CN108593921A - Ampicillin antibody assay kit and its preparation and application - Google Patents
Ampicillin antibody assay kit and its preparation and application Download PDFInfo
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- CN108593921A CN108593921A CN201810418418.2A CN201810418418A CN108593921A CN 108593921 A CN108593921 A CN 108593921A CN 201810418418 A CN201810418418 A CN 201810418418A CN 108593921 A CN108593921 A CN 108593921A
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- 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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- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G01N33/559—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody through a gel, e.g. Ouchterlony technique
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Abstract
The present invention relates to a kind of ampicillin antibody assay kit and its preparation and application, are mainly made of ampicillin antibody test reagent card, ampicillin processing red blood cell, non-ampicillin processing red blood cell, ampicillin antibody positive comparison liquid, ampicillin antibody negative controls liquid.The kit have it is easy to operate, sensitivity is strong with stability, more convenient in clinical application, can to patient progress ampicillin antibody quick detection.
Description
Technical field
The invention belongs to antibiotic antibody detection fields, and in particular to the detection kit of ampicillin antibody, preparation side
Method and application method.
Background technology
With advances in technology with the development of medicinal industry, more and more antibiotics in Clinical practice, to
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, to avoid the haemolysis side effect of 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
The antibody for resisting such drug, to predict whether the antibiotic of selection may occur haemolysis, to instruct the use of clinical rational anti-
Raw element.
Ampicillin is the representative of wide spectrum Aminopenicillin.It is similar to benzyl penicillin to the effect of benzyl penicillin sensitive bacteria.It is right
The effect of gram negative bacilli is more than benzyl penicillin.Bacterium to ampicillin sensitive includes group A β-haemolytic streptococci, pneumonia
Streptococcus, B groups beta hemolytic streptococcus, benzyl penicillin sensitivity staphylococcus aureus, part enterococcus, meningococcus, mould
Plain G sensitivities gonococcus and part haemophilus influenzae.There are part Escherichia coli, unusual deformed rod to ampicillin medium sensitivity
Bacterium, Salmonella, Shigella and a small number of other enterobacterias and the part anaerobic bacteria except bacteroides fragilis.Ampicillin is not
Resistance to-lactamase, thus it is invalid to producing enzyme staphylococcus aureus.Gram negative bacilli also due to generate various beta-lactamases,
The drug resistance of ampicillin is constantly increased, the bacterial strain detached from children such as haemophilus influenzae has 10%-20% drug resistances.
Escherichia coli there are about 50% pair of ampicillin-resistant, the resistant rates such as Salmonella, Shigella, proteus also with Escherichia coli
It is similar to be continuously improved；Enterobacteria, klebsiella bacillus, Serratia, citrobacter, Prudence bacterium, acinetobacter calcoaceticus etc. are most
To ampicillin-resistant；Pseudomonas aeruginosa is then to ampicillin natural drug resistance.Thus ampicillin is as broad-spectrum antibiotic
Value has been decreased obviously.Such as with beta-lactamase inhibitor such as Sulbactam use in conjunction, ampicillin pair can be improved in ampicillin
The antibacterial action of producing enzyme drug-fast bacteria.
When patient uses ampicillin in treatment clinical course, there may be for the anti-of such drug by some patients
Body.Patient once generates the antibody of ampicillin resistant, in the antigen-antibody complex meeting which forms with ampicillin drug
With the drug effect for inhibiting ampicillin drug so that the effect of ampicillin drug cannot fully ensure that.Meanwhile ampicillin medicine
The immunocomplex that the corresponding antibody of object is formed is combined that erythrocyte hemolysis can be caused with human red blood cells, gently then influences treatment effect
Fruit, delay treatment, it is heavy then jeopardize patient vitals safety.Therefore, establish effectively detect the method for internal ampicillin antibody for
Reasonable employment ampicillin drug has important clinical meaning.
Invention content
The purpose of the present invention is to provide a kind of kit of the internal ampicillin antibody of detection, the preparation sides of the kit
Method and application method allow and clinically more simplify fast to the detection of ampicillin antibody.
Technical solution is used by the present invention solves the above problems：A kind of ampicillin antibody assay kit, mainly
It is to be resisted by ampicillin antibody test reagent card, ampicillin processing red blood cell, non-ampicillin processing red blood cell, ampicillin
Body positive control solution, ampicillin antibody negative controls liquid composition.
