Modified composite sealing buffer solution as well as preparation method and detection application thereof

Abstract

The invention belongs to the technical field of immunohistochemical detection, and discloses a modified composite sealing buffer solution as well as a preparation method and detection application thereof. The modified composite blocking buffer solution comprises a basic buffer system, a composite blocking agent, a nonionic detergent, an endogenous interferent blocking agent and an antibody stabilizer, wherein the basic buffer system provides a stable pH environment and ionic strength; the composite sealing agent firstly occupies a shielding site, and the background is lowered; the nonionic detergent destroys hydrophobic interaction, effectively elute and loosen adsorbed molecules, and promote dissolution of components with relatively strong hydrophobicity; the endogenous interferent blocker can be combined with a receptor firstly, so that non-specific combination of an antibody and the receptor is blocked, and the problem of specific background is solved; the antibody stabilizer can protect the activity of the detection reagent and maintain the stability, and the obtained modified composite sealing buffer solution and the kit thereof have the advantages of multiple cooperative sealing mechanisms, prospective blocking of endogenous interference, improvement of the stability, compatibility and high efficiency of the antibody and the like.

Description

A modified composite blocking buffer, its preparation method and detection application Technical Field

[0001] This invention belongs to the field of immunohistochemical detection technology, specifically a modified composite blocking buffer, its preparation method, and its detection application. Background Technology

[0002] Immunohistochemistry is crucial in pathological diagnosis, drug development, and basic research. The widespread adoption of fully automated immunohistochemistry instruments has placed higher demands on the stability, consistency, and specificity of reagent kits. Currently, when using fully automated platforms for detection, especially when using monoclonal antibodies, the problem of non-specific background staining remains common.

[0003] The causes of nonspecificity are complex and mainly include: (1) hydrophobic or ion interactions: antibodies or proteins in the detection system bind nonspecifically to non-target sites in tissue samples; (2) endogenous biotin interference: some tissues (such as liver, kidney, and brain) are rich in endogenous biotin, which will bind to the streptavidin-biotin detection system in the kit, resulting in false positives; (3) antibody aggregation or poor stability: in the fluid lines of automated instruments, antibodies may aggregate slightly due to shear force or repeated freeze-thaw cycles, increasing the probability of nonspecific binding; (4) insufficient blocking: traditional blocking agents (such as BSA and serum) may not be able to effectively block all nonspecific sites on tissues, especially when time is limited in automated programs.

[0004] Patent CN117949647A discloses a composite blocking agent and detection method for single-molecule immunoassay. This patent introduces a compound form of anionic and nonionic surfactants into the blocking agent. By adjusting the pH value of the buffer solution, the charge state of the antibody surface is changed, maintaining the stability of the antigen and the activity of the immune response. The composite blocking agent is only effective for stable antibodies or antigens, with narrow applicability. Moreover, it requires simultaneous optimization of multiple variables such as the ratio of anionic / nonionic surfactants, total concentration, pH value, and blocking agent concentration, making the formulation complex. This technology has many problems and cannot be widely used.

[0005] In summary, while composite blocking agents for immunoassay do have applications in the field of immunohistochemical detection technology, they still suffer from problems such as narrow applicability, complex processes, high optimization requirements, and limited adjustment. Developing a modified composite blocking buffer formulation that can fundamentally inhibit non-specific binding and is compatible with fully automated platforms has significant market value and clinical significance. Summary of the Invention

[0006] The purpose of this invention is to provide a modified composite blocking buffer. This modified composite blocking buffer integrates a basic buffer system, a composite blocking agent, a nonionic detergent, an endogenous interference blocker, and an antibody stabilizer for composite blocking and functional integration. The basic buffer system provides a stable pH environment and ionic strength; the composite blocking agent preemptively occupies the shielding site, reducing background; the nonionic detergent disrupts hydrophobic interactions, effectively eluting loosely adsorbed molecules and promoting the dissolution of strongly hydrophobic components; the endogenous interference blocker binds to the receptor first, thereby blocking the non-specific binding of the antibody to the receptor and solving the specific background problem; the antibody stabilizer protects the activity of the detection reagent and maintains its stability.

[0007] The objective of this invention is achieved through the following scheme: a modified composite blocking buffer, wherein the modified composite blocking buffer comprises the following components in parts by mass: 1.5-4 parts of a basic buffer system, 11-20 parts of a composite blocking agent, 0.8-1.5 parts of a nonionic detergent, 0.05-0.15 parts of an endogenous interferon blocking agent, and 18-25 parts of an antibody stabilizer.

