CN109521192A - A method of latex is improved than turbid reagent detection sensitivity - Google Patents
A method of latex is improved than turbid reagent detection sensitivity Download PDFInfo
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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
- G01N33/5306—Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
Abstract
A kind of method the invention discloses raising latex than turbid reagent detection sensitivity, by between two heavy chains of the antibody for being coupled to carboxylated latex surface, increase an amino acid bracket, to increase the space length between two Fab of antibody, to reduce steric hindrance of the latex than turbid antibody and antigen binding in the process, sensitivity is improved；The present invention is improved to using the reagent after conventional carboxylated latex coupled antibody, and amplification technique is mature, is conducive to promote.
Description
Technical field
The present invention relates to technical field of in vitro diagnostic reagents, more particularly to a kind of raising latex is examined than turbid reagent
The method for surveying sensitivity.
Background technique
In latex immunoturbidimetry reagent, sensitivity height, intuitively show as absorbance signal value change rate height and its
Repetition stability is tested, can indirectly be stated by minimum detection limit LOD, i.e., LOD is lower, and sensitivity is higher.
Currently, conventional method of the latex than turbid detection reagent sensitivity that improve is including improving enhanced sensitivity agent content in reagent, mentioning
High latex particle size uses long spacing arm latex, increase sample size and use more high-affinity antibody.Increase in reagent although improving
Quick agent content is easy to use, and it is obvious to improve sensitivity effect；But sensitizer often selects polyethylene glycol, Tween-20 and Triton X-100
Series etc., polyethylene glycol series of concentrations is excessively high to cause non-specific precipitation, false positive；Tween-20 and Triton X-100 series of concentrations
With sensitivity relation at bell, with its increase of concentration when low concentration, reagent sensitivity is improved, and when excessive concentrations, reduces reagent spirit
Sensitivity.Improve latex particle size: large grain size latex can effectively increase absorbance signal value, significantly improve reagent sensitivity, still
It is excessively high to will lead to reagent blank, and large grain size latex would generally reduce the reagent range of linearity and antigen excess ability, and big grain
Diameter latex production technology is more difficult to control, and reagent storage stability is also problem.Long spacing arm latex: steric hindrance is used, and
It is coupled at the freedom degree of latex surface antibody, the difficulty or ease of antigen-antibody combination are influenced, to influence reagent sensitivity；Although business
The long-chain carboxyl latex of change is also supplied more, but the relatively conventional carboxylated latex of coupling process is easy cohesion, and amplification technique is more tired
It is difficult.Increase sample size: in the constant situation of reagent, reagent sensitivity can be promoted at double by improving sample size, but sacrifice it is linear and
Antigen excess range, and increase matrix effect and interferent concentration, lead to measurement inaccuracy.Use more high-affinity antibody: parent
With power height, can faster stronger combination antigen, to improve absorbance signal change rate and its repetition stability.But it is famous
The commercialized antibody of brand have passed through long-term affinity and improve, and affinity improves difficulty again.
Therefore, outside how independently of the prior art, then improving sensitivity of the latex than turbid reagent is art technology
The problem of personnel's urgent need to resolve.
Summary of the invention
In view of this, the present invention provides a kind of raising latex than the method for turbid reagent detection sensitivity, this method can
Enough improve reagent quality: minimum detection limit LOD is low and reproducible, moreover it is possible to reduce reagent cost: using less antibody
Reach same sensitivity.
To achieve the goals above, the present invention adopts the following technical scheme:
A method of latex is improved than turbid reagent detection sensitivity, the specific steps are as follows:
(1) by double-strand Mono-specific antibodies and carboxylated latex covalent coupling, urea is added in the latex solution before closing,
Urea final concentration in latex solution is set to reach 10-20mM/L；37 degree of constant-temperature tables, 120rpm mildly rotate blending incubation 1 hour,
Disulfide bond of fracture antibody two between heavy chain and light chain and heavy chain, obtains emulsion reagent；
(2) by middle part acidity, the amino acid bracket of cyclic peptide of the both ends containing cysteine, room-temperature dissolution is in bracket lysate
In；The dosage of the amino acid bracket are as follows: every mg antibody corresponds to 0.010-0.020mg amino acid bracket；
For the amino acid stent sequence as shown in formula I, K is lysine, and C is cysteine, and X is 3-4 independent assortment
Acidic amino acid；
Stronger polar acidic, is conducive to that amino acid bracket is avoided to get involved in the disulfide bond between light chain and heavy chain in the middle part of cyclic peptide
It is formed, when amino acid bracket is not serious excessive, less gets involved in light chain and weight interchain disulfide bond.X is too long, may be due to
Steric hindrance problem, it is inefficient between so that it is embedded in heavy chain；Can also antibody structure be caused to destroy, partial denaturation inactivation, sensitivity mentions
It rises not enough, is not so good as 3 and 4 amino acid.X is short, and polarity is not strong, and a large amount of amino acid brackets is caused to participate in light chain and weight interchain disulfide bond
Bridge joint, and amino acid bracket is insufficient between heavy chain of antibody Fab, and the end X extended distance is not big enough, sensitivity enhancement is not as good as 3-4
Amino acid.
