CN109521192B

CN109521192B - Method for improving detection sensitivity of latex turbidimetric reagent

Info

Publication number: CN109521192B
Application number: CN201811426490.6A
Authority: CN
Inventors: 王钊; 王金凤; 蒋欣
Original assignee: Byron Diagnostics Shanghai Co ltd
Current assignee: Byron Diagnostics Shanghai Co ltd
Priority date: 2018-11-27
Filing date: 2018-11-27
Publication date: 2021-07-13
Anticipated expiration: 2038-11-27
Also published as: CN109521192A

Abstract

The invention discloses a method for improving the detection sensitivity of a latex turbidimetric reagent, which increases the space distance between two Fab of an antibody by adding an amino acid bracket between two heavy chains of the antibody coupled on the surface of carboxyl latex, thereby reducing the steric hindrance of the combination of the antibody and the antigen in the turbidimetric process of the latex and improving the sensitivity; the invention improves the reagent after the conventional carboxyl latex is coupled with the antibody, has mature amplification process and is beneficial to popularization.

Description

Method for improving detection sensitivity of latex turbidimetric reagent

Technical Field

The invention relates to the technical field of in-vitro diagnostic reagents, in particular to a method for improving the detection sensitivity of a latex turbidimetric reagent.

Background

In the latex immunoturbidimetric reagent, the sensitivity is high and low, and the visual expression is the variation rate of absorbance signal values and the repeated stability of the test, and can be indirectly expressed by the lowest detection limit LOD, namely the lower the LOD is, the higher the sensitivity is.

At present, conventional methods for improving the sensitivity of latex turbidimetric detection reagents include increasing the content of sensitizer in the reagent, increasing the latex particle size, using long space arm latex, increasing the sample size, and using higher affinity antibodies. Although the content of the sensitizer in the reagent is increased, the sensitivity is improved obviously; however, the sensitizer is usually selected from polyethylene glycol, Tween-20, Triton 100 series and the like, and the concentration of the polyethylene glycol series is too high to cause non-specific precipitation and false positive; the relationship between the Tween-20 and Triton 100 series concentration and sensitivity is bell-shaped, the sensitivity of the reagent is improved along with the increase of the concentration at low concentration, and the sensitivity of the reagent is reduced at overhigh concentration. The particle size of the latex is increased: the latex with large particle size can effectively increase the value of an absorbance signal and improve the sensitivity of the reagent in multiples, but the reagent blank is too high, the linear range and the antigen excess capacity of the reagent are generally reduced by the latex with large particle size, the production process of the latex with large particle size is more difficult to control, and the storage stability of the reagent is also difficult. Long space arm latex was used: steric hindrance and the degree of freedom of an antibody coupled on the surface of the latex influence the difficulty of combining the antigen and the antibody, thereby influencing the sensitivity of the reagent; although commercial long-chain carboxy latex is also available, the coupling process tends to coagulate more easily than conventional carboxy latex, and the scale-up process is difficult. Increasing the sample size: under the condition that the reagent is not changed, the sensitivity of the reagent can be improved in multiples by increasing the sample volume, but the linearity and the antigen surplus range are sacrificed, and the matrix effect and the concentration of interferent are increased, so that the measurement is inaccurate. Using higher affinity antibodies: the affinity is high, and the antigen can be combined more quickly and firmly, so that the change rate of the absorbance signal and the repeated stability of the absorbance signal are improved. But the antibodies of the famous brand commercialization are all subjected to long-term affinity improvement, and the affinity is difficult to improve.

Therefore, how to improve the sensitivity of latex turbidimetric reagents independently of the prior art is a problem that needs to be solved by those skilled in the art.

Disclosure of Invention

In view of the above, the present invention provides a method for improving the detection sensitivity of a latex turbidimetric reagent, which can improve the quality of the reagent: the lowest limit of detection LOD is low, the repeatability is good, and the reagent cost can be reduced: the same sensitivity can be achieved with less antibody.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method for improving the detection sensitivity of a latex turbidimetric reagent comprises the following specific steps:

(1) covalently coupling the double-chain monospecific antibody with carboxyl latex, and adding urea into the latex solution before sealing to ensure that the final concentration of the urea in the latex solution reaches 10-20 mM/L; carrying out gentle rotation and uniform mixing incubation for 1 hour at a constant temperature of 37 ℃ and 120rpm, and breaking two heavy chains of the antibody and disulfide bonds between a light chain and a heavy chain to obtain a latex reagent;

(2) dissolving an amino acid scaffold with the middle part being acidic and the two ends containing cysteine cyclopeptide in a scaffold dissolving solution at room temperature; the dosage of the amino acid scaffold is as follows: 0.010-0.020mg amino acid scaffold per mg antibody;

the amino acid scaffold sequence is shown as a formula I, K is lysine, C is cysteine, and X is 3-4 freely combined acidic amino acids;

the strong acidic polarity in the middle of the cyclopeptide is beneficial to avoiding the amino acid scaffold from being involved in the formation of disulfide bonds between the light chain and the heavy chain, and when the amino acid scaffold is not seriously excessive, the amino acid scaffold is less involved in the disulfide bonds between the light chain and the heavy chain. The efficiency of embedding X between heavy chains is not high due to possible steric hindrance problem because the X is too long; also causes structural damage of the antibody, partial denaturation and inactivation, and the sensitivity is not improved enough, which is not as good as 3 or 4 amino acids. X is short and has low polarity, so that a large number of amino acid scaffolds participate in disulfide bridge connection between a light chain and a heavy chain, while the amino acid scaffold between Fab of a heavy chain of an antibody is insufficient, the distance of the X end is not expanded enough, and the sensitivity is not improved as much as 3-4 amino acids.