[0008] Preferably, the modified composite blocking buffer comprises the following components in parts by weight: 2-3 parts of basic buffer system, 14-18 parts of composite blocking agent, 1-1.2 parts of nonionic detergent, 0.08-0.12 parts of endogenous interference blocker, and 20-23 parts of antibody stabilizer.

[0009] The basic buffer system provides a stable pH environment and ionic strength. The charge-charge interactions between salt ions in the buffer, which are unrelated to the protein surface, prevent protein degradation or precipitation caused by drastic pH changes by stabilizing the pH environment. A single blocking agent cannot cover all types of non-specific sites. Composite blocking agents have broad compatibility, adapt to different tissue types, preemptively occupy shielding sites, reduce background, and facilitate observation and quantification. Non-ionic detergents disrupt hydrophobic interactions, effectively reducing uniform staining background in areas such as cytoplasm and cell membranes, making the signal sharper and clearer. At the same time, they can effectively elute loosely adsorbed molecules and promote the dissolution of strongly hydrophobic components. Endogenous interference blockers can bind to receptors first, thereby blocking the non-specific binding of antibodies to receptors and solving the problem of specific background. Antibody stabilizers can protect the activity of detection reagents, prevent antibody adsorption and aggregation, and maintain conformational stability.

[0010] Preferably, the composite blocking agent comprises the following components at the following concentrations: 1-5% (w / v) casein, 0.5-2% (w / v) purified gelatin, and 0.1-1% (w / v) synthetic peptide blocking agent.

[0011] Casein, at concentrations of 1-5% (w / v), can form a dense, continuous protein network film on the surface of tissue sections, effectively blocking hydrophobic and charged sites in the sample. Gelatin, rich in hydrophilic amino acids, can form a hydrophilic, viscous coating on the tissue surface in solution, reducing non-specific binding based on hydrophobic interactions. Furthermore, gelatin is similar to endogenous Fc receptors in tissue sections; the Fc fragment of antibody molecules binds to Fc receptors on immune cells. In IHC, Fc receptors within the tissue non-specifically capture primary antibodies. The Fc fragment of the secondary antibody can cause false positives in specific cells. Gelatin can act as a pseudo-Fc receptor, preemptively binding weakly to the antibody's Fc fragment, thereby blocking the binding site of endogenous Fc receptors. Synthetic peptides are short-chain peptide mixtures. During tissue preparation, some cell membranes are damaged, leading to leakage of endogenous immunoglobulins, which non-specifically adhere to tissue components and become targets for subsequent secondary antibodies, resulting in high background false positives. Synthetic peptide blocking agents can capture and remove free and adherent IgG. The synergistic effect of these three agents can provide comprehensive coverage and blockade, improving the signal-to-noise ratio.

[0012] Preferably, the synthetic polypeptide blocking agent is a synthetic Fc segment blocking polypeptide with the sequence: SEQ ID NO: 1.

[0013] Traditional synthetic peptide blocking agents mainly rely on charge adsorption, while synthetic Fc-segment blocking peptides mimic the ligand-binding domain of the Fc receptor, preemptively binding to the Fc segment of endogenous IgG in tissue sections, as well as to the Fc segments of primary and secondary antibodies; this can completely eliminate specific false positives caused by endogenous Fc receptors, resulting in clearer immunohistochemical results and significantly improving the signal-to-noise ratio and staining specificity.

[0014] Preferably, the nonionic detergent comprises the following components at the following concentrations: Tween-20 0.05-0.2% (v / v) and Triton X-100 0.01-0.05% (v / v).

[0015] Tween-20 is a mild nonionic detergent. Its molecular structure, with hydrophilic polyoxyethylene groups and hydrophobic long-chain fatty acid esters, allows it to insert into the hydrophobic regions of proteins, preventing non-specific adhesion between proteins via hydrophobic interactions. Tween-20 can continuously dissociate antibodies or contaminating proteins that have reached non-target sites through weak hydrophobic interactions. Triton X-100 has stronger membrane-dissolving capabilities than Tween-20, partially disrupting the lipid bilayer. At low concentrations, it exhibits membrane-breaking properties, providing channels for antibodies to enter the interior and effectively dissolving and dispersing strongly hydrophobic components and protein aggregates. The combination of these two products constitutes a complete background cleaning system from extracellular to intracellular, ensuring low background across the entire tissue section area.

[0016] Preferably, the endogenous interferon blocker comprises one or both of the following concentration components: D-biotin and biotin methyl ester, at a concentration of 0.001-0.01% (w / v).