The preparation method of above-mentioned ampicillin antibody assay kit, mainly by following steps, each step is without precedence relationship：
Step 1: ampicillin antibody test reagent card
(1.1), it measures：Sephadex measurement is transferred to cleaning simultaneously as swelling gum by sephadex is taken in batches
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
Operate 5 times or more, 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 will mix in the gel after antihuman globulin reagent addition washing, be mixed into antigen gel respectively, perform mark
Know for use；
(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 seal.
Step 2: ampicillin 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, repetitive operation 3 times or more；
(2.2), drug sensitization
The ampicillins a drug solution is prepared：By boric acid, potassium chloride is weighed in batches, adds purified water fully to dissolve, be formulated as
± 0.1 borate buffer of 0.1mol/L, pH9.8 is added by the ampicillin weighed in batches, fully dissolves mixing to get ammonia benzyl
XiLin drug solution；
The ampicillins b drug-treated cell：Ampicillin drug solution is measured by batch, is added O-shaped packed cell volume, 37
It DEG C is incubated 1 hour, discontinuity mixing is primary, using sodium chloride solution washing sensitized erythrocyte 3 times or more 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：Ampicillin drug-treated cell is taken, in packing to drop bottle, in 2~8 DEG C of preservations.
Step 3: non-ampicillin 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, is washed with sodium chloride solution, removes supernatant 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 ampicillin drug-treated cell, in packing to drop bottle, in 2~8 DEG C of preservations.
Step 4: ampicillin antibody positive comparison liquid
(4.1), it prepares：The ampicillin monoclonal antibody for taking certain potency, is diluted with BSA；
(4.2), it dispenses：Filtered ampicillin antibody positive is taken to compare, in packing to polyethylene bottle, every bottle of 3ml, in
2~8 DEG C of preservations.
Step 5: ampicillin antibody negative controls liquid
Packing：Filtered ampicillin antibody negative controls are taken, in packing to bottle, in 2~8 DEG C of preservations.
Preferably, a concentration of 0.9wt% of the sodium chloride solution.
Further, 0.9% sodium chloride solution and sephadex volume ratio are 2 in step 1:1；Step 2: three Central Plains
Expect that blood and 0.9% sodium chloride solution volume ratio are 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 ampicillin monoclonal antibody of a price effect of the application kit used in preparation process
Method, steps are as follows
(1) preparation of feeder cells：
6-10 week old Balb/c mouse are taken, neck is drawn to put to death, impregnates in 75% alcohol, sterilizes 3-5min；It is transferred to sterile behaviour
Make in platform, skin is cut off with sterile scissors, its peritonaeum is made fully to expose；With the 10mL serum-frees of asepsis injector injection precooling
1640 culture medium is gently flapped mouse peritoneal with tweezers bottom, and culture solution is sucked out, and is put into 10mL centrifuge tubes, 1200r/min centrifugations
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, per 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, is put 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 to pulverize spleen in ware and filter with copper mesh and prepares B cell suspension, supplement serum-free medium volume to 40mL；B cell is hanged
Liquid centrifuges 5min with 1000r/min, abandons supernatant；Previous step is repeated, then is washed primary；
B. myeloma cell is prepared：Take culture and exponential phase myeloma cell's SP2/0 cells in 250mL culture bottles
1-2 bottles, piping and druming cell adjusts volume to 40mL, is transferred in 50mL sterile centrifugation tubes, and 1000r/min centrifuges 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 cleanings are 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 is dripped 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, and 5min is stood in 37 DEG C,
1000r/min centrifuges 6min；Supernatant is discarded, 20mL complete culture solutions is slowly added to, complete culture is supplied after gentle agitation mixing
1 × HAT (final concentration) is added to 40mL in liquid, in point 96 orifice plates for having completed feeder cells to 4 plates, does not add the hole of fused cell
For negative control；
G.5 the complete culture solution that 50 μ L contain 1 × HT is supplemented behind day per hole；
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 are 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 and one piece of 96 orifice plate is laid on gradient bed board method；
J. according to follow-up ELISA testing results, the screening of 3-5 time cloningizations is carried out, until all, to have the hole of cell be the positive,
Monoclonal hybridoma strain is then obtained, culture is enlarged；
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, is carried out at the same time colonized culture；
L. intersection screening is carried out to Hybridoma Cell Culture supernatant with IELISA methods；
M. 100 holes μ L/ of feeder layer are prepared, 2 × 104/hole, the clone in culture plate is blown and beaten with aseptic straw, is hanged
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 holes L/ make every hole contain 1-2 cell, cultivate 8-12 days, when hybridoma is paved with 1/4 bottom hole, take 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 ampicillin antibody assay kit, steps are as follows
(1), take ampicillin 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 ampicillin 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-ampicillin processing；
(3), it is separately added into sample blood plasma to be checked into the Ith and the IVth hole, positive control solution, the IIIth hole are added in the IIth hole
Middle addition negative controls, mixing set 37 ± 1 DEG C of reagent card couveuse incubation 1 hour or more；
(4), centrifuge is set, result is observed in 30min and is recorded；
(5), result judgement
Positive findings：It is positive reaction that red blood cell, which is located at glue surface or is suspended in glue,；
Negative findings：It is feminine gender that red blood cell, which is all deposited on micropore bottom,.