(3) 50% background buffer is removed to the emulsion reagent that step (1) obtains, that is, changes liquid 50%；Step (2) institute is added
The solution dissolved with amino acid bracket is stated, so that every mg antibody is corresponded to 0.010-0.020mg amino acid bracket and mixes；It is mixed to aforementioned
Closing has the potassium persulfate solution that 50mmol/L is added dropwise in amino acid bracket emulsion reagent, and the mole of the potassium peroxydisulfate is step
(1) 35-50% of urea amount in；
(4) step (3) has been added dropwise to the emulsion reagent of potassium persulfate solution in 37 degree of constant-temperature tables, 120rpm mildly rotates
Blending incubation 30min；Re-form disulfide bond, increase space frame between two heavy chains of antibody, increase two Fab of antibody it
Between space length；Also bracket can be added between part light chain and heavy chain, sensitivity enhancement is influenced little；
(5) emulsion reagent after being incubated for step (4) is closed and changes liquid cleaning removal residual amino acid bracket, urea
And potassium peroxydisulfate, finished product reagent R2.
Preferably, the final concentration of 20mM/L of urea in step (1) described latex solution.
Preferably, step (2) the bracket lysate is 100mM/L pH7.5, and the PBS of the Tween-20 containing 0.1%v/V is slow
Fliud flushing.
Preferably, the dosage of step (2) the amino acid bracket are as follows: every mg antibody corresponds to 0.015mg amino acid bracket.
Preferably, step (2) acidic amino acid is aspartic acid D and/or glutamic acid E.
Preferably, step (2) X be DDD, EEE, DED, EDE, EDD, DDE, EED, DEE, DDDD, EDDD, DEDD,
DDED, DDDE, EEDD, EEED, EEEE, DDEE, DEDE, DEEE, EDEE, EEDE, EDED, DEED or EDDE.
Preferably, step (3) the liquid method of changing is that liquid is changed in centrifugation, dialysis, chromatographic column or ultrafiltration.
Liquid is changed on a small scale, liquid is changed using centrifugation, change liquid on a large scale using ultrafiltration and chromatographic column.
Preferably, the mole of step (3) described potassium peroxydisulfate is 50% of urea amount in step (1).
Preferably, step (3) the 50mmol/L potassium persulfate solution is to dissolve 2.7032g with 200ml bracket lysate
Potassium peroxydisulfate obtains.
Preferably, step (5) it is described closing and change liquid cleaning specific step is as follows: by step (4) be incubated for after latex
Reagent, 22000rpm are centrifuged 15min, remove supernatant, are added in right amount (dependent on latex concentration required for disparity items finished product, usually
For the latex final concentration of 1-4g/L) redissolution of enclosed cleaning liquid, room temperature mixing 2 hours, finished product reagent R2.
Preferably, the enclosed cleaning liquid is the PBS buffer solution of 100mM/L pH7.5, Tween-20 containing 0.1%V/V, 1g/
L BSA, 0.05%v/V preservative PC300.
It can be seen via above technical scheme that compared with prior art, the present disclosure provides a kind of raising latex ratios
The method of turbid reagent detection sensitivity, by increasing an amino between two heavy chains of the antibody for being coupled to carboxylated latex surface
Sour bracket, the space length between two Fab of Lai Zengjia antibody, to reduce latex than antibody during turbid and antigen binding
Steric hindrance improves sensitivity；The present invention is improved to using the reagent after conventional carboxylated latex coupled antibody, amplification technique
Maturation is conducive to promote.
Specific embodiment
The technical scheme in the embodiments of the invention will be clearly and completely described below, it is clear that described implementation
Example is only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, this field is common
Technical staff's every other embodiment obtained without making creative work belongs to the model that the present invention protects
It encloses.
200mM/L, 300mM/L, 400mM/L are prepared, each 100ml of the aqueous solution of urea of 500mM/L, room temperature is spare.
Prepare bracket lysate: 100mM/L PH7.5, the PBS buffer solution of the Tween-20 containing 0.1%v/V, room temperature are spare.
It prepares 50mM/L potassium persulfate solution: dissolving 2.7032g potassium peroxydisulfate using bracket dilution 200ml, 4 DEG C are deposited
It puts spare.
Preparation enclosed cleaning liquid: 100mM/L pH7.5PBS buffer, Tween-20 containing 0.1%v/V, 1g/LBSA,
0.05%v/V preservative PC300, room temperature are spare.
Polyclonal antibody reaction buffer reagent 1-Pab (R1-Pab): 100mM/L pH7.5PBS buffer contains 0.5%
V/V Tween-20,0.05%v/V preservative PC300, final concentration 350mM/L sodium chloride, final concentration 10g/L Macrogol 6000,
Final concentration 2ml/L Scantibody company blocking agent HBR22, room temperature are spare.
Monoclonal antibody reactive buffering agents 1-Mab (R1-Mab): 100mM/L pH7.5PBS buffer contains 0.5%
V/V Tween-20,0.05%v/V preservative PC300, final concentration 150mM/L sodium chloride, final concentration 15g/L Macrogol 6000,
Final concentration 5ml/L Scantibody company blocking agent HBR1, room temperature are spare.
