A method for enhancing single molecule immunoassay signal

Abstract

The application discloses a method for enhancing single-molecule immunodetection signal, and steps are as follows: 1) 100 muL magnetic beads (3 mg) are washed with 25 mM MES (pH 5.0) twice; 50 mg / mL of EDC and NHS are prepared by using 25 mM MES (pH 5.0, pre-cooled), 50 muL of each is added into the washed magnetic beads, and mixing is carried out at room temperature for 30 min; 25 mM MES (pH 5.0) is used for washing twice; and the magnetic beads are re-dissolved into 100 muL; 12-60 muL of capture antibody, 30-3000 pmol of poly-L-tyrosine, 30-3000 pmol of poly-L-tryptophan and 30-3000 pmol of poly-L-phenylalanine are added; mixing is carried out at room temperature for at least 30 min or at 4 DEG C for 2 hours; PBS buffer is used for washing twice; the magnetic beads are re-dissolved into 200 muL of 25 mM Tris (pH 7.2); mixing is carried out at room temperature for 30 min; the magnetic beads are washed for 3 times; and the magnetic beads are re-dissolved into 100 muL of PBS buffer; 2) immunoreaction; 3) enzyme catalysis reaction; and 4) data processing. The application can increase the group of tyramide reagent catalytic precipitation reaction in the tyramine signal amplification method by cross-linking polyaromatic amino acids on the magnetic beads, and finally the sensitivity of single-molecule immunodetection can be increased.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a method for enhancing single-molecule immunodetection signal. BACKGROUND

[0002] Clinical use of protein biomarkers to distinguish between healthy and disease states, and to monitor disease progression, requires the measurement of low concentrations of proteins in complex samples. Current immunoassay methods are generally capable of measuring proteins at concentrations above 10 -12 M, i.e. at a sensitivity of the picomolar level.

[0003] However, in cancer, neurological diseases and early infections, the serum concentrations of most important proteins are considered to be between 10 -16 and 10 -12 M. This places higher demands on the sensitivity of protein immunoassay methods.

[0004] To solve this problem, different solutions have been proposed. The most important of these is the SiMoA (single-molecule arrays) method reported by Rissin et al. in 2010, which is theoretically capable of detecting a single molecule. The theory, practice and algorithms based on this method have become the de facto "gold standard" for single-molecule immunoassay.

[0005] The SiMoA method requires loading a single magnetic bead into a microwell with a volume of 50 femtolitres, and generating a fluorescent signal by enzymatic catalysis. The presence / absence of a fluorescent signal in the microwell is marked as 1 / 0, which serves as the basis for subsequent digital algorithm calculation and analysis. This method has high requirements for the chip carrier and low magnetic bead loading efficiency, making it difficult to meet the needs of high-throughput clinical detection.

[0006] In addition to the signal amplification method of generating a fluorescent signal by enzymatic catalysis in a microwell, there are two common signal amplification paths that can achieve single-molecule immunodetection: one is to directly cross-link fluorescent microspheres with detection antibodies, and generate a fluorescent signal by the large amount of fluorescent substances contained in the fluorescent microspheres; the other is to use tyramide signal amplification (TSA) to catalyze a large amount of tyramide-fluorescent substances to deposit on the magnetic beads, thereby detecting the fluorescent signal.

[0007] The first path requires two different particle sizes of magnetic beads and fluorescent microspheres to be combined by a single antigen-antibody to achieve, and has the problems of low binding efficiency and easy dissociation during the washing process, resulting in reduced sensitivity. In the second path, the aromatic amino acid content of the protein on the magnetic bead is limited, which is the main factor restricting the catalytic deposition of tyramide-fluorescent substances. SUMMARY

[0008] To address the aforementioned technical deficiencies, this invention discloses a method for enhancing single-molecule immunoassay signals. This method involves crosslinking polyaromatic amino acids onto magnetic beads to increase the groups involved in the tyramine reagent-catalyzed precipitation reaction in the tyramine signal amplification (TSA) method, thereby ultimately increasing the sensitivity of single-molecule immunoassay.

[0009] This invention is implemented as follows:

[0010] A method for enhancing single-molecule immune detection signals, characterized in that the method comprises the following steps:

[0011] Step 1: Coating the magnetic beads; specifically:

[0012] 1.1 Cleaning: Remove the supernatant from 100μL magnetic beads (3mg) and wash twice with 25mM MES (pH5.0);

[0013] 1.2 Activation: Prepare 50 mg / mL EDC and NHS using 25 mM MES (pH 5.0, pre-cooled). Add 50 μL of each to the cleaned magnetic beads and mix at room temperature for 30 min.