[0017] D-Biotin is a natural biotin with the same molecular weight as the biotin linked to the biotinylated secondary antibody. This free, small molecule preemptively occupies all binding sites of endogenous biotin in the tissue section, thus interfering with and blocking the binding. Furthermore, D-Biotin's extremely small molecular weight means it does not obscure any antigenic epitopes after binding, protecting the sensitivity of the detection. Biotin methyl ester is a derivative of D-Biotin. After binding, the ester bond undergoes very slow hydrolysis in the aqueous environment of the tissue section, resulting in an irreversible cross-linking complex with the protein, which is more stable and enhances the blocking stability.

[0018] Preferably, the antibody stabilizer comprises the following components at the following concentrations: trehalose 1-5% (w / v) and bovine serum albumin 0.05-0.2% (w / v).

[0019] Trehalose molecules have a strong affinity for water molecules. In solution, they are excluded from the hydration layer of proteins, causing the system to tend towards a tightly folded protein state. This reduces the volume of excluded trehalose, effectively reducing the aggregation of antibody molecules due to hydrophobic interactions. Trehalose can form a glassy, ​​highly viscous protective layer around proteins, acting as a physical barrier to slow molecular motion, effectively preventing collisions and aggregation between proteins, and inhibiting chemical degradation reactions. Bovine serum albumin preemptively adsorbs onto the contact surface to protect antibodies and can act as a carrier protein to reduce the unfolding and inactivation of target antibodies at the interface. The synergy of these two proteins provides comprehensive protection for antibody activity, improving detection sensitivity and repeatability.

[0020] Preferably, the basic buffer system comprises the following components: 1.5-4 parts Tris-base, with HCl added to adjust the pH to 7.2-7.6.

[0021] Compared to phosphate buffer, Tris does not contain cations such as calcium and magnesium, reducing ion-mediated nonspecific binding and improving signal sensitivity. Tris has weak electrostatic interactions at pH 7.2-7.6, improving antibody binding efficiency, and its weaker attraction makes it easier to remove during washing.

[0022] This invention discloses a method for preparing a modified composite blocking buffer, comprising the following steps: adding Tris-base to ultrapure water, adjusting the pH with HCl, sequentially adding a composite blocking agent, an antibody stabilizer, an endogenous interferon blocker, a nonionic detergent, and bringing the ultrapure water to a final volume, filtering for sterilization using a 0.22 μm filter membrane, and then aliquoting and storing at 4°C.

[0023] First, prepare a basic buffer solution to ensure a stable and homogeneous environment for the system. Then, add the composite blocking agent, antibody stabilizer, endogenous interference blocker, and nonionic detergent in sequence to ensure that each component exists in a free and effective form in the solution. This will prevent micelle encapsulation when the nonionic detergent is added last, which would affect the effective concentration.

[0024] The present invention also discloses a detection application of a modified composite blocking buffer, which is used in the reagent kit of a fully automated immunohistochemistry instrument to reduce non-specific background staining and improve the specificity of detection signals.

[0025] The beneficial effects of this invention are as follows: (1) Multiple synergistic blocking mechanism: Casein, gelatin and synthetic Fc blocking peptides are innovatively combined and used to comprehensively cover the main pathways of non-specific binding from three dimensions: charge shielding, steric hindrance and specific competitive inhibition. The effect is far superior to that of a single blocking agent; (2) Prospective blocking of endogenous interference: Trace amounts of biotin analogs are added directly to the blocking buffer to neutralize endogenous biotin at the initial stage of the experiment. The operation is simple and avoids additional pretreatment steps, making it particularly suitable for fully automated processes; (3) Improved antibody stability: Trehalose is added as a stabilizer, which effectively solves the problem of antibody aggregation caused by mechanical force in the fully automated instrument tubing, reducing non-specific staining caused by antibody aggregates from the source; (4) Compatibility and high efficiency: All components of this formula are compatible with existing fully automated immunohistochemistry instrument platforms and standard operating procedures. They can be applied directly without changing the existing procedures and can significantly improve the accuracy and reliability of the detection results. Detailed Implementation

[0026] Example 1: This example provides a modified composite blocking buffer, which specifically contains the following components in parts by weight: 2.42 parts of basic buffer system, 16 parts of composite blocking agent, 1.1 parts of nonionic detergent, 0.1 parts of endogenous interference blocker, and 22 parts of antibody stabilizer.