Embodiment 1
Myoglobins MB project, import rabbit polyclonal antibody, centrifugation change liquid, reagent is not transformed is compared with compareing
Compared with, while comparing amount of urea influence
(1) latex cleaning (disposable Japanese JSR company latex can be without this step): the MES- of 100mM/L is used
NaOH buffer, PH5.5, centrifugation are changed liquid latex microsphere 2 times；Take 10ml, the Agilent PL6115138nm glue of 10% mass concentration
Newborn microballoon 22000rpm is centrifuged 15min, removes supernatant, is redissolved using the above-mentioned buffer of 10ml, is then centrifuged again, supernatant is gone to redissolve,
It is spare to complete cleaning；
(2) latex activates: by taking the above-mentioned latex cleaned of 10ml as an example, weighing 50mg NHS, (N- hydroxysuccinimidyl acyl is sub-
Amine), 30mg EDC (1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride), with the buffer of the above-mentioned cleaning of 5ml
After dissolution, takes 1ml that 10ml is added and cleaned in microballoon, room temperature magnetic agitation mixes 10min；
(3) it is cleaned after activating: the latex 22000rpm after step (2) activation being centrifuged 15min, supernatant is removed, 20ml is added
The PBS buffer solution of 100mM/L pH7.5 is redissolved spare；
(4) antibody coupling: taking Dako company article No. is the myoglobins antibody 2ml (12mg/ml) of OA044, is added to
It states in the latex solution of activation redissolved with PBS buffer solution, room temperature magnetic agitation 3 hours, completes antibody coupling；
(5) R2 is transformed:
1) latex for completing antibody coupling is divided into 5 parts, every group of 4ml (being named as A/B/C/D/E group), extra is useless
Abandon (2ml antibody is added in 20ml PBS, since different liquids dissolve each other principle, final volume only point slightly more more than 20ml, not
It is to obtain 22ml solution)；
2) A group is control group, B group urea final concentration 10mM/L, C group urea final concentration 15mM/L, D group urea final concentration
20mM/L, E group urea final concentration 25mM/L；
Concrete operations: placebo 200ul purified water is added in A group, and B/C/D/E group is separately added into 200ul 200mM/L,
The aqueous solution of urea of 300mM/L, 400mM/L, 500mM/L, i.e. B/C/D/E group correspond to the final concentration of 10/15/20/25mM/ of urea
L；
3) A, B, C, D, the equal 22000rpm 15min centrifugation of component E, A group remove whole supernatants, and B/C/D/E group removes 2ml supernatant,
" change liquid 50% "；
4) X sequence is molten for amino acid bracket (Sangon Biotech's synthesis) 1mg of EDED
Solution can be calculated in every group of experiment, the quality of antibody is about 2*12*4/20=4.8mg in 20.8ml bracket lysate；
Each solution that 2ml is added and is dissolved with amino acid bracket of B/C/D/E group, redissolve, and can calculate to obtain antibody and bracket
Dosage is that 1mg antibody corresponds to 0.020mg amino acid bracket；
5) B/C/D/E group is separately added into 50mM/L potassium persulfate solution 0.4ml, 0.6ml, 0.8ml, 1.0ml, i.e. persulfuric acid
Potassium mole is about 50% of urea amount in step 2).
6) 37 degree of constant-temperature tables of B/C/D/E group, 120rpm mildly rotate blending incubation 30min；
(6) it closes and cleans:
A group is added enclosed cleaning liquid 8ml and redissolves, and removes whole supernatants after B/C/D/E group 22000rpm is centrifuged 15min, adds
Enter enclosed cleaning liquid 8ml redissolution, room temperature mixes 2 hours spare, as finished product R2.
(7) examination with computer
Instrument: Hitachi 7180, set factors, the R1-Pab of the aforementioned preparation of sample size 6ul, 180ul, 60ul R2,570/
800nm dual wavelength, Two point end assay, 18-34 read point calculate Δ ABS (absorbance change value)；
MB calibration object (S1 20ng/ml, S2 100ng/ml, S3 200ng/ml, S4 400ng/ml) is diagnosed using Bai Rong,
Zero point uses physiological saline, by S1 using normal saline dilution to 5ng/ml, 7.5ng/ml, 10ng/ml, 15ng/ml together with S1,
It is tested for minimum detection limit；
Testing scheme: the A/B/C/D/E group mating above-mentioned pre-prepd identical R1-Pab of difference, identical parameters, phase are used
It is calibrated with calibration object, more each calibration point Δ ABS, while measuring 5ng/ml respectively, 7.5ng/ml, 10ng/ml, 15ng/ml connect
Each 10 times with S1, mean value is calculated separately, STDEV obtains precision CV using STDEV/ mean value；Note that from high concentration when testing
Start, precision is too poor, then accident lower concentration, and N/A is indicated.
Each experimental group calibration data are as follows:
From calibration data as can be seen that compared to control group, B/C/D/E group sensitivity Δ ABS has different degrees of mention
Height, wherein D group increase rate is most obvious, promotes about 148%, B/C/D/E group compared to control and corresponds to urea final concentration of 10/15/
20/25mM/L；The result shows that improving with urea concentration, sensitivity enhancement first rises and declines afterwards, and preferably urea concentration is
20mM/L。
Each experimental group detection limit experiment is as follows:
As a result as can be seen that detection limit LOD (concentration of specimens at CV20%), compares A group between 15-20ng/ml, estimate
Meter is in 16ng/ml or so；B group between 7.5-10ng/ml, estimate about 8.5ng/ml or so, C group also 7.5-10ng/ml it
Between, but each concentration precision is substantially better than B group, estimation LOD is 7.4ng/ml or so, and D group is optimal, and LOD is in 5-7.5ng/ml
Between, it is estimated as 6ng/ml or so, E group LOD raising occurs compared to B/C/D group, but better than control A group, LOD is in 10-15ng/
Between ml, estimate in 10ng/ml or so；As a result as can be seen that preferably D group, LOD drop to about 37.5%, the B of control A group
46.3%, the D group that 53.1%, the C group that group drops to control group drops to control group drops to the 62.5% of control；B/C/D/E
The corresponding final concentration of 10/15/20/25mM/L of urea of group, it can be seen that as urea concentration improves, LOD rises after falling before,
Preferably concentration is 20mM/L.