[0014] Wash twice (pH 5.0) and reconstitute to 100 μL;

[0015] 1.3 Antibody Coating: Add 12-60 μg of capture antibody and polyaromatic amino acids, mix at room temperature for at least 30 min or at 4°C for 2 h, and wash twice with PBS buffer; the polyaromatic amino acids are 30-3000 pmol poly-L-tyrosine and 30-3000 pmol poly-L-tryptophan.

[0016] 30-3000 pmol poly-L-phenylalanine;

[0017] 1.4 Blocking: Redissolve in 200 μL 25 mM Tris (pH 7.2), mix at room temperature for 30 min, wash 3 times, and redissolve in 100 μL PBS buffer to obtain coated magnetic beads.

[0018] In the magnetic bead coating process of this invention, the capture antibody added per 1 mg of magnetic bead is 4-20 μg, poly-L-tyrosine is 10-1000 pmol, poly-L-tryptophan is 10-1000 pmol, and poly-L-phenylalanine is 10-1000 pmol.

[0019] Step Two: Immune Response; Step Two specifically includes:

[0020] 2.1 Add 10 μL of 0.3 mg / mL magnetic beads coated with polyaromatic amino acids to the reaction tube, add 100 μL of antigen (the antigen corresponding to the capture antibody in step 1.3), and mix well;

[0021] 2.2 Add 10 μL of 0.3 μg / mL biotin-labeled detection antibody to the reaction tube, mix at room temperature for 1 hour, wash 3 times with 200 μL PBS buffer, and resuspend in 100 μL PBS buffer.

[0022] 2.3. Add 10 μL of 0.5 μg / mL SA-poly-HRP working solution to the reaction tube, mix well at room temperature for 30 min, wash three times with 200 μL PBS buffer, and discard the supernatant.

[0023] Step 3: Enzyme-catalyzed reaction; Step 3 specifically includes:

[0024] 3.1 Add 100 μL of tyramide working solution to the magnetic beads after removing the washing solution, and incubate at room temperature for 1 hour;

[0025] 3.2 Add 50 μL of stop solution to the reaction tube, mix at room temperature for 2 min, wash 6 times with 200 μL of PBS buffer, and resuspend in 100 μL of PBS buffer before testing.

[0026] Step 4: Data Processing; Step 4 specifically involves: Administering the sample to be tested into a flow cytometer, acquiring signals from the FSC, SSC, and FITC channels, performing gating statistics after the test, and processing the data as follows:

[0027] 4.1 When the percentage of active microspheres is below 70%, the numerical model AEB (average enzyme per bead) is calculated using microsphere counting and Poisson statistics. AEB is calculated using the following formula:

[0028] μ=AEB digital = -ln[1-f on ]

[0029] Where digital represents a number, f on This indicates the percentage of active microspheres.

[0030] 4.2 When the percentage of active microspheres is above 70%, the Poisson curve deviates from linearity. The simulated AEB is calculated by measuring the average fluorescence intensity of the microspheres using the following two formulas:

[0031]

[0032]

[0033] Where digital represents a number, f on This indicates the percentage of active microspheres. This represents the average fluorescence intensity of the active microspheres. This represents the average fluorescence intensity produced by a single enzyme;

[0034] 4.3 Using the calibrator concentration as the X-axis and the AEB calculated from the calibrator detection as the Y-axis, a calibration curve is fitted using four or five parameters. The AEB of the sample to be tested is then substituted into the calibration curve to calculate the sample concentration.

[0035] Furthermore, the polyaromatic amino acid in step one can also be any combination of monomeric tyrosine, tryptophan, and phenylalanine.

[0036] Furthermore, the polyaromatic amino acids may also include polyamino acids combined with other non-aromatic amino acids.

[0037] Furthermore, the polyamino acid contains monomeric or polymeric groups, including PEG, carbon chains, oligonucleotides, fluorescein, quenching groups, amino groups, carboxyl groups, aldehyde groups, acrylamide groups, azides, alkynyl groups, diphenylcyclooctyne, maleimide, biotin, desulfurized biotin, thiol groups, dithiol, ferrocene, and other common crosslinking and modifying groups; the polyamino acid can be linear, branched, cyclic, or any combination of these structures.

