 200 cells with gradient bed board method and is laid on one piece of 96 orifice plate；
J. according to subsequent ELISA testing result, the screening of 3-5 time cloningization is carried out, until all holes for having cell are the positive, then
To monoclonal hybridoma strain, expand culture；
K. the clone of the antibody-secreting positive is transferred to 24 orifice plates, further transferred species, which enters, expands culture in culture bottle, freeze part gram
Grand cell, while carrying out colonized culture；
L. intersection screening is carried out to Hybridoma Cell Culture supernatant with IELISA method；
M. 100 hole μ L/ of feeder layer is prepared, 2 × 104/hole, with the clone in aseptic straw piping and druming culture plate, it is suspended in
Complete culture solution, adjustment cell concentration to 10-20/mL, adds 1 × HT, is added in the culture plate containing feeder cells, 100 μ L/
Hole makes every hole contain 1-2 cell, cultivates 8-12 days in the incubator, when hybridoma is paved with 1/4 bottom hole, takes supernatant
Detection；It is a large amount of to expand culture until positive hole carries out colonized culture to 100% clones secrete specific antibody again, and mark
It freezes well with liquid nitrogen container, record freezes position.
7. the application method of Amoxicillin antibody assay kit described in a kind of claim 1, it is characterised in that：Operating procedure is such as
Under
(1), Amoxicillin antibody test reagent card, every part of 4 hole of clinical samples label to be checked, respectively I, II, III, IV are taken；
(2), the red cell suspension of Amoxicillin processing is added into every hole in the I, the II, III hole, be added into the IVth hole non-Ah
The red cell suspension of Amdinocillin processing；
(3), it is separately added into sample to be examined blood plasma into the Ith and the IVth hole, positive control solution is added in the IIth hole, adds in the IIIth hole
Enter negative controls, mix, sets 37 ± 1 DEG C of reagent card couveuse incubations 1 hour or more；
(4), centrifuge centrifugation is set, result is observed in 30min and is recorded；
(5), result judgement
Positive findings：It is positive reaction in glue that red blood cell, which is located at glue surface or is suspended in,；
Negative findings：Red blood cell is deposited on micropore bottom all as feminine gender.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101735987A (en) * | 2008-11-04 | 2010-06-16 | 中华人民共和国北京出入境检验检疫局 | Mouse monoclonal antibody cell strain for resisting amoxicillin and ampicillin |
| CN107132323A (en) * | 2017-07-07 | 2017-09-05 | 江苏中济万泰生物医药有限公司 | Piperacillin induction hemolysis test kit and preparation method thereof |
-
2018
- 2018-05-04 CN CN201810418420.XA patent/CN108845116A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101735987A (en) * | 2008-11-04 | 2010-06-16 | 中华人民共和国北京出入境检验检疫局 | Mouse monoclonal antibody cell strain for resisting amoxicillin and ampicillin |
| CN107132323A (en) * | 2017-07-07 | 2017-09-05 | 江苏中济万泰生物医药有限公司 | Piperacillin induction hemolysis test kit and preparation method thereof |
Non-Patent Citations (1)
| Title |
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| 李敬宽等: "阿莫西林胶囊口服致自身免疫性溶血性贫血1例", 《中国药业》 * |
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