The above results show in urea concentration 10-20mM/L concentration, and in MB project, improving reagent R2 can be effective
Improve the detection sensitivity of reagent.But urea cannot be excessive, and otherwise may cause antibody activity reduces, under sensitivity enhancement rate
Drop；Urea amount is very little, and it is insufficient to may cause antibody disulfide bonds, and sensitivity enhancement is also not significant enough；Preferably urea is dense
Degree is 20mM/L.
Embodiment 2
AFP AFP project, import rabbit polyclonal antibody, centrifugation change liquid, reagent is not transformed is compared with compareing
Compared with, while comparing amino acid bracket dosage influences
(1) latex cleans: using the MES-NaOH buffer of 100mM/L, pH5.5, centrifugation is changed liquid latex microsphere 2 times；It takes
10ml, the Polymicrospheres CB0230D 230nm latex microsphere of 10% mass concentration, 22000rpm are centrifuged 10min,
Supernatant is removed, is redissolved using the above-mentioned buffer of 10ml, is then centrifuged again, supernatant is gone to redissolve, it is spare to complete cleaning；
(2) latex activates: by taking the above-mentioned latex cleaned of 10ml as an example, weighing 50mg NHS, (N- hydroxysuccinimidyl acyl is sub-
Amine), 30mg EDC (1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride), with the buffer of the above-mentioned cleaning of 5ml
After dissolution, takes 1ml that 10ml is added and cleaned in microballoon, room temperature magnetic agitation mixes 10min；
(3) it is cleaned after activating: latex 22000rpm after step (2) activation being centrifuged 10min, supernatant is removed, 36ml is added
The PBS buffer solution of 100mM/L pH7.5 is redissolved spare；
(4) antibody coupling: taking Dako company article No. is the alpha-fetoprotein antibody 15ml (2.5mg/ml) of A0008, is added to
In the above-mentioned latex solution of activation redissolved with PBS buffer solution, room temperature magnetic agitation 3 hours, antibody coupling is completed；
(5) R2 is transformed:
1) latex for completing antibody coupling is divided into 5 parts, every group of 10ml (being named as A2/B2/C2/D2/E2 group) is more
It is remaining that discarded (15ml antibody is added in 36ml PBS, and since different liquids dissolve each other principle, final volume is only slightly more more than 50ml
Point is not to obtain 51ml solution).
2) A2 group is that each urea that 500mM/L is added of placebo purified water 400ul, B2/C2/D2/E2 group is added in control group
Aqueous solution 400ul, until urea final concentration about 20mM/L；
3) the equal 22000rpm 10min centrifugation of A2, B2, C2, D2, E2 component, A2 group go whole supernatants, B2/C2/D2/E2 group
5ml supernatant is removed, i.e., " changes liquid 50% "；
4) amino acid bracket (Sangon Biotech's synthesis) 1mg that X sequence is DED is dissolved
In 20ml bracket lysate, it can be calculated in every group of experiment, the quality of antibody is about 15*2.5*10/50=7.5mg；
The 0.75ml of solution containing bracket, bracket lysate 4.25ml is added in B2 group, and it is about corresponding can to calculate 1mg antibody in B2 group
0.005mg bracket；
The 1.5ml of solution containing bracket, bracket lysate 3.5ml is added in C2 group, and it is about corresponding can to calculate 1mg antibody in C2 group
0.010mg bracket；
The 2.25ml of solution containing bracket, bracket lysate 2.75ml is added in D2 group, and it is about corresponding can to calculate 1mg antibody in D2 group
0.015mg bracket；
The 3.0ml of solution containing bracket, bracket lysate 2.0ml is added in E2 group, and it is about corresponding can to calculate 1mg antibody in E2 group
0.020mg bracket；
5) B2/C2/D2/E2 group is separately added into 50mM/L potassium persulfate solution 2.0ml, i.e. potassium peroxydisulfate mole about walks
It is rapid 2) in urea amount 50%；
6) 37 degree of constant-temperature tables of B2/C2/D2/E2 group, 120rpm mildly rotate blending incubation 30min.
(6) it closes and cleans:
A2 group is added enclosed cleaning liquid 10ml and redissolves, and will remove whole supernatants after B2/C2/D2/E2 group 22000rpm centrifugation, adds
Enter enclosed cleaning liquid 10ml redissolution, room temperature mixes 2 hours spare, as finished product R2；
(7) examination with computer
Instrument: Hitachi 7180, set factors, the R1-Pab of the aforementioned preparation of sample size 6ul, 180ul, 45ul R2,700nm
Single wavelength, Two point end assay, 19-34 read point calculate Δ ABS；
AFP calibration object (S1 10ng/ml, S2 20ng/ml, S3 63ng/ml, S4 315ng/ml) is diagnosed using Bai Rong,
Zero point uses physiological saline, by S1 using normal saline dilution to 1ng/ml, 2.5ng/ml, 5ng/ml, 7.5ng/ml together with S1,
It is tested for minimum detection limit；
Testing scheme: the A2/B2/C2/D2/E2 group mating above-mentioned pre-prepd identical R1-Pab of difference, identical ginseng are used
Number, the calibration of same calibration product, more each calibration point Δ ABS, while 1ng/ml is measured respectively, 2.5ng/ml, 5ng/ml, 7.5ng/
Ml each 10 times, calculates separately mean value, STDEV obtains precision CV using STDEV/ mean value together with S1；Note that test Shi Conggao
Concentration starts, and precision is too poor, then accident lower concentration.