[0038] The advantages of this invention compared to the prior art are as follows:

[0039] This invention increases the groups involved in the tyramine reagent-catalyzed precipitation reaction in the tyramine signal amplification (TSA) method by crosslinking poly-L-tyrosine, poly-L-tryptophan, and poly-L-phenylalanine onto magnetic beads. This has two effects: first, it enhances the fluorescence signal intensity on the magnetic beads after the reaction; second, it ensures a uniform distribution of fluorescent material around the magnetic beads. Firstly, the intensity of the fluorescence signal is fundamental to the sensitivity of single-molecule immunoassay; a stronger fluorescence signal means higher sensitivity. Secondly, both imaging and flow cytometry can only detect fluorescence signals in a specific direction around the magnetic beads; a uniform distribution of fluorescent material around the beads indicates a higher positive detection rate. Ultimately, these two effects increase the sensitivity of single-molecule immunoassay.

[0040] Furthermore, the polyaromatic amino acids in this invention are not limited to poly-L-tyrosine, poly-L-tryptophan, or poly-L-phenylalanine. They can be any combination of monomeric tyrosine, tryptophan, and phenylalanine, or combinations with other non-aromatic amino acids. Moreover, the polyamino acids may contain monomeric or polymeric groups, including PEG, carbon chains, oligonucleotides, fluorescein, quenching groups, amino groups, carboxyl groups, aldehyde groups, acrylamide groups, azides, alkynyl groups, diphenylcyclooctyne, maleimide, biotin, desulfurized biotin, thiol groups, dithiols, ferrocene, and other common crosslinking and modifying groups. The polyamino acids can be linear, branched, cyclic, or any combination of these structures. Attached Figure Description

[0041] Figure 1 This is a schematic diagram of uncrosslinked polyaromatic amino acids in magnetic beads in an embodiment of a method for enhancing single-molecule immune detection signals according to the present invention.

[0042] Figure 2 This is a schematic diagram of magnetic beads crosslinking with polyaromatic amino acids in an embodiment of a method for enhancing single-molecule immune detection signals according to the present invention. Detailed Implementation

[0043] To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the following examples provide a more detailed description of the invention. It should be noted that the specific embodiments described herein are merely illustrative and not intended to limit the scope of the invention.

[0044] In this embodiment, a method for enhancing single-molecule immune detection signals is specifically as follows:

[0045] Example 1:

[0046] Step 1: Coating the magnetic beads:

[0047] 1.1 Cleaning: 3 mg of 100 μL magnetic beads (Dynabeads M270 can be used) were removed from the supernatant and washed twice with 25 mM MES (pH 5.0);

[0048] 1.2 Activation: Prepare 50 mg / mL EDC and NHS with 25 mM MES (pH 5.0, pre-cooled), take 50 μL of each and add them to the cleaned magnetic beads, mix at room temperature for 30 min, wash twice with 25 mM MES (pH 5.0), and reconstitute to 100 μL.

[0049] 1.3 Antibody coating: Add 12 μg CEA capture antibody, 30 pmol poly-L-tyrosine, 30 pmol poly-L-tryptophan, and 30 pmol poly-L-phenylalanine. Mix at room temperature for at least 30 min or at 4 °C for 2 h. Wash twice with PBS buffer.

[0050] 1.4 Blocking: Redissolve in 200 μL 25 mM Tris (pH 7.2), mix at room temperature for 30 min, wash 3 times, and redissolve in 100 μL PBS buffer to form magnetic beads coated with CEA capture antibody.

[0051] Step 2, Immune Response:

[0052] 2.1 Add 10 μL of 0.3 mg / mL magnetic bead-CEA capture antibody-coated magnetic beads to the reaction tube, and add 100 μL of CEA antigen and mix well;

[0053] 2.2 Add 10 μL of 0.3 μg / mL biotin-labeled CEA detection antibody to the reaction tube, mix at room temperature for 1 hour, wash 3 times with 200 μL PBS buffer, and resuspend in 100 μL PBS buffer.

[0054] 2.3 Add 10 μL of 0.5 μg / mL SA-poly-HRP working solution to the reaction tube, mix at room temperature for 30 min, wash 3 times with 200 μL PBS buffer, and discard the supernatant.

[0055] Step 3: Enzyme-catalyzed reaction:

[0056] 3.1 Add 100 μL of tyramide working solution to the magnetic beads after removing the washing solution, and incubate at room temperature for 1 hour;

[0057] 3.2 Add 50 μL of stop solution to the reaction tube, mix at room temperature for 2 min, wash 6 times with 200 μL of PBS buffer, and resuspend in 100 μL of PBS buffer before testing.