(3) Removing 50% of background buffer solution from the latex reagent obtained in the step (1), namely, replacing the buffer solution by 50%; adding the solution dissolved with the amino acid scaffold in the step (2) to ensure that each mg of antibody corresponds to 0.010-0.020mg of amino acid scaffold, and uniformly mixing; dropwise adding 50mmol/L potassium persulfate solution into the amino acid stent latex mixed reagent, wherein the molar weight of the potassium persulfate is 35-50% of the urea amount in the step (1);

(4) dropwise adding the latex reagent of the potassium persulfate solution in the step (3) into a constant temperature shaking table at 37 ℃, and carrying out gentle, uniform-rotation and incubation at 120rpm for 30 min; a disulfide bond is reformed, a space scaffold is added between two heavy chains of the antibody, and the space distance between two Fab of the antibody is increased; a scaffold is added between part of light chains and heavy chains, so that the sensitivity is not greatly influenced;

(5) and (4) sealing the latex reagent incubated in the step (4), changing the liquid, cleaning and removing residual amino acid bracket, urea and potassium persulfate to obtain a finished product reagent R2.

Preferably, the final concentration of urea in the latex solution in the step (1) is 20 mM/L.

Preferably, the scaffold lysis solution in step (2) is 100mM/L pH7.5, PBS buffer containing 0.1% V/V Tween-20.

Preferably, the dosage of the amino acid scaffold in the step (2) is as follows: 0.015mg of amino acid scaffold per mg of antibody.

Preferably, the acidic amino acid of step (2) is aspartic acid D and/or glutamic acid E.

Preferably, in step (2), X is DDD, EEE, DED, EDE, EDD, DDE, EED, DEE, DDDD, EDDD, DED, DDDE, EEDD, EEED, EEEE, DDEE, DEDE, DEEE, EDEE, EEDE, EDED, DEED or EDDE.

Preferably, the liquid exchange method in the step (3) is centrifugation, dialysis, chromatographic column or ultrafiltration liquid exchange.

The small-scale liquid exchange adopts centrifugation liquid exchange, and the large-scale liquid exchange adopts ultrafiltration and chromatographic columns.

Preferably, the molar amount of the potassium persulfate in the step (3) is 50% of the amount of the urea in the step (1).

Preferably, the 50mmol/L potassium persulfate solution in step (3) is obtained by dissolving 2.7032g of potassium persulfate in 200ml of a scaffold dissolving solution.

Preferably, the specific steps of the sealing and liquid-changing cleaning in the step (5) are as follows: centrifuging the latex reagent incubated in the step (4) at 22000rpm for 15min, removing supernatant, adding a proper amount of closed cleaning solution (the concentration of latex is usually 1-4g/L depending on the concentration of the finished product of different projects), redissolving, and uniformly mixing at room temperature for 2 hours to obtain the finished product reagent R2.

Preferably, the blocking wash is 100mM/L PBS buffer pH7.5, containing 0.1% V/V Tween-20, 1g/L BSA, 0.05% V/V preservative PC 300.

According to the technical scheme, compared with the prior art, the invention discloses a method for improving the detection sensitivity of a latex turbidimetric reagent, and the spatial distance between two Fab of an antibody is increased by adding an amino acid bracket between two heavy chains of the antibody coupled to the surface of carboxyl latex, so that the steric hindrance of the combination of the antibody and an antigen in the turbidimetric process of the latex is reduced, and the sensitivity is improved; the invention improves the reagent after the conventional carboxyl latex is coupled with the antibody, has mature amplification process and is beneficial to popularization.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

100ml of 200mM/L, 300mM/L, 400mM/L, 500mM/L aqueous urea solution was prepared and kept at room temperature.

Preparing a scaffold dissolving solution: 100mM/L pH7.5, PBS buffer containing 0.1% V/V Tween-20, at room temperature for use.

Preparing 50mM/L potassium persulfate solution: 2.7032g of potassium persulfate was dissolved in 200ml of the stent diluent and stored at 4 ℃ until use.

Preparing a closed cleaning solution: 100mM/L pH7.5PBS buffer solution, containing 0.1% V/V Tween-20, 1g/LBSA, 0.05% V/V preservative PC300, room temperature for use.

Polyclonal antibody reaction buffer reagent 1-Pab (R1-Pab): 100mM/L pH7.5PBS buffer solution, containing 0.5% V/V Tween-20, 0.05% V/V preservative PC300, final concentration of 350mM/L sodium chloride, final concentration of 10g/L polyethylene glycol 6000, final concentration of 2ml/L ScANTIBody company blocking agent HBR22, room temperature standby.