[0027] The basic buffer system includes 2.42 parts Tris-base; the composite blocking agent includes 10 parts casein, 5 parts purified gelatin, and 1 part synthesized Fc segment blocking peptide; the nonionic detergent includes 1 part Tween-20 and 0.1 parts Triton X-1000; the endogenous interference blocker includes 0.1 parts D-biotin; and the antibody stabilizer includes 20 parts trehalose and 2 parts bovine serum albumin.

[0028] This embodiment also provides a method for preparing a modified composite blocking buffer, specifically including the following steps: Tris-base is added to 900 mL of ultrapure water, and the pH is adjusted to 7.4 with HCl. Then, the following are added sequentially and fully dissolved: casein, purified gelatin, synthesized Fc-blocking polypeptide (sequence: SEQ ID NO: 1), trehalose, BSA, and D-biotin, followed by Tween-20 and Triton X-100. Finally, the volume is brought to 1 L with ultrapure water, filtered through a 0.22 μm filter membrane for sterilization, aliquoted, and stored at 4 °C.

[0029] Example 2: 1.5 parts of basic buffer system, 16 parts of composite blocking agent, 1.1 parts of nonionic detergent, 0.1 parts of endogenous interference blocker, and 22 parts of antibody stabilizer.

[0030] The basic buffer system includes 1.5 parts Tris-base; the composite blocking agent includes 10 parts casein, 5 parts purified gelatin, and 1 part synthesized Fc segment blocking peptide; the nonionic detergent includes 1 part Tween-20 and 0.1 parts Triton X-100; the endogenous interference blocker includes 0.1 parts D-biotin; and the antibody stabilizer includes 20 parts trehalose and 2 parts bovine serum albumin.

[0031] The preparation method of the modified composite blocking buffer is the same as that in Example 1.

[0032] Example 3: 4 parts of basic buffer system, 16 parts of composite blocking agent, 1.1 parts of nonionic detergent, 0.1 parts of endogenous interference blocker, and 22 parts of antibody stabilizer.

[0033] The basic buffer system includes 4 parts Tris-base; the composite blocking agent includes 10 parts casein, 5 parts purified gelatin, and 1 part synthesized Fc segment blocking peptide; the nonionic detergent includes 1 part Tween-20 and 0.1 parts Triton X-100; the endogenous interference blocker includes 0.1 parts D-biotin; and the antibody stabilizer includes 20 parts trehalose and 2 parts bovine serum albumin.

[0034] The preparation method of the modified composite blocking buffer is the same as that in Example 1.

[0035] Example 4: 2.42 parts of basic buffer system, 11 parts of composite blocking agent, 1.1 parts of nonionic detergent, 0.1 parts of endogenous interference blocker, and 22 parts of antibody stabilizer.

[0036] The basic buffer system includes 2.42 parts Tris-base; the composite blocking agent includes 7 parts casein, 3 parts purified gelatin, and 1 part synthesized Fc segment blocking peptide; the nonionic detergent includes 1 part Tween-20 and 0.1 parts Triton X-100; the endogenous interference blocker includes 0.1 parts D-biotin; and the antibody stabilizer includes 20 parts trehalose and 2 parts bovine serum albumin.

[0037] The preparation method of the modified composite blocking buffer is the same as that in Example 1.

[0038] Example 5: 2.42 parts of basic buffer system, 21 parts of composite blocking agent, 1.1 parts of nonionic detergent, 0.1 parts of endogenous interference blocker, and 22 parts of antibody stabilizer.

[0039] The basic buffer system includes 2.42 parts Tris-base; the composite blocking agent includes 13 parts casein, 7 parts purified gelatin, and 1 part synthesized Fc segment blocking peptide; the nonionic detergent includes 1 part Tween-20 and 0.1 parts Triton X-1000; the endogenous interference blocker includes 0.1 parts D-biotin; and the antibody stabilizer includes 20 parts trehalose and 2 parts bovine serum albumin.

[0040] The preparation method of the modified composite blocking buffer is the same as that in Example 1.

[0041] Example 6: 2.42 parts of basic buffer system, 16 parts of composite blocking agent, 0.8 parts of nonionic detergent, 0.1 parts of endogenous interference blocker, and 22 parts of antibody stabilizer.

[0042] The basic buffer system includes 2.42 parts Tris-base; the composite blocking agent includes 10 parts casein, 5 parts purified gelatin, and 1 part synthesized Fc segment blocking peptide; the nonionic detergent includes 0.7 parts Tween-20 and 0.1 parts Triton X-100; the endogenous interference blocker includes 0.1 parts D-biotin; and the antibody stabilizer includes 20 parts trehalose and 2 parts bovine serum albumin.