Compared with the prior art, the advantages of the present invention are as follows：The application can be used for quickly detecting ampicillin antibody,
Easy to operate, time-consuming short, sensitivity is strong with stability, more convenient in clinical application.
Specific implementation mode
This application involves ampicillin antibody assay kits, include mainly ampicillin antibody test reagent card, ammonia benzyl
XiLin handles red blood cell, non-ampicillin processing red blood cell, ampicillin antibody positive comparison liquid, ampicillin 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 ampicillin antibody assay kit (column agglutination)
Step 1: ampicillin 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：Wash 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 centrifuging 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 being added after washing, is uniformly mixed respectively with utensil
At antigen gel, it is for use 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
It on the reaction cell wall of micro-pipe upper end).
(1.5), it seals：After centrifugation, card, which is transferred on sealing machine, to seal.Closed condition is：0.3~0.35MPa is pressed
158 ± 2 DEG C of power, temperature, 3 seconds time/card.
Step 2: ampicillin 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
O-shaped raw material erythrocyte mixing, is washed with 0.9% sodium chloride solution, and raw material blood and 0.9% sodium chloride solution volume ratio are 1:4,
3000rpm removes supernatant, repetitive operation 3 times after centrifuging 1 minute.
(2.2), drug sensitization
The ampicillins a drug solution is prepared：By boric acid, potassium chloride is weighed in batches, adds purified water fully to dissolve, be formulated as
± 0.1 borate buffers of 0.1mol/L pH9.8 are added by the ampicillin weighed in batches, fully dissolve mixing to get ammonia benzyl
XiLin drug solution.
The ampicillins b drug-treated cell：Ampicillin drug solution is measured by batch, is added O-shaped packed cell volume, 37
DEG C be incubated 1 hour, it is primary every 15 minutes mixings.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：Ampicillin drug-treated cell is taken, in packing to drop bottle, every bottle of 5ml, in 2~8 DEG C of preservations.
Step 3: non-ampicillin 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 centrifuging 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 ampicillin drug-treated cell, in packing to drop bottle, every bottle of 2ml, in 2~8 DEG C of guarantors
It deposits.
Step 4: ampicillin antibody positive comparison liquid
(4.1), it prepares：The ampicillin monoclonal antibody for taking certain potency, is diluted with BSA；
(4.2), it dispenses：Filtered ampicillin antibody positive is taken to compare, in packing to polyethylene bottle, every bottle of 3ml, in
2~8 DEG C of preservations.
Step 5: ampicillin antibody negative controls liquid
Packing：Filtered ampicillin antibody negative controls are taken, in packing to polyethylene bottle, every bottle of 3ml, in 2~8 DEG C
It preserves.
