Sulfydryl modified gold nanoparticle covalent coupling-based pathogenic microorganism antigen passive agglutination semi-quantitative detection reagent and method

Abstract

The invention discloses a pathogenic microorganism antigen passive agglutination semi-quantitative detection reagent and method based on sulfydryl modified gold nanoparticle covalent coupling, and belongs to the field of medical detection. According to the method, 50nm sulfydryl modified gold nanoparticles are used as a carrier, the 50nm sulfydryl modified gold nanoparticles and pathogenic microorganism antigens are subjected to sulfydryl-amino covalent coupling according to the mass ratio of 1: 50, accurate sealing through 0.5% BSA and centrifugal purification to prepare a 0.02 mg / mL liquid reagent, the antigen shedding rate is smaller than or equal to 5% after the liquid reagent is stored in a dark place at the temperature of 4 DEG C for 3 months, and the activity retention rate is larger than or equal to 97%. During detection, after serum is subjected to complement inactivation and dilution treatment and is incubated with the reagent at 37 DEG C for 1 hour, objective interpretation is realized by determining a semi-quantitative spectrum criterion that A650nm / A520nm is greater than or equal to 1.5, the sensitivity reaches 98.2%, and the specificity reaches 99.2%. The method can be popularized and applied to antibody detection of various pathogenic microorganisms such as mycoplasma pneumoniae and respiratory syncytial viruses, and the detection stability and objectivity are remarkably improved.

Description

Technical Field

[0001] This invention belongs to the field of medical detection technology, specifically relating to a semi-quantitative detection reagent and method for passive agglutination of pathogenic microbial antigens based on covalently coupled thiol-modified gold nanoparticles (AuNPs-SH). This technology utilizes the covalent bonding between 50nm AuNPs-SH and pathogenic microbial-specific antigens, combined with passive agglutination reaction and dual-wavelength spectroscopy (A... 650nm / A 520nm ≥1.5), to achieve stable conjugation of pathogenic microbial antigens and highly specific semi-quantitative detection of antibodies. Background Technology

[0002] This invention relates to the passive agglutination detection of pathogenic microorganism antibodies, particularly for the detection of Treponema pallidum antibodies. Syphilis is a chronic infectious disease caused by Treponema pallidum infection, and clinical diagnosis is highly dependent on serological testing. Passive agglutination assay (TPPA) is widely used due to its ease of operation, but its traditional carriers (gelatin particles or red blood cells) encapsulate antigens through physical adsorption, which has the following inherent defects: (1) Unstable antigen binding Physical adsorption depends on van der Waals forces (bond energy <10kcal / mol), with an antigen shedding rate of 20-30%, resulting in batch-to-batch variability (CV) >15%; signal attenuation >40% after 5 days of liquid storage. (2) Severe non-specific interference The non-specific adsorption rate of gelatin particles to serum albumin is 15-25%, the cross-reactivity rate of rheumatoid factor is 12.8%, and the risk of false positives is significant. (3) Lack of objective judgment ability: The interpretation of results depends on visual observation of agglutination morphology (such as button-like or flocculent), and the consistency among testers is poor (K=0.62). In particular, it is difficult to distinguish low-titer samples (1:10-1:40), which makes it difficult to meet the quantitative needs of efficacy monitoring; (4) Poor scenario adaptability: The general nanoparticle detection scheme is not optimized for specific pathogen serum samples. For example, complement components in syphilis serum are prone to non-specific agglutination, resulting in a false positive rate of >10%, which is not specific enough. Existing technologies in related fields have failed to effectively solve the above problems. For example, Chinese invention patent CN101363848A (publication date: February 11, 2009) discloses a double-antigen sandwich method for antibody detection using indirectly labeled nanoparticles. Although it involves the application of nanoparticles in the detection of antibodies against various pathogens, including Treponema pallidum, it does not optimize the stability of antigen-carrier binding, non-specific interference, and quantitative criteria. The paper "DNAzymes and gold nanoparticles used to detect infectious diseases" (News-Medical.net, 2013) reported a method for detecting Treponema pallidum gene fragments based on DNAzymes and gold nanoparticles. However, it relies on nucleic acid amplification and cannot directly detect antibodies in serum. Furthermore, it does not solve the problems of complement interference and quantification. Compared with CN101363848A, this invention reduces the antigen drop rate from 20-30% to ≤5% and the false positive rate from 10% to 0.8% through a combination of covalent coupling, precise blocking, and serum optimization. The passive agglutination test kits for Treponema pallidum produced by Fujibio Co., Ltd. of Japan (such as the TPPA kit) still use gelatin particle carriers and only support semi-quantitative visual grading (± to ++++). Traditional passive agglutination methods (such as TPPA) mainly rely on visual qualitative interpretation, which can only achieve limited semi-quantitative grading, is highly subjective, and cannot be quantified. This invention measures A... 650nm / A 520nmA semi-quantitative spectral criterion of ≥1.5 (1.5-2.0 for weak positive, >2.0 for strong positive) enables objective grading and interpretation, significantly improving the scientific rigor of semi-quantitative analysis. In summary, existing passive agglutination detection technologies for pathogenic microorganism antibodies have significant shortcomings in antigen binding stability, anti-interference ability, quantitative accuracy, and scenario specificity. There is an urgent need for a universal technical solution based on antigen covalent coupling, low non-specific interference, and spectral semi-quantitative analysis to meet the precision requirements of clinical testing. Summary of the Invention