[0043] The preparation method of the modified composite blocking buffer is the same as that in Example 1.

[0044] Example 7: 2.42 parts of basic buffer system, 16 parts of composite blocking agent, 1.5 parts of nonionic detergent, 0.1 parts of endogenous interference blocker, and 22 parts of antibody stabilizer.

[0045] The basic buffer system includes 2.42 parts Tris-base; the composite blocking agent includes 10 parts casein, 5 parts purified gelatin, and 1 part synthesized Fc segment blocking peptide; the nonionic detergent includes 1.2 parts Tween-20 and 0.3 parts Triton X-100; the endogenous interference blocker includes 0.1 parts D-biotin; and the antibody stabilizer includes 20 parts trehalose and 2 parts bovine serum albumin.

[0046] The preparation method of the modified composite blocking buffer is the same as that in Example 1.

[0047] Example 8: 2.42 parts of basic buffer system, 16 parts of composite blocking agent, 1.1 parts of nonionic detergent, 0.05 parts of endogenous interference blocker, and 22 parts of antibody stabilizer.

[0048] The basic buffer system includes 2.42 parts Tris-base; the composite blocking agent includes 10 parts casein, 5 parts purified gelatin, and 1 part synthesized Fc segment blocking peptide; the nonionic detergent includes 1 part Tween-20 and 0.1 parts Triton X-1000; the endogenous interference blocker includes 0.05 parts D-biotin; and the antibody stabilizer includes 20 parts trehalose and 2 parts bovine serum albumin.

[0049] The preparation method of the modified composite blocking buffer is the same as that in Example 1.

[0050] Example 9: 2.42 parts of basic buffer system, 16 parts of composite blocking agent, 1.1 parts of nonionic detergent, 0.15 parts of endogenous interference blocker, and 22 parts of antibody stabilizer.

[0051] The basic buffer system includes 2.42 parts Tris-base; the composite blocking agent includes 10 parts casein, 5 parts purified gelatin, and 1 part synthesized Fc segment blocking peptide; the nonionic detergent includes 1 part Tween-20 and 0.1 parts Triton X-1000; the endogenous interference blocker includes 0.15 parts D-biotin; and the antibody stabilizer includes 20 parts trehalose and 2 parts bovine serum albumin.

[0052] The preparation method of the modified composite blocking buffer is the same as that in Example 1.

[0053] Example 10: 2.42 parts of basic buffer system, 16 parts of composite blocking agent, 1.1 parts of nonionic detergent, 0.1 parts of endogenous interference blocker, and 18 parts of antibody stabilizer.

[0054] The basic buffer system includes 2.42 parts Tris-base; the composite blocking agent includes 10 parts casein, 5 parts purified gelatin, and 1 part synthesized Fc segment blocking peptide; the nonionic detergent includes 1 part Tween-20 and 0.1 parts Triton X-1000; the endogenous interference blocker includes 0.1 parts D-biotin; and the antibody stabilizer includes 17 parts trehalose and 1 part bovine serum albumin.

[0055] The preparation method of the modified composite blocking buffer is the same as that in Example 1.

[0056] Example 11: 2.42 parts of basic buffer system, 16 parts of composite blocking agent, 1.1 parts of nonionic detergent, 0.1 parts of endogenous interference blocker, and 25 parts of antibody stabilizer.

[0057] The basic buffer system includes 2.42 parts Tris-base; the composite blocking agent includes 10 parts casein, 5 parts purified gelatin, and 1 part synthesized Fc segment blocking peptide; the nonionic detergent includes 1 part Tween-20 and 0.1 parts Triton X-1000; the endogenous interference blocker includes 0.1 parts D-biotin; and the antibody stabilizer includes 22 parts trehalose and 3 parts bovine serum albumin.

[0058] The preparation method of the modified composite blocking buffer is the same as that in Example 1.

[0059] Experimental Example 1: This experimental example is a performance test of the modified composite blocking buffer prepared in Examples 1-11. The specific steps and results are as follows.

[0060] Signal-to-noise ratio analysis: Human liver cancer tissue sections were selected. On the same TMA section, the modified composite blocking buffer of Examples 1-11 was used as the primary antibody dilution and blocking buffer. The sections were incubated at room temperature for 30 min using universal secondary antibody dilution. After washing, freshly prepared chromogenic solution was added for counterstaining and mounting. Images were acquired and quantitatively analyzed. The average optical density was measured to calculate the signal-to-noise ratio. The experiment was repeated three times.