Embodiment 2
The present embodiment is related to the preparation method of ampicillin monoclonal antibody
(1) preparation of feeder cells：
6-10 week old Balb/c mouse are taken, neck is drawn to put to death, impregnates in 75% alcohol, sterilizes 3-5min；It is transferred to sterile behaviour
Make in platform, skin is cut off with sterile scissors, its peritonaeum is made fully to expose；With the 10mL serum-frees of asepsis injector injection precooling
1640 culture medium is gently flapped mouse peritoneal with tweezers bottom, and culture solution is sucked out, and is put into 10mL centrifuge tubes, 1200r/min centrifugations
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, per 100 μ L of hole, is then placed in 37 DEG C of CO2Incubator culture；
(2) hybridoma cell fusion and limiting dilution assay subclone screening
A. immunized B cells are prepared：Mouse after will be immune plucks eyeball bloodletting, is put 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 to pulverize spleen in ware and filter with copper mesh and prepares B cell suspension, supplement serum-free medium volume to 40mL；B cell is hanged
Liquid centrifuges 5min with 1000r/min, abandons supernatant；Previous step is repeated, then is washed primary；
B. myeloma cell is prepared：Take culture and exponential phase myeloma cell's SP2/0 cells in 250mL culture bottles
1-2 bottles, piping and druming cell adjusts volume to 40mL, is transferred in 50mL sterile centrifugation tubes, and 1000r/min centrifuges 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 cleanings are 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 is dripped 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, and 5min is stood in 37 DEG C,
1000r/min centrifuges 6min；Supernatant is discarded, 20mL complete culture solutions is slowly added to, complete culture is supplied after gentle agitation mixing
1 × HAT (final concentration) is added to 40mL in liquid, in point 96 orifice plates for having completed feeder cells to 4 plates, does not add the hole of fused cell
For negative control；
G.5 the complete culture solution that 50 μ L contain 1 × HT is supplemented behind day per hole；
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 are 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 and one piece of 96 orifice plate is laid on gradient bed board method；
J. according to follow-up ELISA testing results, the screening of 3-5 time cloningizations is carried out, until all, to have the hole of cell be the positive,
Monoclonal hybridoma strain is then obtained, culture is enlarged；
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, is carried out at the same time colonized culture；
L. intersection screening is carried out to Hybridoma Cell Culture supernatant with IELISA methods；
M. 100 holes μ L/ of feeder layer are prepared, 2 × 104/hole, the clone in culture plate is blown and beaten with aseptic straw, is hanged
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 holes L/ make every hole contain 1-2 cell, cultivate 8-12 days, when hybridoma is paved with 1/4 bottom hole, take 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 ampicillin antibody assay kit
1, ampicillin antibody test reagent card, every part of 4 hole of clinical samples label (being respectively I, II, III, IV) to be checked are taken.
2, each 25 μ l of red cell suspension of ampicillin processing are 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-ampicillin processing are added.
3, it is separately added into 50 μ l of sample blood plasma to be checked into the Ith and the IVth hole, 50 μ l of positive control solution are added in the IIth hole,
50 μ l of negative controls are added in IIIth hole.Mixing is set 37 ± 1 DEG C of reagent card couveuse and is incubated 1 hour.
4, LB-3000 medical centrifuges centrifugation 5min (900rpm × 2min, 1500rpm × 3min) is set, is observed in 30min
As a result it and records.
5, result judgement
Positive findings：It is positive reaction that red blood cell, which is located at glue surface or is suspended in glue,.
Negative findings：It is feminine gender that red blood cell, which is all deposited on micropore bottom,.
Claims (7)
1. a kind of ampicillin antibody assay kit, it is characterised in that：Mainly by ampicillin antibody test reagent card, ammonia
Benzyl XiLin handles red blood cell, non-ampicillin processing red blood cell, ampicillin antibody positive comparison liquid, ampicillin negative antibody
Comparison liquid forms.
2. a kind of method preparing ampicillin antibody assay kit described in claim 1, it is characterised in that：Including walking as follows
Suddenly, between each step without precedence,
Step 1: ampicillin antibody test reagent card
(1.1), it measures：Sephadex measurement is transferred to cleaning and is sterilized as swelling gum by sephadex is taken 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 batch3D), it is 1 by antibody and gel volume ratio:3 ratio
Example will mix in the gel after antihuman globulin reagent addition washing, be mixed into antigen gel respectively, perform mark and wait for
With；
(1.4), filling, centrifugation：Continuous sample-adding gun is transferred to accurate calibration, quality control officer's monitoring is fixed micro- according to 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 seal；
Step 2: ampicillin 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
Raw material erythrocyte mixes, and is washed with sodium chloride solution, removes supernatant after centrifugation, repetitive operation 3 times or more；
(2.2), drug sensitization
The ampicillins a drug solution is prepared：By boric acid, potassium chloride is weighed in batches, adds purified water fully to dissolve, be formulated as
± 0.1 borate buffer of 0.1mol/L, pH9.8 is added by the ampicillin weighed in batches, fully dissolves mixing to get ammonia benzyl
XiLin drug solution；
The ampicillins b drug-treated cell：Ampicillin drug solution is measured by batch, O-shaped packed cell volume is added, 37 DEG C incubate
It educates 1 hour, discontinuity mixing is primary, above or until supernatant shows without haemolysis using sodium chloride solution washing sensitized erythrocyte 3 times
As with blood cell analysis diluted to 2~2.5% concentration, 2~8 DEG C of preservations；
(2.3), it dispenses：Ampicillin drug-treated cell is taken, in packing to drop bottle, in 2~8 DEG C of preservations；
Step 3: non-ampicillin 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
Raw material erythrocyte mixes, and is washed with sodium chloride solution, removes supernatant after centrifugation, repetitive operation 3 times or more, with haemocyte point
To 2~2.5% concentration, 2~8 DEG C preserve analysis diluted；
(3.2), it dispenses：Negated ampicillin drug-treated cell, in packing to drop bottle, in 2~8 DEG C of preservations；
Step 4: ampicillin antibody positive comparison liquid
(4.1), it prepares：The ampicillin monoclonal antibody for taking certain potency, is diluted with BSA；
(4.2), it dispenses：Filtered ampicillin antibody positive is taken to compare, in packing to polyethylene bottle, every bottle of 3ml, in 2~8
DEG C preserve；
Step 5: ampicillin antibody negative controls liquid
Packing：Filtered ampicillin antibody negative controls are taken, in packing to bottle, in 2~8 DEG C of preservations.