Monoclonal antibody reaction buffer reagent 1-Mab (R1-Mab): 100mM/L pH7.5PBS buffer solution, containing 0.5% V/V Tween-20, 0.05% V/V preservative PC300, final concentration of 150mM/L sodium chloride, final concentration of 15g/L polyethylene glycol 6000, final concentration of 5ml/L ScANTIBody company blocking agent HBR1, room temperature standby.

Example 1

Myoglobin MB project, imported rabbit polyclonal antibody, liquid change in a centrifugal mode, comparison with a control unmodified reagent and comparison of the influence of urea dosage

(1) Latex washing (no-wash JSR corporation latex may not go through this step): using 100mM/L MES-NaOH buffer solution, pH5.5, centrifuging to change the latex microspheres for 2 times; centrifuging 10ml of agilent PL6115138nm latex microspheres with the mass concentration of 10% at 22000rpm for 15min, removing supernatant, redissolving by using 10ml of the buffer solution, then centrifuging again, removing supernatant, redissolving, and finishing cleaning for later use;

(2) latex activation: taking 10ml of the washed latex as an example, 50mg of NHS (N-hydroxysuccinimide) and 30mg of EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) are weighed, dissolved by 5ml of the buffer solution for washing, 1ml of the solution is added into 10ml of the washed microspheres, and the mixture is magnetically stirred and uniformly mixed for 10min at room temperature;

(3) and (3) cleaning after activation: centrifuging the activated latex in the step (2) at 22000rpm for 15min, removing supernatant, and adding 20ml of 100mM/L PBS buffer solution with pH7.5 for re-dissolution for later use;

(4) antibody coupling: adding 2ml (12mg/ml) of myoglobin antibody with the product number of OA044 of Dako company into the activated latex solution redissolved by the PBS buffer solution, and magnetically stirring for 3 hours at room temperature to complete antibody coupling;

(5) modification of R2:

1) the latex after antibody coupling was divided into 5 parts on average, each 4ml (named as A/B/C/D/E group), and the excess was discarded (2ml antibody was added to 20ml PBS, the final volume was only slightly more than 20ml due to the different liquid mutual solution principle, and 22ml solution was not obtained);

2) the group A is a control group, the final concentration of urea in the group B is 10mM/L, the final concentration of urea in the group C is 15mM/L, the final concentration of urea in the group D is 20mM/L, and the final concentration of urea in the group E is 25 mM/L;

the method comprises the following specific operations: 200ul of placebo purified water is added into the group A, 200ul of 200mM/L, 300mM/L, 400mM/L and 500mM/L urea aqueous solution is respectively added into the group B/C/D/E, namely the group B/C/D/E corresponds to the final concentration of 10/15/20/25mM/L urea;

3) centrifuging the components A, B, C, D and E at 22000rpm for 15min, removing all supernatant from the component A, and removing 2ml supernatant from the component B/C/D/E, namely 'changing the liquid by 50%';

4) the mass of the antibody in each experiment was calculated to be about 2 × 12 × 4/20 mg to 4.8mg by dissolving 1mg of an amino acid scaffold (synthesized by bio-engineering (shanghai) ltd) with the sequence X as EDED in 20.8ml of a scaffold solution;

adding 2ml of solution dissolved with the amino acid scaffold into each group B/C/D/E, and redissolving to obtain 0.020mg of amino acid scaffold corresponding to 1mg of antibody in the amount of the antibody and the scaffold;

5) 0.4ml, 0.6ml, 0.8ml and 1.0ml of 50mM/L potassium persulfate solution, namely, the molar amount of potassium persulfate is about 50 percent of the amount of urea in the step 2), are respectively added into the B/C/D/E component.

6) B/C/D/E group 37-degree constant temperature shaking table, 120rpm gently rotates, mixes evenly and incubates for 30 min;

(6) sealing and cleaning:

adding 8ml of closed cleaning liquid into the group A for redissolution, centrifuging the group B/C/D/E at 22000rpm for 15min, removing all supernatant, adding 8ml of closed cleaning liquid for redissolution, and uniformly mixing at room temperature for 2 hours for later use to obtain a finished product R2.

(7) On-machine testing

The instrument comprises the following steps: hitachi 7180, computer parameters, sample size of 6ul and 180ul of the prepared R1-Pab, 60ul of R2 and 570/800nm double-wavelength, a two-point end point method and a method for calculating delta ABS (absorbance change value) by reading points from 18 to 34;

the MB calibrator for diagnosis of Pieron (S120 ng/ml, S2100 ng/ml, S3200 ng/ml, S4400 ng/ml) is used, physiological saline is used in zero point, and S1 is diluted to 5ng/ml, 7.5ng/ml, 10ng/ml and 15ng/ml together with S1 by using the physiological saline for the lowest detection limit test;

the test scheme is as follows: respectively matching the A/B/C/D/E groups with the same previously prepared R1-Pab, the same parameters and the same calibrators to calibrate, comparing each calibration point delta ABS, simultaneously respectively measuring 5ng/ml, 7.5ng/ml, 10ng/ml, 15ng/ml and S1 for 10 times respectively, respectively calculating the mean value, STDEV, and obtaining precision CV by using the STDEV/mean value; note that when the test is started with a high concentration, the accuracy is too poor, and a lower concentration is not measured, N/A.

