Application of polyethyleneimine biological sealing agent in IVD detection reagent

Abstract

The invention discloses an application of a polyethyleneimine biological sealing agent in an IVD detection reagent. The applied polymer is of a block structure, a main chain segment is polyethyleneimine, polyether is used for modification, and the sealing requirements of different systems are met. Polyamine reactive groups (-NH2 and-NH-) are provided on the basis of PEI, activated carboxyl, which is not coupled with an antibody, on the solid-phase surface is effectively combined, and PEI can also provide positive charges to adjust the charge attribute of the solid-phase surface. PEI is modified with a polyether segment, so that a hydrophilic PEG chain is introduced to the solid-phase surface, electrostatic interaction is shielded, surface hydrophilicity is enhanced, and non-specific adsorption with impure protein can be inhibited; an amphiphilic PPG-PEG chain segment can also be introduced into the solid phase surface, so that the interaction between the PEI segment and a hydrophobic region on the solid phase surface to be sealed is enhanced, and the sealing efficiency is improved. The polyether-modified sealing agent taking PEI as a main chain can obtain a stable and excellent sealing effect on the surfaces of latex, magnetic beads, 96-well plates and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of in vitro diagnosis, and more particularly to the application of a polyethyleneimine biological sealant in an IVD detection reagent. Background Art

[0002] In the field of in vitro diagnostics (IVD), the characteristics and highlights of reagents mainly revolve around reducing nonspecific binding and improving detection sensitivity and specificity. For example: blocking nonspecific binding sites, that is, covering the surface of the solid phase carrier (such as microplates, nanoparticles, nitrocellulose membranes) with blocking agents to fill unbound sites and prevent nonspecific adsorption of interfering substances (such as miscellaneous proteins, lipids) or detection reagents (such as antibodies, probes) in the sample; stabilizing the solid phase surface, that is, preventing the aggregation or denaturation of reagents caused by hydrophobic or charge effects on the carrier surface; reducing background signals, reducing false positives or high background caused by non-target molecules, and improving the signal-to-noise ratio. Therefore, substances with moderate molecular weight, good stability, strong binding ability, low immunogenicity, and steric hindrance effects (proteins, synthetic polymers, small molecule compounds, inert biological molecules, etc.) are often needed to play the role of blocking agents and stabilizers in the entire reagent.

[0003] Currently, the most commonly used blocking and stabilizing agent is bovine serum albumin (BSA). Its disadvantages include: 1. Risk of animal origin: cross-translation; 2. Batch testing: heterogeneity due to natural sources; 3. Background interference and signal suppression; and low coverage on highly hydrophobic and positively charged surfaces. Summary of the Invention

[0004] In view of this, the present invention provides an application of a polyethyleneimine biological sealant in an IVD detection reagent.

[0005] The present invention uses a block polymer as a blocking agent, with a main chain segment of polyethyleneimine (PEI) modified with a polyether segment, which is suitable for the blocking requirements of different systems. Based on the polyamine reactive groups (-NH2, -NH-) provided by PEI, it effectively binds to the activated carboxyl groups of uncoupled antibodies on the solid surface. PEI can also provide positive charges to adjust the charge properties of the solid surface. At the same time, through the modification of the polyether segment on PEI, (1) a hydrophilic PEG chain can be introduced into the blocking agent, which can, on the one hand, shield the electrostatic interaction of the solid surface, enhance its surface hydrophilicity, weaken the self-coagulation phenomenon, and on the other hand, inhibit the nonspecific adsorption of foreign proteins; (2) an amphiphilic PPG-PEG segment can also be introduced into the blocking agent to enhance the interaction between the PEI segment and the hydrophobic region of the solid surface to be blocked, thereby improving the blocking effect on surfaces such as latex, magnetic beads, and 96-well plates. In summary, with PEI as the main chain, through polyether modification, the coupling efficiency, charge properties, hydrophilicity, and hydrophobicity of the solid surface are optimized, so that the product can obtain a stable and excellent blocking effect.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A use of a polyethyleneimine bioblocking agent in an IVD detection reagent, wherein the polyethyleneimine polymer is selected from one or more of the following structures:

[0008] Structure 1: The main chain is linear PEI, modified with a hydrophilic chain PEG, for example:

[0009]

[0010] Structure 2: The main chain is linear PEI, modified with an amphiphilic polyether chain PPG-PEG, for example:

[0011]

[0012] Structure 3: The main chain is branched PEI, modified with a hydrophilic chain PEG, for example:

[0013]

[0014] Structure 4: The main chain is branched PEI, modified with an amphiphilic polyether chain PPG-PEG, for example:

[0015]

[0016] Preferably, the molecular weight of the polyether-modified polyethyleneimine block polymer is 1000-50000 Da.