[0061] Microplate adsorption model: The well plates were coated with simulated proteins, and the wells were blocked with the modified composite blocking buffer of each embodiment. The enzyme-labeled secondary antibody dilution solution was added directly, and the reaction was terminated by adding substrate. The absorbance value of each well was measured with an ELISA reader.

[0062] Surface plasmon resonance experiment: Bovine serum albumin was fixed on the surface of the SPR chip, and the modified composite blocking liquid of different embodiments was flowed through the chip surface and the signal was measured.

[0063] Table 1: Test Results of Examples 1-11 Example 1 (Tris 2.42 parts, Trehalose 20 parts, BSA 2 parts, Casein 10 parts, Gelatin 5 parts, Fc peptide 1 part, Tween-20 1 part, Triton X-100 0.1 part, D-Biotin 0.1 part): Optimal signal-to-noise ratio, absorbance, and RU change value. Tris provides buffer capacity, ensuring that the antigen-antibody reaction takes place under optimal pH conditions, maximizing antibody affinity, ensuring that both the antibody complementarity-determining region and the antigen epitope are in an optimal ionized state, maximizing electrostatic attraction and hydrogen bonding efficiency, and stabilizing the antibody's three-dimensional conformation, avoiding inactivation or aggregation caused by pH fluctuations. High concentrations of trehalose form a protective layer around the antibody molecules through a preferential exclusion mechanism, stabilizing the native conformation and preventing denaturation or aggregation during incubation. BSA acts as a sacrificial protein, preemptively adsorbing onto the tube wall and pipette tip surface, avoiding antibody reagent loss. Casein forms a dense physical membrane covering most of the hydrophobic and charged non-specific sites on the tissue section, synthesizing Fc fragment peptides to neutralize and remove leaked and adhered endogenous IgG on the tissue. Tween-20 continuously dissociates impurities bound by weak hydrophobic interactions, and biotin preemptively saturates endogenous biotin molecules in the tissue, avoiding false positive background.

[0064] Example 2 (Tris 1.5 parts, trehalose 20 parts, BSA 2 parts, casein 10 parts, gelatin 5 parts, Fc peptide 1 part, Tween-20 1 part, Triton X-100 0.1 part, D-biotin 0.1 part): The low concentration of Tris resulted in insufficient buffering capacity, causing the pH of the reaction environment to deviate from the optimal range. This altered the charged state of the ionized groups, leading to a weakening of the specific signal intensity and a decrease in the signal-to-noise ratio. The unstable pH altered the surface charge of various proteins and solid-phase carriers in the tissue sections, making them more susceptible to non-specific electrostatic adsorption with antibody molecules, resulting in increased background staining. The blocking agent protein, due to the charge change, could not adsorb at non-specific sites in its optimal conformation, resulting in a non-dense blocking layer and increased absorbance and RU value.

[0065] Example 3 (Tris 4 parts, trehalose 20 parts, BSA 2 parts, casein 10 parts, gelatin 5 parts, Fc peptide 1 part, Tween-20 1 part, Triton X-100 0.1 part, D-biotin 0.1 part): The high concentration of Tris increases the ionic strength of the system. Through the electrostatic shielding effect, it slightly weakens the electrostatic attraction between the antigen and antibody complementary determinant regions, reduces the apparent affinity of the antibody, and limits the ultimate upper limit of the signal intensity. Due to the non-specific and transient adsorption of more antibody molecules in the buffer system, the absorbance and RU response value increase.

[0066] Example 4 (Tris 2.42 parts, Trehalose 20 parts, BSA 2 parts, Casein 7 parts, Gelatin 3 parts, Fc peptide 1 part, Tween-20 1 part, Triton X-100 0.1 part, D-Biotin 0.1 part): The concentration of the composite blocking agent was insufficient, and a complete and dense blocking layer could not be formed. A large number of hydrophobic and potential sites were exposed. The primary and secondary antibodies non-specifically bound to the sites, generating a very strong background signal, which severely phagocytosed the weak specific signal, resulting in a decrease in the signal-to-noise ratio. Due to the insufficient concentration of blocking agent, the blocking layer failed, and the antibody generated a non-specific adsorption signal, resulting in the highest absorbance and RU value.

[0067] Example 5 (Tris 2.42 parts, Trehalose 20 parts, BSA 2 parts, Casein 13 parts, Gelatin 7 parts, Fc peptide 1 part, Tween-20 1 part, Triton X-100 0.1 parts, D-Biotin 0.1 parts): Excessive blocking agent, while covering the background, caused over-blocking, resulting in an excessively thick protein layer that created steric hindrance, hindering the antibody macromolecules from approaching and binding to the target antigen epitope, thus reducing the signal-to-noise ratio.