Each experimental group calibration data are as follows:
From calibration data as can be seen that compared to control group, B2/C2/D2/E2 group sensitivity Δ ABS has in various degree
Raising, wherein D2 and E2 group increase rate is most obvious, compared to control promoted about 150%, B2/C2/D2/E2 group in antibody 1mg
Orresponding amino acid bracket amount is 0.005/0.010/0.015/0.020mg；It can be seen that sensitivity mentions as bracket amount improves
It rises first to rise and tend to be steady afterwards, consider bracket cost, it is about 0.015mg that preferably 1mg antibody, which corresponds to bracket amount,.
Each experimental group detection limit experiment is as follows:
As a result as can be seen that detection limit LOD (concentration of specimens at CV20%), compares A2 group between 5-7.5ng/ml,
Estimation is in 6.7ng/ml or so；B2 group is between 5-7.5ng/ml, but each concentration precision is substantially better than A2 group, and estimation is about
5ng/ml or so, C2 group are also between 2.5-5ng/ml, and estimation LOD is 3.3ng/ml or so, D2 and E2 group is optimal, and LOD is in 1-
Between 2.5ng/ml, it is estimated as 2.4ng/ml or so, preferably B2 group, LOD drops under about 74.6%, the C2 group of control A2 group
49.3%, D2 and the E2 group for dropping to control group drop to the 35.8% of control group.B2/C2/D2/E2 group antibody 1mg corresponds to amino
Sour bracket is 0.005/0.010/0.015/0.020mg, it can be seen that as bracket concentration improves, LOD tends to be flat after falling before
Steady 0.015 and 0.020mg effect be not much different, but bracket higher cost considers that cost problem, preferably every mg antibody are corresponding
Amino acid bracket is 0.015mg.
When the above results show that the corresponding bracket amount of every mg antibody is between 0.010-0.020mg, in AFP project, change
The detection sensitivity of reagent is effectively improved into scheme；Consider that cost problem bracket cannot be improved infinitely, preferably every mg antibody pair
Answering amino acid bracket is 0.015mg.
Embodiment 3
Ferritin FER project, domestic rabbit polyclonal antibody, centrifugation change liquid, and reagent is not transformed with control and is compared,
Compare the influence of potassium peroxydisulfate concentration simultaneously
(1) latex cleaning (disposable Japanese JSR company latex can be without this step): the MES- of 100mM/L is used
NaOH buffer, pH5.5, centrifugation are changed liquid latex microsphere 2 times；Take 10ml, the Polymicrospheres of 10% mass concentration
CB0297C 297nm latex microsphere is centrifuged 6min using 22000rpm, removes supernatant, is redissolved using the above-mentioned buffer of 10ml, then
It is centrifuged again, supernatant is gone to redissolve, it is spare to complete cleaning；
(2) latex activates: by taking the above-mentioned latex cleaned of 10ml as an example, weighing 50mg NHS, (N- hydroxysuccinimidyl acyl is sub-
Amine), 30mg EDC (1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride), with the buffer of the above-mentioned cleaning of 5ml
After dissolution, takes 1ml that 10ml is added and cleaned in microballoon, room temperature magnetic agitation mixes 10min；
(3) it is cleaned after activating: latex 22000rpm after step (2) activation being centrifuged 6min, supernatant is removed, 46ml is added
The PBS buffer solution of 100mM/L pH7.5 is redissolved spare；
(4) antibody coupling: taking ZenBio company article No. is 301067 ferritin antibody 5ml (13mg/ml), is added to
It states in the latex solution of activation redissolved with PBS buffer solution, room temperature magnetic agitation 3 hours, completes antibody coupling；
(5) R2 is transformed:
1) latex for completing antibody coupling is divided into 5 parts, every group of 10ml (being named as A3/B3/C3/D3/E3 group) is more
It is remaining that discarded (5ml antibody is added in 46ml PBS, and since different liquids dissolve each other principle, final volume is only slightly more more than 50ml
Point is not to obtain 51ml solution)；
2) A3 group is that each urea that 500mM/L is added of placebo purified water 400ul, B3/C3/D3/E3 group is added in control group
Aqueous solution 400ul, until urea final concentration about 20mM/L；
3) the equal 22000rpm 10min centrifugation of A3, B3, C3, D3, E3 component, A3 group go whole supernatants, B3/C3/D3/E3 group
5ml supernatant is removed, i.e., " changes liquid 50% "；
4) amino acid bracket (Sangon Biotech's synthesis) 1mg that X sequence is EDE is dissolved
In 20ml bracket lysate, it can be calculated in every group of experiment, the quality of antibody is about 5*13*10/50=13mg；
B3/C3/D3/E3 group is separately added into the standoff each 3.9ml of bracket lysate of dissolution, can calculate and learn, every mg
The corresponding bracket of antibody is 0.015mg；
5) 50mM/L concentration potassium persulfate solution 1.0ml is added in B3 group, i.e. potassium peroxydisulfate mol amount is urea in step 4.2
About the 25% of amount；
50mM/L concentration potassium persulfate solution 1.4ml is added in C3 group, i.e. potassium peroxydisulfate mol amount is urea amount in step 4.2
About 35%；
50mM/L concentration potassium persulfate solution 1.6ml is added in D3 group, i.e. potassium peroxydisulfate mol amount is urea amount in step 4.2
About 40%；
50mM/L concentration potassium persulfate solution 2.0ml is added in E3 group, i.e. potassium peroxydisulfate mol amount is urea amount in step 4.2
About 50%；
6) 37 degree of constant-temperature tables of B3/C3/D3/E3 group, 120rpm mildly rotate blending incubation 30min；
(6) it closes and cleans:
A3 group is added enclosed cleaning liquid 12ml and redissolves, and will remove whole supernatants after B3/C3/D3/E3 group 22000rpm centrifugation, adds
Enter enclosed cleaning liquid 12ml redissolution, room temperature mixes 2 hours spare, as finished product R2.