[0017] It can be seen from the above technical solutions that compared with the prior art, the present invention has the following beneficial effects:

[0018] 1. No animal protein, virus, or biological contamination; no batch-to-batch differences.

[0019] 2. Optimized nonlinear structure, higher binding efficiency than similar products, can replace composite sealers.

[0020] 3. Small molecular weight (customizable), electrically neutral after binding, and will not affect the immune response due to charge.

[0021] 4. It is beneficial to the dispersion and stability of microsphere particles. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0023] Figure 1 This is a line graph of the thermal stability change of structure three under the SAA project.

[0024] Figure 2 This is the clinical sample correlation diagram of structure three under the fluorescent immunochromatography project. DETAILED DESCRIPTION

[0025] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0026] The reagents used in the examples of the present invention are as follows:

[0027] SA magnetic beads (Suzhou Pujia), carboxyl fluorescent microspheres (thermo)

[0028] Test strip components:

[0029] Sample pad: glass fiber (pre-treated with a buffer solution containing Triton X-100 + BSA).

[0030] Conjugate pad: detection antibody (anti-PCT monoclonal antibody MAB2) labeled with fluorescent microspheres (Eu microspheres / FITC quantum dots).

[0031] Nitrocellulose membrane (NC membrane):

[0032] Detection line (T line): spray anti-PCT monoclonal antibody MAB1 (capture antibody, 1 mg / mL).

[0033] Quality control line (line C): spraying of anti-mouse IgG secondary antibody (to verify the chromatography process).

[0034] Absorbent pad: absorbent paper.

[0035] Auxiliary reagents:

[0036] Diluent: PBS (pH 7.4) containing 0.5% BSA and 0.1% Tween-20.

[0037] Buffer1: HEPES (0.1M, PH 7.0).

[0038] Buffer2: TRIS-HCl (0.5M, pH 8.0).

[0039] Calibrator: recombinant human PCT (0, 0.1, 0.5, 2, 10, 50 ng / mL).

[0040] Example 1: Application of polyacetimide blocking agent in latex turbidimetry

[0041] 1.1 Experimental steps

[0042] 1. Latex particle activation:

[0043] Take a 5mL centrifuge tube and add the following components: (This experiment is a 1.5mL system, and the amount of microspheres is 1%)

[0044] a. Add the following components to the centrifuge tube:

[0045] Water, ml 0.177

[0046] Buffer, ml 0.600

[0047] Latex particles, ml 0.150

[0048] b. Prepare EDC immediately (within 30 minutes before use) and quickly add the calculated volume (carboxyl:EDC = 1:5, 287ul) to the reaction system and mix repeatedly with a pipette

[0049] 2. Coupling:

[0050] After the reaction is complete, immediately add the antibody (100 μg / mL) and mix on a mixer or other device in a constant temperature incubator or shaker for 8 hours.

[0051] 3. Closure:

[0052] Add the same volume of Structures 1 to 5 to the reaction solution and mix for 0.5 h as in step 2.

[0053] Example 2: Application of polyacetimide derivative blocking agents in chemiluminescence

[0054] 2.1: Solid phase coating

[0055] Biotinylated PCT antibody (Hangzhou Yibaixin) (1 mg / mL) was mixed with streptavidin magnetic beads (1 mg / mL) at a volume ratio of 1:100 and incubated with shaking at 37°C for 2 hours. The supernatant was discarded after magnetic separation and the cells were washed three times with washing buffer.

[0056] Coating time (1-4 hours) and temperature (25°C vs 37°C).

[0057] 2.2: Closed

[0058] The coated magnetic beads were resuspended in blocking solution (1 mL / mg magnetic beads) and incubated at 37°C for 1 hour.

[0059] After magnetic separation, the samples were stored in a 0.1% ProClin TM300% PBS (set aside at 4°C).

[0060] Blocking agent selection (BSA, PPG-PEG-PEI (molecular weight of 1500, 5000, 25000)).