The calibration data for each experimental group is as follows:

as can be seen from the calibration data, the sensitivity Δ ABS of the B/C/D/E group is improved to different degrees compared with the control group, wherein the improvement of the D group is most obvious, the improvement is about 148% compared with the control group, and the corresponding final urea concentration of the B/C/D/E group is 10/15/20/25 mM/L; the result shows that the sensitivity is improved and then is reduced along with the increase of the urea concentration, and the better urea concentration is 20 mM/L.

The detection limit experiment of each experimental group is as follows:

as can be seen from the results, the limit of detection LOD (sample concentration at CV 20%) was between 15 and 20ng/ml for control group A, estimated around 16 ng/ml; the group B is between 7.5 and 10ng/ml, and is estimated to be about 8.5ng/ml, the group C is also between 7.5 and 10ng/ml, but the precision of each concentration is obviously better than that of the group B, the LOD is estimated to be about 7.4ng/ml, the group D is optimal, the LOD is between 5 and 7.5ng/ml, and is estimated to be about 6ng/ml, the group E has LOD increase compared with the group B/C/D, but is better than that of the group A, the LOD is between 10 and 15ng/ml, and is estimated to be about 10 ng/ml; as can be seen from the results, the LOD of the superior group D is reduced to about 37.5% of that of the control group A, 53.1% of that of the control group B, 46.3% of that of the control group C and 62.5% of that of the control group D; the final concentration of urea in the B/C/D/E group was 10/15/20/25mM/L, and it can be seen that the LOD decreased first and then increased as the concentration of urea increased, with a preferred concentration of 20 mM/L.

The above results show that the improved reagent R2 is effective in improving the detection sensitivity of the reagent in the MB project at a urea concentration of 10-20 mM/L. But the urea cannot be excessive, otherwise, the antibody activity can be reduced, and the sensitivity improvement rate is reduced; the urea amount is too small, so that the breakage of the disulfide bond of the antibody is possibly insufficient, and the sensitivity is not improved remarkably enough; the preferred urea concentration is 20 mM/L.

Example 2

An alpha-fetoprotein AFP project, imported rabbit polyclonal antibody, liquid change in a centrifugal mode, comparison with a control unmodified reagent, and comparison of influence of the using amount of an amino acid scaffold

(1) Latex cleaning: using 100mM/L MES-NaOH buffer solution, pH5.5, centrifuging to change the latex microspheres for 2 times; 10ml of 10 mass percent Polymicrospheres CB0230D 230nm latex microspheres are taken, centrifuged for 10min at 22000rpm, the supernatant is removed, 10ml of the buffer solution is used for redissolving, then, the centrifugation is carried out, the supernatant is removed for redissolving, and the washing is finished for standby application;

(3) and (3) cleaning after activation: centrifuging the activated latex in the step (2) at 22000rpm for 10min, removing supernatant, and adding 36ml of 100mM/L PBS buffer solution with pH7.5 for re-dissolution for later use;

(4) antibody coupling: 15ml (2.5mg/ml) of alpha-fetoprotein antibody (product number A0008) from Dako was added to the activated latex solution reconstituted with PBS buffer, and magnetic stirring was carried out at room temperature for 3 hours to complete antibody coupling;

(5) modification of R2:

1) the latex after antibody coupling was divided into 5 aliquots, 10ml each (named group A2/B2/C2/D2/E2), and the excess was discarded (15ml of antibody was added to 36ml PBS, the final volume was only slightly more than 50ml due to the different liquid miscibility principle, and no 51ml solution was obtained).

2) The group A2 was a control group to which 400ul of placebo purified water was added, and the group B2/C2/D2/E2 each added 400ul of 500mM/L aqueous urea solution to a final urea concentration of about 20 mM/L;

3) centrifuging the components A2, B2, C2, D2 and E2 at 22000rpm for 10min, removing all supernatant from the component A2, and removing 5ml of supernatant from the component B2/C2/D2/E2, namely changing liquid by 50%;

4) the mass of the antibody in each experiment was calculated to be about 15 × 2.5 × 10/50 — 7.5mg by dissolving 1mg of an amino acid scaffold (synthesized by bio-engineering (shanghai) ltd) with the sequence X as DED in 20ml of scaffold solution;

0.75ml of the solution containing the scaffold and 4.25ml of the scaffold solution are added into the group B2, and the 1mg of the antibody in the group B2 can be calculated to correspond to about 0.005mg of the scaffold;

adding 1.5ml of the solution containing the scaffold into the C2 group and 3.5ml of the scaffold dissolving solution, and calculating that 1mg of the antibody in the C2 group approximately corresponds to 0.010mg of the scaffold;

2.25ml of the solution containing the scaffold and 2.75ml of the solution containing the scaffold are added into the D2 group, and the 1mg of antibody in the D2 group can be calculated to correspond to 0.015mg of the scaffold;

3.0ml of the solution containing the scaffold and 2.0ml of the scaffold solution are added into the group E2, and the 1mg of the antibody in the group E2 can be calculated to correspond to about 0.020mg of the scaffold;

5) 2.0ml of 50mM/L potassium persulfate solution is respectively added into the B2/C2/D2/E2 group, namely the molar weight of the potassium persulfate is about 50 percent of the urea in the step 2);

6) B2/C2/D2/E2 group 37 degree constant temperature shaking table, 120rpm gently rotate mixing incubation for 30 min.