(7) examination with computer
Instrument: Hitachi 7180, set factors, sample size 10ul, 120ul R1,60ul R2,570nm Single wavelength, two o'clock are whole
Point method, 18-34 read point calculate Δ ABS；
FER calibration object (S1 20ng/ml, S2 100ng/ml, S3 200ng/ml, S4 500ng/ is diagnosed using Bai Rong
Ml), zero point uses physiological saline, and S1 is connected using normal saline dilution to 5ng/ml, 7.5ng/ml, 10ng/ml, 15ng/ml
Same S1 is tested for minimum detection limit；
Testing scheme: the A3/B3/C3/D3/E3 group mating above-mentioned pre-prepd identical R1-Pab of difference, identical ginseng are used
Number, the calibration of same calibration product, more each calibration point Δ ABS, while 5ng/ml is measured respectively, 7.5ng/ml, 10ng/ml, 15ng/
Ml each 10 times together with S1, mean value is calculated separately, STDEV obtains precision CV using STDEV/ mean value；Note that test Shi Conggao
Concentration starts, and precision is too poor, then accident lower concentration.
Each experimental group calibration data are as follows:
As a result as can be seen that compared to control A3 group, with the raising of potassium peroxydisulfate concentration, FER reagent sensitivity Δ ABS is aobvious
It writes and is promoted, wherein when potassium peroxydisulfate concentration is relative to urea concentration 50%, i.e. E3 group, sensitivity highest, but compare E3 and D3
It was found that urea concentration rises to 50% from 40%, and sensitivity enhancement has eased up although D3 to E3.And potassium peroxydisulfate is dense
Height is spent, reagent stability and non-specific responding are adversely affected.Therefore preferably potassium peroxydisulfate addition concentration is about
The 50% of preceding step urea concentration.
The following are the minimum detection limit experimental datas of embodiment 3:
As a result as can be seen that detection limit LOD (concentration of specimens at CV20%), compares A3 group between 10-15ng/ml,
Estimation is in 15ng/ml or so；B3 group is between 10-15ng/ml, but each concentration precision is substantially better than A3 group, and estimation is about
10ng/ml or so, C3 group are also between 7.5-10ng/ml, and estimation LOD is 7.5ng/ml or so, and D3 group, LOD is in 7.5-5ng/
Between ml, it is estimated as 6.5ng/ml or so, preferably D3 group, LOD is estimated as 6.1ng/ml or so between 7.5-5ng/ml,
50%, D3 and the E3 group that about 66.7%, the C3 group that B3 group LOD drops to control A2 group drops to control group drop to control group
43.3% and 40.7%.The corresponding potassium peroxydisulfate concentration of B3/C3/D3/E3 is respectively the 25% of preceding step urea concentration,
35%, 40% and 50%, it can be seen that as potassium peroxydisulfate concentration improves, LOD becomes after 40% in the trend that is gradually reduced
Gesture slows down.
The above results show that in FER project, as potassium peroxydisulfate concentration improves, improvement project effectively improves reagent
Detection sensitivity.And potassium peroxydisulfate excessive concentration, reagent stability and non-specific responding are adversely affected.Over cure
The sour more excellent concentration of potassium is the 50% of previous step urea concentration.
Embodiment 4
D dimer D-Dimer project, import mouse monoclonal antibody, centrifugation change liquid, and reagent progress is not transformed with compareing
Compare, while comparing the influence of X amino acid quantity variance；
(1) latex cleaning (disposable Japanese JSR company latex can be without this step): the MES- of 100mM/L is used
NaOH buffer, pH5.5, centrifugation are changed liquid latex microsphere 2 times；Take 10ml, the Agilent PL6115138nm glue of 10% mass concentration
Newborn microballoon, 22000rpm are centrifuged 15min, remove supernatant, are redissolved using the above-mentioned buffer of 10ml, are then centrifuged again, supernatant is gone to redissolve,
It is spare to complete cleaning；
(2) latex activates: by taking the above-mentioned latex cleaned of 10ml as an example, weighing 50mg NHS, (N- hydroxysuccinimidyl acyl is sub-
Amine), 30mg EDC (1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride), with the buffer of the above-mentioned cleaning of 5ml
After dissolution, takes 1ml that 10ml is added and cleaned in microballoon, room temperature magnetic agitation mixes 10min；
(3) it is cleaned after activating: latex 22000rpm after step (2) activation being centrifuged 15min, supernatant is removed, 37ml is added
The PBS buffer solution of 100mM/L pH7.5 is redissolved spare；
(4) antibody coupling: each 2ml of D dimer mouse monoclonal antibody that Hytest company article No. is DD41 and DD02 is taken (
For 5mg/ml), it is added in the above-mentioned latex solution of activation redissolved with PBS buffer solution, room temperature magnetic agitation 3 hours, completes
Antibody coupling；
(5) R2 is transformed:
1) latex for completing antibody coupling is divided into 5 parts, every group of 8ml (being named as A4/B4/C4/D4/E4 group) is more
Remaining is discarded.