[0058] Step 4: Data Processing. The sample is then fed into a flow cytometer for detection. Signals from the FSC, SSC, and FITC channels are acquired. After detection, gating statistics are performed, and the data processing is as follows:

[0059] 4.1 When the percentage of active microspheres is below 70%, the numerical model AEB (average enzyme per bead, the average number of enzyme molecules per microsphere) is calculated by microsphere counting and Poisson statistics. AEB is calculated using the following formula:

[0060] μ=AEB digital = -ln[1-f on ]

[0061] Where digital represents a number, f on This indicates the percentage of active microspheres.

[0062] 4.2 When the percentage of active microspheres is above 70%, the Poisson curve deviates from linearity. The simulated AEB is calculated by measuring the average fluorescence intensity of the microspheres using the following two formulas:

[0063]

[0064]

[0065] Where digital represents a number, f on This indicates the percentage of active microspheres. This represents the average fluorescence intensity of the active microspheres. This represents the average fluorescence intensity produced by a single enzyme.

[0066] 4.3 Using the calibrator concentration as the X-axis and the AEB calculated from the calibrator detection as the Y-axis, a calibration curve is fitted using four or five parameters. The AEB of the sample to be tested is then substituted into the calibration curve to calculate the sample concentration.

[0067] 4.4. Using the zero-concentration calibrator as the sample, the test was repeated 20 times, and the AEB values ​​of the 20 measurements were obtained. The mean (M) and standard deviation (SD) of these 20 measurements were calculated, and the AEB value corresponding to M + 2.5SD was obtained. A two-point regression was performed on the concentration-AEB values ​​between the zero-concentration calibrator and adjacent concentration calibrators to obtain a linear equation. The AEB value of M + 2.5SD was then substituted into the equation to calculate the corresponding concentration value, which is the limit of detection. The data are shown in Table 1.

[0068] Compare with Example 1:

[0069] The steps were similar to those in Example 1 for the detection of Control Example 1, except that poly-L-tyrosine, poly-L-tryptophan, and poly-L-phenylalanine were not added to the antibody coating.

[0070] The minimum detection limit data are shown in Table 1.

[0071] Example 2:

[0072] The detection in Example 2 was performed similarly to that in Example 1, except that 60 μg of CEA capture antibody, 3000 pmol of poly-L-tyrosine, 3000 pmol of poly-L-tryptophan, and 3000 pmol of poly-L-phenylalanine were added to the antibody coating.

[0073] The minimum detection limit data are shown in Table 1.

[0074] Compare with Example 2:

[0075] The steps were similar to those in Example 2 for the detection of Control Example 2, except that poly-L-tyrosine, poly-L-tryptophan, and poly-L-phenylalanine were not added to the antibody coating.

[0076] The minimum detection limit data are shown in Table 1.

[0077] Table 1. Results of CEA minimum detection limit detection

[0078] Examples Example One Comparative Example One Example Two Comparative Example Two Lowest detection limit 0.473 pg / mL 2.015 pg / mL 0.486 pg / mL 2.006 pg / mL

[0079] like Figures 1-2 As shown, Figure 1 Uncrosslinked poly-L-tyrosine, poly-L-tryptophan, and poly-L-phenylalanine on the magnetic beads Figure 2 Cross-linking of poly-L-tyrosine, poly-L-tryptophan, and poly-L-phenylalanine onto the magnetic beads increases the groups involved in the tyramine reagent-catalyzed precipitation reaction in the tyramine signal amplification (TSA) method. This produces two effects: first, it enhances the fluorescence signal intensity on the magnetic beads after the reaction; second, it ensures a uniform distribution of fluorescent material around the magnetic beads. These two effects ultimately increase the sensitivity of single-molecule immunoassay.

[0080] As shown in Table 1, the limit of detection (LOD) is an important indicator of the sensitivity of immunological detection methods; a lower value indicates higher sensitivity. Examples 1 and 2, which crosslinked polyaromatic amino acids onto magnetic beads, showed comparable sensitivity, as did Comparative Examples 1 and 2, which did not crosslink polyaromatic amino acids onto magnetic beads. Crosslinking polyaromatic amino acids onto magnetic beads in single-molecule immunoassay significantly increases detection sensitivity.

[0081] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements can be made without departing from the principle of the present invention, and these improvements should also be considered within the scope of protection of the present invention.

Claims

1. A method for enhancing single-molecule immune detection signals, characterized in that, The method includes the following steps: Step 1: Coating the magnetic beads; specifically: 1.1 Cleaning: Remove the supernatant with 100μL and 3mg magnetic beads, and wash twice with pH5.0 and 25mM MME. 1.2 Activation: Prepare 50 mg / mLEDC and NHS with pre-cooled 25 mM MMEES at pH 5.