[0061] Blocking time (30 minutes to overnight).

[0062] Example 3: Application of polyacetimide derivative blocking agent in fluorescence immunochromatography

[0063] Activation of fluorescent microspheres:

[0064] Carboxylated Eu microspheres (200 nm) were taken and the surface carboxyl groups were activated by EDC / NHS method. The unreacted reagents were removed by centrifugation (10,000×g, 10 min).

[0065] Antibody conjugation:

[0066] Anti-PCT MAB2 (0.1 mg / mL) was mixed with the activated microspheres (pH 6.0, room temperature for 2 h).

[0067] BSA was added to block unbound sites (final concentration 1%), purified by centrifugation, and resuspended in storage buffer (containing 0.1% NaN3).

[0068] Step 2: Test strip assembly

[0069] NC membrane treatment:

[0070] T-line spray coating: MAB1 (1 mg / mL) was sprayed onto the NC membrane at a rate of 1 μL / cm.

[0071] C line spraying: anti-mouse IgG (0.5 mg / mL) was sprayed in parallel.

[0072] Drying: Dry at 37°C for 2 hours and store in a sealed container (humidity < 30%).

[0073] Conjugate Pad Treatment:

[0074] A solution of fluorescently labeled MAB2 microspheres (0.5% solid content) was sprayed onto glass fibers, dried at 37° C., and then cut into 5 mm wide mats.

[0075] Test strip assembly:

[0076] The sample pad, conjugate pad, NC membrane, and absorption pad were stacked in sequence, adhered to a PVC base plate, and cut into 4 mm wide test strips.

[0077] Effect experiment

[0078] 1. Latex turbidimetric data analysis:

[0079] 1.1 Sensitivity and high value accuracy test:

[0080] Three samples, one negative, one low-value positive, and one high-value positive, were tested on a Hitachi 7180. A gradient was observed in all modified PEI samples, with Structure 3 showing the highest sensitivity. Thermal stability testing of the four samples at 50°C revealed a deviation of less than 10%, meeting stability requirements.

[0081]

[0082] 1.2 Thermal stability test:

[0083]

[0084] 2. Chemiluminescence data analysis:

[0085] On the Yingkai i2000 luminometer, two groups of reagents, strong negative and strong positive, were taken and blocked with structures 4 of different molecular weights. The absorbance and CV of the strong positive were better than those of traditional BSA, and 5000 was the optimal value under comprehensive evaluation.

[0086]

[0087]

[0088] 3. Fluorescence immunochromatography data analysis:

[0089] S0-S9 CTNI standards were used for detection on the Wondfo fluorescence analyzer, and buffer 1 was significantly better than buffer 2 as a diluent.

[0090] Twenty clinical samples (Beckmann's value) were collected for correlation analysis with the buffer1 group, with K>1 and r2>0.999.

[0091] 3.1 Sensitivity analysis

[0092]

[0093]

[0094] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. Reference can be made to the common and similar parts between the various embodiments. For the devices disclosed in the embodiments, since they correspond to the methods disclosed in the embodiments, the description is relatively simple, and the relevant parts can be referred to the method description.

[0095] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Application of a polyethyleneimine biological sealant in IVD detection reagents, characterized in that: The polyethyleneimines are selected from one or more of the following structures: Structure 1: The main chain is linear PEI, modified with a hydrophilic polyether chain PEG; Structure 2: The main chain is linear PEI, modified with amphiphilic polyether chain PPG-PEG; Structure 3: The main chain is branched PEI, modified with a hydrophilic polyether chain PEG; Structure 4: The main chain is branched PEI, modified with amphiphilic polyether chain PPG-PEG.

2. The use of a polyethyleneimine biological sealant in IVD detection reagents according to claim 1, characterized in that: The structure 1 is:

3. The use of a polyethyleneimine biological sealant in IVD detection reagents according to claim 1, characterized in that: The structure 2 is:

4. The use of a polyethyleneimine biological sealant in IVD detection reagents according to claim 1, characterized in that: The structure 3 is:

5. The use of a polyethyleneimine biological sealant in IVD detection reagents according to claim 1, characterized in that: The structure 4 is:

6. Use of a polyethyleneimine biological sealant according to any one of claims 1 to 5 in IVD detection reagents, characterized in that: The molecular weight of the polyethyleneimine polymer is 1000-50000 Da.

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