[0068] Example 6 (Tris 2.42 parts, Trehalose 20 parts, BSA 2 parts, Casein 10 parts, Gelatin 5 parts, Fc peptide 1 part, Tween-20 0.7 parts, Triton X-100 0.1 parts, D-Biotin 0.1 parts): The low concentration of Tween-20 disrupted hydrophobic interactions and reduced continuous washing ability. Proteins and antibodies loosely bound by hydrophobic interactions in tissue sections could not be effectively washed away, resulting in increased background residue and decreased signal-to-noise ratio. The cleaning ability was insufficient, and it could not effectively prevent antibodies from adsorbing onto the experimental surface through hydrophobic interactions, resulting in high absorbance and RU value.

[0069] Example 7 (Tris 2.42 parts, Trehalose 20 parts, BSA 2 parts, Casein 10 parts, Gelatin 5 parts, Fc peptide 1 part, Tween-20 1.2 parts, Triton X-100 0.3 parts, D-Biotin 0.1 parts): Excessive detergent concentration leads to extremely low background. Excessive concentration of Triton X-100 slightly damages cell membrane structure and some fragile antigenic epitopes, thereby limiting the upper limit of specific signal intensity.

[0070] Example 8 (Tris 2.42 parts, trehalose 20 parts, BSA 2 parts, casein 10 parts, gelatin 5 parts, Fc peptide 1 part, Tween-20 1 part, Triton X-100 0.1 parts, D-biotin 0.05 parts): The D-biotin concentration was insufficient, which could not saturate all endogenous biotin sites, resulting in strong, localized false positive background, which lowered the signal-to-noise ratio. In addition, the protein coated on the microplate and the BSA on the SPR chip contained trace amounts of biotinylated impurities, resulting in high absorbance and RU value.

[0071] Example 9 (Tris 2.42 parts, trehalose 20 parts, BSA 2 parts, casein 10 parts, gelatin 5 parts, Fc peptide 1 part, Tween-20 1 part, Triton X-100 0.1 part, D-biotin 0.15 parts) and Example 11 (Tris 2.42 parts, trehalose 22 parts, BSA 3 parts, casein 10 parts, gelatin 5 parts, Fc peptide 1 part, Tween-20 1 part, Triton X-100 0.1 part, D-biotin 0.1 part): The results are similar to those of Example 1, but considering cost-effectiveness, the formulation of Example 1 is less expensive.

[0072] Example 10 (Tris 2.42 parts, Trehalose 17 parts, BSA 1 part, Casein 10 parts, Gelatin 5 parts, Fc peptide 1 part, Tween-20 1 part, Triton X-100 0.1 part, D-Biotin 0.1 part): Insufficient stabilizer concentration led to more antibodies becoming inactive due to denaturation, aggregation, or adsorption to the tube wall during incubation and storage. This resulted in a decrease in the effective antibody concentration participating in the specific reaction, causing the final signal intensity to fail to reach its peak value. Insufficient antibody stability indirectly affected the durability of the blocking effect, reduced background control ability, and increased absorbance and RU value.

[0073] Example 11 (basic buffer system 2.42 parts, composite blocking agent 16 parts, nonionic detergent 1.1 parts, endogenous interferon blocker 0.1 parts, antibody stabilizer 25 parts): performance is similar to Example 1, but the stabilizer concentration is too high, and the performance is redundant compared to Example 1.

[0074] In summary, the optimal concentration ratio for Example 1 is as follows: Tris 2.42 parts, trehalose 20 parts, BSA 2 parts, casein 10 parts, gelatin 5 parts, Fc peptide 1 part, Tween-20 1 part, Triton X-100 0.1 parts, and D-biotin 0.1 parts.

[0075] Experimental Example 2: This experimental example is a verification of the application effect of the modified composite blocking buffer prepared in Example 1.

[0076] Human liver cancer tissue sections (rich in endogenous biotin) and tonsil tissue sections (rich in Fc receptors) were selected. The sections were divided into two groups and used as follows: Group A: control group (commercial conventional kit, blocking solution was TBS buffer containing 1% BSA).

[0077] Group B: Experimental group (using the composite blocking buffer prepared in this invention as the primary antibody dilution and blocking solution).

[0078] CD3 (monoclonal antibody) staining was performed on a fully automated immunohistochemistry instrument according to the same procedure. The results are as follows: Liver tissue: In group A, obvious brownish background staining (endogenous biotin interference) was visible in areas such as hepatic sinusoids; In group B, the background was extremely clean, with only specific cells showing clear, specific membrane staining.