(6) Sealing and cleaning:

adding 10ml of closed cleaning liquid into the group A2 for redissolution, centrifuging the group B2/C2/D2/E2 at 22000rpm, removing all supernatant, adding 10ml of closed cleaning liquid for redissolution, and uniformly mixing at room temperature for 2 hours for later use to obtain a finished product R2;

(7) on-machine testing

The instrument comprises the following steps: hitachi 7180, computer parameters, sample size of 6ul, 180ul, R1-Pab prepared in the previous step, 45ul R2, single wavelength of 700nm, a two-point end point method, and calculating delta ABS by reading points from 19 to 34;

using a Pieron diagnosis AFP calibrator (S110 ng/ml, S220 ng/ml, S363 ng/ml, S4315 ng/ml), using normal saline at zero point, and using the normal saline to dilute S1 to 1ng/ml, 2.5ng/ml, 5ng/ml, 7.5ng/ml together with S1 for the lowest detection limit test;

the test scheme is as follows: respectively matching the A2/B2/C2/D2/E2 groups with the same R1-Pab prepared in advance, respectively using the same parameters and the same calibrators to calibrate, comparing each calibration point delta ABS, simultaneously respectively measuring 1ng/ml, 2.5ng/ml, 5ng/ml and 7.5ng/ml together with S1, respectively 10 times, respectively calculating a mean value and STDEV, and respectively using the STDEV/mean value to obtain precision CV; note that the test starts with a high concentration, and if the accuracy is too poor, no lower concentration is measured.

The calibration data for each experimental group is as follows:

as can be seen from calibration data, the sensitivity Δ ABS of the B2/C2/D2/E2 groups is improved to different degrees compared with the control group, wherein the improvement of the D2 and E2 groups is most obvious, and is improved by about 150 percent compared with the control group, and the amount of the amino acid scaffold corresponding to 1mg of the antibody in the B2/C2/D2/E2 group is 0.005/0.010/0.015/0.020 mg; it can be seen that, with the increase of the amount of the scaffold, the sensitivity increases first and then becomes stable, and considering the cost of the scaffold, the amount of the preferred 1mg antibody corresponding to the scaffold is about 0.015 mg.

The detection limit experiment of each experimental group is as follows:

as can be seen from the results, the limit of detection LOD (sample concentration at CV 20%), the control A2 group was between 5 and 7.5ng/ml, estimated around 6.7 ng/ml; the B2 group was between 5-7.5ng/ml, but the precision of each concentration was significantly better than the A2 group, estimated around 5ng/ml, the C2 group was also between 2.5-5ng/ml, the LOD was estimated around 3.3ng/ml, the D2 and E2 groups were optimal, the LOD was between 1-2.5ng/ml, estimated around 2.4ng/ml, the superior B2 group, the LOD decreased to around 74.6% of the control A2 group, the C2 group decreased to 49.3% of the control group, and the D2 and E2 groups decreased to 35.8% of the control group. The 1mg corresponding amino acid scaffold of the B2/C2/D2/E2 group antibody is 0.005/0.010/0.015/0.020mg, and it can be seen that along with the increase of the scaffold concentration, the LOD tends to be stable after being reduced, the effects of 0.015 and 0.020mg are not greatly different, but the cost of the scaffold is higher, and considering the cost problem, the optimal amino acid scaffold corresponding to each mg of antibody is 0.015 mg.

The result shows that when the amount of the scaffold corresponding to each mg of the antibody is between 0.010 and 0.020mg, the detection sensitivity of the reagent is effectively improved by the improved scheme in the AFP project; considering the cost problem, the scaffold can not be infinitely improved, and the preferable amino acid scaffold per mg of antibody is 0.015 mg.

Example 3

Ferritin FER project, domestic rabbit polyclonal antibody, changing liquid by centrifugation, comparing with control unmodified reagent, and comparing influence of potassium persulfate concentration

(1) Latex washing (no-wash JSR corporation latex may not go through this step): using 100mM/L MES-NaOH buffer solution, pH5.5, centrifuging to change the latex microspheres for 2 times; centrifuging 10ml of 10% mass concentration Polymicrospheres CB0297C 297nm latex microspheres at 22000rpm for 6min, removing supernatant, redissolving by using 10ml of the buffer solution, then centrifuging again, removing supernatant, redissolving, and finishing cleaning for later use;

(3) and (3) cleaning after activation: centrifuging the activated latex of the step (2) at 22000rpm for 6min, removing supernatant, adding 46ml of 100mM/L PBS buffer solution with pH7.5 for re-dissolution for later use;

(4) antibody coupling: 5ml (13mg/ml) of ferritin antibody 301067 from ZenBio was added to the activated latex solution reconstituted with PBS buffer and magnetically stirred at room temperature for 3 hours to complete antibody coupling;