2) A4 group is control group, and placebo 335ul purifying is added in B4/C4/D4/E4 group urea final concentration 20mM/L, A group
Water, B4/C4/D4/E4 group are separately added into the aqueous solution of urea 335ul of 500mM/L, i.e. it is dense eventually to correspond to urea for B4/C4/D4/E4 group
Degree is 20mM/L；
3) the equal 22000rpm 15min centrifugation of A4, B4, C4, D4,4E component, A4 group go whole supernatants, B4/C4/D4/E4 group
4ml supernatant is removed, i.e., " changes liquid 50% "；
It 4) is amino acid bracket (the raw limited public affairs of work bioengineering (Shanghai) share of EE, EEE, EEEE, EEEEE by X sequence
Department's synthesis) each 1mg is dissolved in 25ml bracket lysate, can be calculated in every group of experiment, and the quality of antibody is about (5*2+
5*2) * 8/40=4mg；
B4/C4/D4/E4 group is each to be added the standoff solution of corresponding 2ml dissolution, and B4 group is EE bracket, and C4 group is EEE branch
Frame, D4 group are EEEE bracket, and E4 group is EEEEE bracket, is redissolved, can calculate antibody corresponds to 0.020mg than bracket ratio for 1mg
Amino acid bracket；
5) B4/C4/D4/E4 group is separately added into 50mM/L concentration potassium persulfate solution 1.6ml, i.e. potassium peroxydisulfate mol amount is
About the 50% of urea amount in step 4.2；
6) 37 degree of constant-temperature tables of B4/C4/D4/E4 group, 120rpm mildly rotate blending incubation 30min；
(6) it closes and cleans:
A4 group is added enclosed cleaning liquid 10ml and redissolves, and goes in whole after B4/C4/D4/E4 group 22000rpm is centrifuged 15min
Clearly, enclosed cleaning liquid 10ml is added to redissolve, room temperature mixes 2 hours spare, as finished product R2.
(7) examination with computer
Instrument: Hitachi 7180, set factors, the R1-Mab of the aforementioned preparation of sample size 4ul, 180ul, 60ul R2,700nm
Single wavelength, Two point end assay, 20-34 read point calculate Δ ABS；
D-Dimer calibration object (S1 0.500mg/L, S2 2.000mg/L, S3 8.000mg/L, S4 are diagnosed using Bai Rong
16.000mg/L), zero point uses physiological saline, and by S2 using normal saline dilution to 1.000mg/L, S1 is used physiological saline
It is diluted to 0.100mg/L, 0.250mg/L, together with S1, S2, is tested for minimum detection limit；
Testing scheme: the A4/B4/C4/D4/E4 group mating above-mentioned pre-prepd identical R1-Mab of difference, identical ginseng are used
Number, the calibration of same calibration product, more each calibration point Δ ABS, while 0.100mg/L is measured respectively, 0.250mg/L, 0.500mg/
L, 1.000mg/L, 2.000mg/L, calculate separately mean value, and STDEV obtains precision CV using STDEV/ mean value；Note that test
When since high concentration, precision is too poor, then accident lower concentration.
Each experimental group calibration data are as follows:
As a result as can be seen that compared to control A4 group, four groups of sensitivity for increasing bracket are significantly increased, and X is represented in bracket
Acidic amino acid quantity increase to 4 from 2, sensitivity is gradually increased, and 3 and 4 difference are smaller, and equal relative comparison A4 group sensitivity mentions
Rise about 145% or so；When amino acid quantity increases to 5, sensitivity is dropped by a relatively large margin compared to 3 and 4 amino acid appearance
It is low；Illustrate that acidic amino acid quantity is preferably 3 and 4 in amino acid bracket X.
The following are 4 minimum detection limit data of embodiment:
From data as can be seen that control A4 group, LOD is between 0.500-1.000mg/L, and estimation is on the left side 0.550mg/L
It is right；B4 group, for LOD between 0.250-0.500mg/L, estimation is about 0.375mg/L or so；C4 group, LOD is between 0.100-
Between 0.250, about 0.240mg/L；D4 group, LOD is also between 0.100-0.250, but each concentration C V is slightly better than C4 group,
LOD is about 0.195mg/L；E4 group, for LOD between 0.250-0.500mg/L, estimation is about 0.41mg/L；C4 and D4 group, point
Other relative comparison group LOD drops to 43.6% and 35.4%.
The above results show that the acidic amino acid quantity of X in amino acid bracket is preferably 3-4.
Reagent R2 is transformed by increasing amino acid bracket between two heavy chains of antibody in the present invention, the reagent R2 of transformation and often
Rule reagent R1 (above-mentioned R1-Pab or R1-Mab) are used cooperatively, and improve the sensitivity of latex immunoturbidimetry.
Present invention is generally directed to traditional two chain Mono-specific antibodies raw materials, to use the latex of these raw materials more logical than turbid reagent
Use technology.The present invention is not suitable for single-chain antibody, is not suitable for physisorphtion to quick latex, is not suitable for not being coupled anti-
Body, reason may be that disulfide bonds will lead to non-coupled antibody and fall apart and can not restore in situ.
The foregoing description of the disclosed embodiments enables those skilled in the art to implement or use the present invention.
Various modifications to these embodiments will be readily apparent to those skilled in the art, as defined herein
General Principle can be realized in other embodiments without departing from the spirit or scope of the present invention.Therefore, of the invention
It is not intended to be limited to the embodiments shown herein, and is to fit to and the principles and novel features disclosed herein phase one
The widest scope of cause.