3. the method for ampicillin antibody assay kit according to claim 2, it is characterised in that：The sodium chloride is molten
A concentration of 0.9wt% of liquid.
4. the method for ampicillin antibody assay kit according to claim 3, it is characterised in that：In step 1
0.9% sodium chloride solution is 2 with sephadex volume ratio:1；Step 2: raw material blood and 0.9% sodium chloride solution volume in three
Than being 1:4.
5. the method for ampicillin 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 ampicillin antibody assay kit according to claim 2, it is characterised in that：The ampicillin
The preparation method of monoclonal antibody, steps are as follows
(1) preparation of feeder cells：
6-10 week old Balb/c mouse are 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, its peritonaeum is made fully to expose；It is trained with the 10mL serum-frees 1640 of asepsis injector injection precooling
Nutrient solution is gently flapped mouse peritoneal with tweezers bottom, and culture solution is sucked out, and is put into 10mL centrifuge tubes, and 1200r/min centrifuges 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, per 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, is put 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 filtered with copper mesh and prepare B cell suspension, supplement serum-free medium volume is to 40mL；By B cell suspension with
1000r/min centrifuges 5min, abandons supernatant；Previous step is repeated, then is washed primary；
B. myeloma cell is prepared：Take culture and exponential phase myeloma cell's SP2/0 cells 1-2 in 250mL culture bottles
Bottle, piping and druming cell adjust volume to 40mL, are transferred in 50mL sterile centrifugation tubes, and 1000r/min centrifuges 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 cleanings are 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 37 DEG C of pre-temperatures of 0.8mL, slowly drips
Enter in pipe, the rotation of side edged, is dripped 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, and 5min is stood in 37 DEG C,
1000r/min centrifuges 6min；Supernatant is discarded, 20mL complete culture solutions is slowly added to, complete culture is supplied after gentle agitation mixing
1 × HAT (final concentration) is added to 40mL in liquid, in point 96 orifice plates for having completed feeder cells to 4 plates, does not add the hole of fused cell
For negative control；
G.5 the complete culture solution that 50 μ L contain 1 × HT is supplemented behind day per hole；
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 are 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 and is laid on one piece of 96 orifice plate with gradient bed board method；
J. according to follow-up ELISA testing results, the screening of 3-5 time cloningizations is carried out, to have the hole of cell be the positive to all, then must
To monoclonal hybridoma strain, it is enlarged 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, is carried out at the same time colonized culture；
L. intersection screening is carried out to Hybridoma Cell Culture supernatant with IELISA methods；
M. 100 holes μ L/ of feeder layer are prepared, 2 × 104/hole, the clone in culture plate is blown and beaten with aseptic straw, 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 ampicillin antibody assay kit described in a kind of claim 1, it is characterised in that：Operating procedure is such as
Under
(1), ampicillin antibody test reagent card is taken, every part of 4 hole of clinical samples label to be checked, respectively I, II, III, IV；
(2), the red cell suspension of ampicillin processing is added into every hole in the I, the II, III hole, non-ammonia is added into the IVth hole
The red cell suspension of benzyl XiLin processing；
(3), it is separately added into sample blood plasma to be checked into the Ith and the IVth hole, positive control solution is added in the IIth hole, adds in the IIIth hole
Enter negative controls, mixing sets 37 ± 1 DEG C of reagent card couveuse incubation 1 hour or more；
(4), centrifuge is set, result is observed in 30min and is recorded；
(5), result judgement
Positive findings：It is positive reaction that red blood cell, which is located at glue surface or is suspended in glue,；
Negative findings：It is feminine gender that red blood cell, which is all deposited on micropore bottom,.
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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 CN201810418418.2A patent/CN108593921A/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 |
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| Title |
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| 王瑞等: "氨苄西林致溶血性贫血死亡一例", 《临床误诊误治》 * |
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