(5) modification of R2:

1) the latex after antibody coupling was divided into 5 aliquots in 10ml each (named group A3/B3/C3/D3/E3), and the excess was discarded (5ml of antibody was added to 46ml of PBS, the final volume was only slightly more than 50ml due to the different liquid mutual solution principle, and no 51ml solution was obtained);

2) the group A3 was a control group to which 400ul of placebo purified water was added, and the group B3/C3/D3/E3 each added 400ul of 500mM/L aqueous urea solution to a final urea concentration of about 20 mM/L;

3) centrifuging the components A3, B3, C3, D3 and E3 at 22000rpm for 10min, removing all supernatant from the component A3, and removing 5ml of supernatant from the component B3/C3/D3/E3, namely changing liquid by 50%;

4) the mass of the antibody in each experiment was calculated to be about 5 × 13 × 10/50 — 13mg by dissolving 1mg of an amino acid scaffold (synthesized by bio-engineering (shanghai) ltd) with the sequence of X as EDE in 20ml of a scaffold solution;

the B3/C3/D3/E3 components are respectively added into 3.9ml of scaffold dissolving solution dissolved with the scaffold, and the calculated result shows that the scaffold corresponding to each mg of antibody is 0.015 mg;

5) b3 set was added 1.0ml of a 50mM/L potassium persulfate solution, i.e., potassium persulfate in an amount of about 25% by mol based on the amount of urea in step 4.2;

c3 group was added 1.4ml of a 50mM/L potassium persulfate solution, i.e., the molar amount of potassium persulfate was about 35% of the amount of urea in step 4.2;

d3 set was added 1.6ml of a 50mM/L potassium persulfate solution, i.e., the molar amount of potassium persulfate was about 40% of the amount of urea in step 4.2;

e3 group was added 2.0ml of a 50mM/L potassium persulfate solution, i.e., the molar amount of potassium persulfate was about 50% of the amount of urea in step 4.2;

6) B3/C3/D3/E3 group 37 degree constant temperature shaking table, 120rpm gently rotate to mix evenly and incubate for 30 min;

(6) sealing and cleaning:

adding 12ml of closed cleaning solution into the group A3 for redissolution, centrifuging the group B3/C3/D3/E3 at 22000rpm, removing all supernatant, adding 12ml of closed cleaning solution for redissolution, and uniformly mixing at room temperature for 2 hours for later use to obtain a finished product R2.

(7) On-machine testing

The instrument comprises the following steps: hitachi 7180, computer parameters, sample size of 10ul, 120ul R1, 60ul R2, single wavelength of 570nm, a two-point end point method, and delta ABS calculation by reading points 18-34;

using boor diagnosis FER calibrator (S120 ng/ml, S2100 ng/ml, S3200 ng/ml, S4500 ng/ml), using normal saline at zero point, diluting S1 to 5ng/ml, 7.5ng/ml, 10ng/ml, 15ng/ml together with S1 using normal saline, and using for lowest detection limit test;

the test scheme is as follows: respectively matching the A3/B3/C3/D3/E3 groups with the same R1-Pab prepared in advance, respectively matching the same parameters and the same calibrators, calibrating the same calibration points, comparing the calibration points delta ABS, simultaneously respectively measuring 5ng/ml, 7.5ng/ml, 10ng/ml, 15ng/ml and S1 for 10 times, respectively calculating the mean value and STDEV, and respectively obtaining the precision CV by using the STDEV/mean value; note that the test starts with a high concentration, and if the accuracy is too poor, no lower concentration is measured.

The calibration data for each experimental group is as follows:

as a result, it can be seen that FER reagent sensitivity Δ ABS was significantly increased with the increase in potassium persulfate concentration compared to the control a3 group, where the sensitivity was highest at a potassium persulfate concentration of 50% relative to the urea concentration, i.e., the E3 group, but the comparison of E3 and D3 found that the increase in sensitivity was already gradual, although the urea concentration increased from 40% to 50% from D3 to E3. And the potassium persulfate concentration is too high, which has adverse effects on reagent stability and non-specific reaction. The preferred potassium persulfate addition is about 50% of the urea concentration from the previous step.

The following is the minimum detection limit experimental data for example 3:

as can be seen from the results, the limit of detection LOD (sample concentration at CV 20%) was between 10 and 15ng/ml in the control A3 group, estimated around 15 ng/ml; the B3 group is 10-15ng/ml, but the precision of each concentration is obviously better than that of the A3 group, the estimated concentration is about 10ng/ml, the C3 group is also 7.5-10ng/ml, the estimated LOD is about 7.5ng/ml, the D3 group is 7.5-5ng/ml, the estimated LOD is about 6.5ng/ml, the preferred D3 group is 7.5-5ng/ml, the estimated LOD is about 6.1ng/ml, the LOD of the B3 group is reduced to about 66.7% of the control A2 group, the C3 group is reduced to 50% of the control group, and the D3 and E3 groups are reduced to 43.3% and 40.7% of the control group. The concentrations of potassium persulfate corresponding to B3/C3/D3/E3 were 25%, 35%, 40% and 50% of the urea concentration in the preceding step, respectively, and it was observed that LOD gradually decreased as the concentration of potassium persulfate increased, but the trend was gradually decreased after 40%.