Claims (10)
1. a kind of improve method of the latex than turbid reagent detection sensitivity, which is characterized in that specific step is as follows:
(1) by double-strand Mono-specific antibodies and carboxylated latex covalent coupling, urea is added in the latex solution before closing, makes glue
Urea final concentration reaches 10-20mM/L in milk solution；37 degree of constant-temperature tables, 120rpm mildly rotate blending incubation 1 hour, obtain
Emulsion reagent；
(2) by middle part acidity, the amino acid bracket of cyclic peptide of the both ends containing cysteine, room-temperature dissolution is in bracket lysate；Institute
State the dosage of amino acid bracket are as follows: every mg antibody corresponds to 0.010-0.020mg amino acid bracket；
For the amino acid stent sequence as shown in formula I, K is lysine, and C is cysteine, and X is the acidity of 3-4 independent assortment
Amino acid；
(3) 50% background buffer is removed to the emulsion reagent that step (1) obtains, that is, changes liquid 50%, it is described molten that step (2) are added
Solution has the solution of amino acid bracket, so that every mg antibody is corresponded to 0.010-0.020mg amino acid bracket and mixes；It is mixed with to aforementioned
The potassium persulfate solution of 50mmol/L is added dropwise in the emulsion reagent of amino acid bracket, the mole of the potassium peroxydisulfate is step
(1) 35-50% of urea amount in；
(4) step (3) has been added dropwise to the emulsion reagent of potassium persulfate solution in 37 degree of constant-temperature tables, 120rpm mildly rotates mixing
It is incubated for 30min；
(5) emulsion reagent after being incubated for step (4) is closed and changes liquid cleaning removal residual amino acid bracket, urea and mistake
Potassium sulfate, finished product reagent R2.
2. a kind of raising latex according to claim 1 is than the method for turbid reagent detection sensitivity, which is characterized in that step
(1) the final concentration of 20mM/L of urea in the latex solution.
3. a kind of raising latex according to claim 1 is than the method for turbid reagent detection sensitivity, which is characterized in that step
(2) the bracket lysate is 100mM/L pH7.5, the PBS buffer solution of the Tween-20 containing 0.1%v/V.
4. a kind of raising latex according to claim 1 is than the method for turbid reagent detection sensitivity, which is characterized in that step
(2) dosage of the amino acid bracket are as follows: every mg antibody corresponds to 0.015mg amino acid bracket.
5. a kind of raising latex according to claim 1 is than the method for turbid reagent detection sensitivity, which is characterized in that step
(2) acidic amino acid is aspartic acid D and/or glutamic acid E.
6. a kind of raising latex according to claim 5 is than the method for turbid reagent detection sensitivity, which is characterized in that step
(2) X be DDD, EEE, DED, EDE, EDD, DDE, EED, DEE, DDDD, EDDD, DEDD, DDED, DDDE, EEDD, EEED,
EEEE, DDEE, DEDE, DEEE, EDEE, EEDE, EDED, DEED or EDDE.
7. a kind of raising latex according to claim 1 is than the method for turbid reagent detection sensitivity, which is characterized in that step
(3) the liquid method of changing is that liquid is changed in centrifugation, dialysis, chromatographic column or ultrafiltration.
8. a kind of raising latex according to claim 1 is than the method for turbid reagent detection sensitivity, which is characterized in that step
(3) mole of the potassium peroxydisulfate is 50% of urea amount in step (1).
9. a kind of raising latex according to claim 1 is than the method for turbid reagent detection sensitivity, which is characterized in that step
(3) the 50mmol/L potassium persulfate solution is to dissolve 2.7032g potassium peroxydisulfate with 200ml bracket lysate to obtain.
10. a kind of raising latex according to claim 1 is than the method for turbid reagent detection sensitivity, which is characterized in that step
Suddenly (5) it is described closing and change liquid cleaning specific step is as follows: by step (4) be incubated for after emulsion reagent, 22000rpm centrifugation
15min removes supernatant, and enclosed cleaning liquid is added and redissolves, and makes the final concentration of 1-4g/L of latex, and room temperature mixes 2 hours, finished product examination
Agent R2.
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| CN114217071A (en) * | 2021-12-01 | 2022-03-22 | 柏荣诊断产品（上海）有限公司 | High-specificity transmission-scattering integrated ferritin latex turbidimetric detection kit |
| CN114235749A (en) * | 2021-12-17 | 2022-03-25 | 柏荣诊断产品（上海）有限公司 | Sensitivity enhancing method for latex turbidimetric reagent by transmission-scattering integrated method |
| CN116908265A (en) * | 2023-09-11 | 2023-10-20 | 常州先趋医疗科技有限公司 | Preparation method of electrochemical biosensor for detecting LAMP amplification products of nucleic acids |
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| CN114217071B (en) * | 2021-12-01 | 2023-11-14 | 柏荣诊断产品（上海）有限公司 | High-specificity integrated transmission and scattering ferritin latex turbidimetry detection kit |
| CN114235749A (en) * | 2021-12-17 | 2022-03-25 | 柏荣诊断产品（上海）有限公司 | Sensitivity enhancing method for latex turbidimetric reagent by transmission-scattering integrated method |
| CN116908265A (en) * | 2023-09-11 | 2023-10-20 | 常州先趋医疗科技有限公司 | Preparation method of electrochemical biosensor for detecting LAMP amplification products of nucleic acids |
| CN116908265B (en) * | 2023-09-11 | 2023-12-12 | 常州先趋医疗科技有限公司 | Preparation method of electrochemical biosensor for detecting LAMP amplification products of nucleic acids |
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