0. Take 50 μL of each and add them to the cleaned magnetic beads. Mix at room temperature for 30 min. Wash twice with pH 5.0 and 25 mM MMEES, and reconstitute to 100 μL. 1.3 Antibody coating: Add 12-60 μg of capture antibody and polyaromatic amino acids, mix at room temperature for at least 30 min or at 4°C for 2 h, and wash twice with PBS buffer. 1.4 Blocking: Redissolve in 200 μL of 25 mM Tris at pH 7.2, mix at room temperature for 30 min, and wash 3 times. Redissolve in 100 μL PBS buffer to obtain coated magnetic beads; The polyaromatic amino acid contains monomeric or polymeric groups, including PEG, carbon chains, oligonucleotides, fluorescein, quenching groups, amino groups, carboxyl groups, aldehyde groups, acrylamide groups, azides, alkynyl groups, diphenylcyclooctyne, maleimide, biotin, desulfurized biotin, thiol groups, dithiols, and common crosslinking and modifying groups of ferrocene; the polyaromatic amino acid has a linear, branched, cyclic structure, or any combination of these structures.

2. The method for enhancing single-molecule immune detection signals according to claim 1, characterized in that, The polyaromatic amino acids mentioned are 30-3000 pmol poly-L-tyrosine, 30-3000 pmol poly-L-tryptophan, and 30-3000 pmol poly-L-phenylalanine.

3. The method for enhancing single-molecule immune detection signals according to claim 1, characterized in that, The aforementioned polyaromatic amino acids are polyamino acids formed by any combination of monomers tyrosine, tryptophan, and phenylalanine.

4. The method for enhancing single-molecule immune detection signals according to claim 3, characterized in that, The aforementioned polyaromatic amino acids include polyamino acids combined with aromatic amino acids and other non-aromatic amino acids.

5. The method for enhancing single-molecule immune detection signals according to claim 1, characterized in that, The steps described in step one also include the following steps: Step 2: Immune response; Step 3: Enzyme-catalyzed reaction; Step 4: Data Processing.

6. The method for enhancing single-molecule immune detection signals according to claim 5, characterized in that, The second step is specifically as follows: 2.1 Add 10 μL of 0.3 mg / mL magnetic beads coated with polyaromatic amino acids to a reaction tube and add 100 μL of antigen and mix well; 2.2 Add 10 μL of 0.3 μg / mL biotin-labeled detection antibody to the reaction tube, mix at room temperature for 1 hour, wash 3 times with 200 μL PBS buffer, and resuspend in 100 μL PBS buffer. 2.3 Add 10 μL of 0.5 μg / mL SA-poly-HRP working solution to the reaction tube, mix well at room temperature for 30 min, wash three times with 200 μL PBS buffer, and discard the supernatant.

7. The method for enhancing single-molecule immune detection signals according to claim 5, characterized in that, Step three specifically refers to: 3.1 Add 100 μL of tyramide working solution to the magnetic beads after removing the washing solution, and incubate at room temperature for 1 hour; 3.2 Add 50 μL of stop solution to the reaction tube, mix at room temperature for 2 min, wash 6 times with 200 μL of PBS buffer, and resuspend in 100 μL of PBS buffer before testing.

8. The method for enhancing single-molecule immune detection signals according to claim 5, characterized in that, Step four specifically involves: introducing the sample into a flow cytometer for detection, acquiring signals from the FSC, SSC, and FITC channels, performing gating statistics after detection, and processing the data as follows: 4.1 When the percentage of active microspheres is below 70%, the numerical model AEB (average enzyme per bead) is calculated using microsphere counting and Poisson statistics. AEB is calculated using the following formula: μ=AEB digital =-ln[1-f on ] in, digital represents a number, f on This indicates the percentage of active microspheres. 4.2 When the percentage of active microspheres is above 70%, the Poisson curve deviates from linearity. The simulated AEB is calculated by measuring the average fluorescence intensity of the microspheres using the following two formulas: in, digital represents a number, f on This indicates the percentage of active microspheres. This represents the average fluorescence intensity of the active microspheres. This represents the average fluorescence intensity produced by a single enzyme; 4.3 Using the calibrator concentration as the X-axis and the AEB calculated from the calibrator detection as the Y-axis, a calibration curve is fitted using four or five parameters. The AEB of the sample to be tested is then substituted into the calibration curve to calculate the sample concentration.

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