The results show that in the FER project, the detection sensitivity of the reagent is effectively improved by the improved scheme along with the increase of the concentration of the potassium persulfate. And the potassium persulfate concentration is too high, which has adverse effects on reagent stability and non-specific reaction. The preferable concentration of potassium persulfate is 50% of the concentration of urea in the previous step.

Example 4

D Dimer D-Dimer project, namely, importing mouse monoclonal antibody, changing liquid in a centrifugal mode, comparing with a control unmodified reagent, and simultaneously comparing the influence of X amino acid quantity difference;

(1) latex washing (no-wash JSR corporation latex may not go through this step): using 100mM/L MES-NaOH buffer solution, pH5.5, centrifuging to change the latex microspheres for 2 times; taking 10ml of agilent PL6115138nm latex microspheres with the mass concentration of 10%, centrifuging at 22000rpm for 15min, removing supernatant, redissolving by using 10ml of the buffer solution, then centrifuging again, removing supernatant, redissolving, and finishing cleaning for later use;

(2) latex activation: taking 10ml of the washed latex as an example, 50mg of NHS (N-hydroxysuccinimide) and 30mg of EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) are weighed, dissolved by 5ml of the buffer for washing, 1ml of the solution is added into 10ml of the washed microspheres, and the mixture is magnetically stirred and uniformly mixed for 10min at room temperature;

(3) and (3) cleaning after activation: centrifuging the activated latex in the step (2) at 22000rpm for 15min, removing supernatant, adding 37ml of 100mM/L PBS buffer solution with pH7.5 for redissolving for later use;

(4) antibody coupling: taking 2ml of each D dimer mouse monoclonal antibody (5 mg/ml) with the product numbers DD41 and DD02 of Hytest company, adding the D dimer mouse monoclonal antibodies into the activated latex solution redissolved by the PBS buffer solution, and magnetically stirring for 3 hours at room temperature to complete antibody coupling;

(5) modification of R2:

1) the latex subjected to antibody coupling was divided into 5 portions on average, each of which was 8ml (designated as A4/B4/C4/D4/E4), and the excess was discarded.

2) The A4 group is a control group, the B4/C4/D4/E4 group has 20mM/L of final concentration of urea, the A group is added with 335ul of placebo purified water, and the B4/C4/D4/E4 group is respectively added with 500mM/L of urea aqueous solution 335ul, namely the B4/C4/D4/E4 group has 20mM/L of corresponding final concentration of urea;

3) centrifuging the components A4, B4, C4, D4 and 4E at 22000rpm for 15min, removing all supernatant from the component A4, and removing 4ml supernatant from the component B4/C4/D4/E4, namely changing 50% ";

4) the mass of the antibody in each experiment was calculated to be about (5 × 2+5 × 2) × 8/40-4 mg by dissolving 1mg each of the amino acid scaffolds (synthesized by bio-engineering (shanghai) corporation) with the X sequences EE, EEE, EEEE and eeeeee in 25ml of scaffold solution;

adding 2ml of corresponding scaffold-dissolved solution into each of groups B4/C4/D4/E4, wherein the group B4 is an EE scaffold, the group C4 is an EEE scaffold, the group D4 is an EEEE scaffold, the group E4 is an EEEEEEEEE scaffold, and redissolving, wherein the ratio of the antibody to the scaffold is calculated to be 1mg and corresponds to 0.020mg of amino acid scaffold;

5) adding 1.6ml of 50mM/L potassium persulfate solution into the B4/C4/D4/E4 groups respectively, wherein the mol amount of the potassium persulfate is about 50 percent of the amount of the urea in the step 4.2;

6) B4/C4/D4/E4 group 37 degree constant temperature shaking table, 120rpm gently rotate to mix evenly and incubate for 30 min;

(6) sealing and cleaning:

adding 10ml of closed cleaning solution into the group A4 for redissolution, centrifuging the group B4/C4/D4/E4 at 22000rpm for 15min, removing all supernatant, adding 10ml of closed cleaning solution for redissolution, and uniformly mixing at room temperature for 2 hours for later use to obtain a finished product R2.

(7) On-machine testing

The instrument comprises the following steps: hitachi 7180, computer parameters, sample size of 4ul, 180ul, R1-Mab prepared in the previous step, 60ul R2, 700nm single wavelength, two-point end point method, and delta ABS calculation by 20-34 reading points;

the diagnostic D-Dimer calibrator (S10.500mg/L, S22.000mg/L, S38.000mg/L and S416.000mg/L) for Berry is used, normal saline is used in zero point, S2 is diluted to 1.000mg/L with normal saline, S1 is diluted to 0.100mg/L and 0.250mg/L with normal saline, and S1 and S2 are used for the lowest detection limit test;

the test scheme is as follows: respectively matching the A4/B4/C4/D4/E4 groups with the same R1-Mab prepared in advance, calibrating the same calibration substances with the same parameters and the same calibrators, comparing the calibration points delta ABS, simultaneously respectively measuring 0.100mg/L, 0.250mg/L, 0.500mg/L, 1.000mg/L and 2.000mg/L, respectively calculating the mean value and STDEV, and respectively obtaining the precision CV by using the STDEV/mean value; note that the test starts with a high concentration, and if the accuracy is too poor, no lower concentration is measured.