[0079] Tonsil tissue: In group A, a slight diffuse background was visible in some cellular areas; in group B, the background was clean, the positive signal was accurately located, and the signal-to-noise ratio was significantly improved.

[0080] Traditional blocking solutions (Group A) contain only 1% BSA and lack the ability to block endogenous biotin. When using a streptavidin detection system, the abundant endogenous biotin in liver tissue strongly binds to streptavidin, resulting in severe false-positive background. The buffer solution of this invention (Group B) contains 0.1% D-biotin, preemptively saturating all endogenous biotin binding sites and eradicating background. Group A BSA has limited blocking effect on Fc receptors, and Fc receptors on the cell surface in tissues can still non-specifically capture the Fc fragments of primary or secondary antibodies, leading to diffuse background. Group B contains synthetic Fc fragment blocking peptides that mimic Fc receptors, actively binding to the antibody Fc fragments and occupying the binding sites of endogenous Fc receptors for precise blocking. The composite blocking agent in the buffer solution of this invention provides physical shielding and a hydrophilic coating of more than 1% BSA, while the non-ionic detergent continuously removes hydrophobic interactions during incubation and washing. The high buffer capacity and antibody stabilizer ensure that the entire reaction proceeds under optimal conditions and protects antibody activity.

[0081] In summary, the modified composite blocking buffer provided by this invention achieves comprehensive shielding against various common endogenous interfering substances and non-specific bindings during immunohistochemistry through the synergistic effect of multiple components and multiple targets, ultimately significantly improving the specificity and signal-to-noise ratio of the detection.

Claims

1. A modified composite blocking buffer solution, characterized in that, The modified composite blocking buffer comprises the following components in parts by weight: 1.5-4 parts of basic buffer system, 11-20 parts of composite blocking agent, 0.8-1.5 parts of nonionic detergent, 0.05-0.15 parts of endogenous interference blocker, and 18-25 parts of antibody stabilizer.

2. The modified composite blocking buffer solution according to claim 1, characterized in that, The modified composite blocking buffer comprises the following components in parts by weight: 2-3 parts of basic buffer system, 14-18 parts of composite blocking agent, 1-1.2 parts of nonionic detergent, 0.08-0.12 parts of endogenous interference blocker, and 20-23 parts of antibody stabilizer.

3. The modified composite blocking buffer solution according to claim 1 or 2, characterized in that, The composite blocking agent comprises the following components at the following concentrations: casein 1-5% (w / v), purified gelatin 0.5-2% (w / v), and synthetic peptide blocking agent 0.1-1% (w / v).

4. The modified composite blocking buffer according to claim 3, wherein the synthetic polypeptide blocking agent is a synthetic Fc segment blocking polypeptide with the sequence: SEQ ID NO:

1.

5. The modified composite blocking buffer solution according to claim 1 or 2, characterized in that, The nonionic detergent comprises the following components at the following concentrations: Tween-20 0.05-0.2% (v / v) and Triton X-100 0.01-0.05% (v / v).

6. The modified composite blocking buffer solution according to claim 1 or 2, characterized in that, The endogenous interference blocker comprises one or both of the following concentrations: D-biotin and biotin methyl ester, at a concentration of 0.001-0.01% (w / v).

7. The modified composite blocking buffer solution according to claim 1 or 2, characterized in that, The antibody stabilizer comprises the following components at the following concentrations: trehalose 1-5% (w / v) and bovine serum albumin 0.05-0.2% (w / v).

8. The modified composite blocking buffer solution according to claim 1 or 2, characterized in that, The basic buffer system comprises the following components: 1.5-4 parts Tris-base, with HCl added to adjust the pH to 7.2-7.

6.

9. A method for preparing the modified composite blocking buffer solution according to any one of claims 1-8, characterized in that, The process includes the following steps: adding Tris-base to ultrapure water, adjusting the pH with HCl, then sequentially adding a composite blocking agent, an antibody stabilizer, an endogenous interferon blocker, and a nonionic detergent. After bringing the ultrapure water to a final volume, the mixture is filtered through a 0.22μm filter membrane for sterilization, dispensed, and stored at 4°C.

10. A detection application of the modified composite blocking buffer solution according to any one of claims 1-9, characterized in that, The modified composite blocking buffer is used in the kits of fully automated immunohistochemistry instruments to reduce non-specific background staining and improve the specificity of detection signals.

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