The calibration data for each experimental group is as follows:

the results show that compared with the control A4 group, the four groups of increased scaffolds have significantly improved sensitivity, the number of acidic amino acids represented by X in the scaffold is increased from 2 to 4, the sensitivity is gradually improved, the difference between 3 and 4 is small, and the sensitivity of the control A4 group is improved by about 145 percent; when the number of amino acids was increased to 5, a large reduction in sensitivity occurred compared to 3 and 4 amino acids; the number of acidic amino acids in the amino acid scaffold X is preferably 3 and 4.

The following is the lowest detection limit data for example 4:

as can be seen from the data, the LOD of the control group A4 was between 0.500-1.000mg/L, estimated at around 0.550 mg/L; group B4, LOD between 0.250-0.500mg/L, estimated around 0.375 mg/L; group C4, LOD between 0.100-0.250, about 0.240 mg/L; the LOD of the D4 group is also between 0.100 and 0.250, but each concentration CV is slightly better than that of the C4 group, and the LOD is about 0.195 mg/L; group E4, LOD between 0.250-0.500mg/L, estimated at about 0.41 mg/L; the LOD of the C4 and D4 groups decreased to 43.6% and 35.4%, respectively, relative to the control group.

The above results indicate that the number of acidic amino acids in X in the amino acid scaffold is preferably 3 to 4.

According to the invention, an amino acid scaffold is added between two heavy chains of an antibody to modify a reagent R2, and the modified reagent R2 is matched with a conventional reagent R1 (the R1-Pab or R1-Mab) for use, so that the sensitivity of latex immunoturbidimetry is improved.

The invention mainly aims at the conventional double-chain monospecific antibody raw materials, and is a general technique of latex turbidimetric reagents using the raw materials. The invention is not suitable for single-chain antibodies, physical adsorption methods to sensitive latex, and unconjugated antibodies, as the breakage of disulfide bonds may cause the unconjugated antibodies to fall apart and not to recover in situ.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for improving the detection sensitivity of a latex turbidimetric reagent is characterized by comprising the following specific steps:

(1) covalently coupling the double-chain monospecific antibody with carboxyl latex, and adding urea into the latex solution before sealing to ensure that the final concentration of the urea in the latex solution reaches 10-20 mM/L; carrying out gentle, uniform-rotating and incubation for 1 hour at a constant temperature of 37 ℃ and 120rpm to obtain a latex reagent;

(3) removing 50% of background buffer solution from the latex reagent obtained in the step (1), namely, changing the solution by 50%, adding the solution dissolved with the amino acid scaffold in the step (2), and uniformly mixing 0.010-0.020mg of the amino acid scaffold corresponding to each mg of the antibody; dropwise adding 50mmol/L potassium persulfate solution into the latex reagent mixed with the amino acid stent, wherein the molar weight of the potassium persulfate is 35-50% of the amount of the urea in the step (1);

(4) dropwise adding the latex reagent of the potassium persulfate solution in the step (3) into a constant temperature shaking table at 37 ℃, and carrying out gentle, uniform-rotation and incubation at 120rpm for 30 min;

2. The method of claim 1, wherein the final concentration of urea in the latex solution of step (1) is 20 mM/L.

3. The method of claim 1, wherein the scaffold lysis solution in step (2) is 100mM/L PBS buffer solution with pH7.5 and Tween-20 of 0.1% V/V.

4. The method for improving the detection sensitivity of the latex turbidimetric reagent according to claim 1, wherein the amino acid scaffold in the step (2) is used in an amount of: 0.015mg of amino acid scaffold per mg of antibody.

5. The method of claim 1, wherein the acidic amino acid in step (2) is aspartic acid D and/or glutamic acid E.

6. The method of claim 5, wherein X in step (2) is DDD, EEE, DED, EDE, EDD, DDE, EED, DEE, DDDD, EDDD, DDED, DDDE, EEDD, EEED, EEEE, DDEE, DEEE, EDEE, EEDE, EDED, DEED or EDDE.

7. The method of claim 1, wherein the fluid exchange method in step (3) is centrifugation, dialysis, chromatography column or ultrafiltration.

8. The method of claim 1, wherein the molar amount of potassium persulfate in step (3) is 50% of the amount of urea in step (1).

9. The method for improving the detection sensitivity of latex turbidimetric reagents according to claim 1, wherein said 50mmol/L potassium persulfate solution of step (3) is obtained by dissolving 2.7032g of potassium persulfate in 200ml of a scaffold dissolving solution.

10. The method for improving the detection sensitivity of the latex turbidimetric reagent according to claim 1, wherein the specific steps of blocking, replacing and washing in the step (5) are as follows: and (4) centrifuging the latex reagent incubated in the step (4) at 22000rpm for 15min, removing the supernatant, adding a closed cleaning solution for redissolving to enable the final concentration of the latex to be 1-4g/L, and uniformly mixing at room temperature for 2 hours to obtain a finished product reagent R2.