[0003] The purpose of this invention is to overcome the shortcomings of existing passive agglutination detection technologies, such as insufficient antigen binding stability, severe non-specific interference, lack of objective judgment criteria, and poor adaptability to various scenarios. This invention provides a semi-quantitative detection reagent and method for pathogenic microorganism antigen passive agglutination based on AuNPs-SH covalent coupling. Taking Treponema pallidum as an example, the core technology of this invention can be extended to the detection of antibodies against other pathogens, achieving highly stable, highly specific, and semi-quantitative serological detection.

[0004] The technical problem solved by this invention is to address the four core defects of the prior art described in the background (insufficient antigen binding stability, serious non-specific interference, lack of objective judgment criteria, and poor scenario adaptability), and proposes a targeted solution: (1) For insufficient antigen-carrier binding stability: Traditional carriers (gelatin particles or physically adsorbed gold nanoparticles) rely on van der Waals forces (bond energy <10kcal / mol), the antigen drop rate is ≥20%, the batch-to-batch variation CV is >15%, and the liquid shelf life is ≤5 days. This invention significantly improves the antigen binding stability through thiol-amino covalent bonds (bond energy 30-50kcal / mol). Specifically, 50nm AuNPs-SH is used as the carrier, and the antigen and AuNPs-SH are directionally coupled at a mass ratio of 1:50. After centrifugation purification, a ready-to-use liquid reagent with a concentration of 0.02mg / mL is prepared. It can be stably stored at 4℃ in the dark for 3 months, with an antigen drop rate ≤5%, an activity retention of ≥97%, and an intra-batch coefficient of variation (CV) of 4.8%, breaking through the stability bottleneck of traditional physical adsorption. (2) Addressing severe non-specific interference: Existing carriers exhibit a non-specific adsorption rate of 15-25% for serum proteins and a cross-reactivity rate of 12.8% for rheumatoid factor, resulting in a high risk of false positives. This invention reduces background interference through a precise blocking process, using a final concentration of 0.5% BSA for blocking (the blocking solution is prepared based on 0.1M PBS buffer (pH 7.4±0.1) containing 0.1% BSA). Simultaneously, 5%-10% glycerol or 2% trehalose is added to the preservation buffer as a stabilizer (with an ionic strength equivalent to 0.15M NaCl). This reduces the non-specific adsorption rate from 15-25% to <5%, lowers background interference by more than 40%, balances blocking efficiency and antigen activity, and significantly reduces the risk of false positives. (3) Addressing the lack of objective judgment criteria: Traditional visual interpretation is highly subjective (K=0.62) and cannot distinguish between positive and negative results for low-titer samples (1:10-1:40). This invention establishes a dual-wavelength spectral semi-quantitative criterion to achieve objective quantification. When specific antibodies are present in the sample, the aggregation of AuNPs-SH particles causes the solution color to change from red to blue-purple, with a decrease in absorbance at 520 nm (dispersed state) and an increase in absorbance at 650 nm (aggregated state). The absorbance at both wavelengths is detected using a UV-Vis spectrophotometer, and A is calculated. 650nm / A 520nm≥1.5 is considered positive (1.5-2.0 is weakly positive, >2.0 is strongly positive). The area under the receiver operating characteristic curve (ROC-AUC) of this criterion is 0.98. The detection rate of low-titer samples (1:10-1:40) is 26.6% higher than that of traditional visual interpretation, and the intra-assay CV is <4.8%, filling the gap in objective semi-quantitative analysis. (4) Poor adaptability to scenarios: The general detection scheme is not optimized for pathogen serum, and complement interference leads to a false positive rate of >10%. This invention improves specificity through serum processing technology. Specifically, the serum sample is inactivated in a 56℃ water bath for 30 min to remove complement interference. After cooling, it is diluted with PBS at a ratio of 1:10. This treatment can reduce the false positive rate caused by complement interference from 10% to 0.8%, making the detection specificity 99.2% (sensitivity: 95% CI: 98.5-99.8%), forming a dedicated technical path for pathogen serum.

[0005] The technical solution of the present invention includes the construction of the detection reagent, the detection process and the supporting kit, as follows: (1) The detection reagent is constructed using 50nm AuNPs-SH as the carrier, and the antigen and AuNPs-SH are coupled with the pathogen-specific antigen (using Treponema pallidum antigen as an example) through a "thiol-amino" covalent bond (bond energy 30-50kcal / mol) at a mass ratio of 1:50; (2) 0.5% BSA is the final concentration of the blocking solution, which is used to block the free sites of unbound antigen on the surface of AuNPs-SH particles, reduce non-specific adsorption, and centrifuge and purify to prepare a ready-to-use liquid reagent with a concentration of 0.02mg / mL. The reagent is stored at 4℃ in the dark for 3 months, and the activity is maintained at ≥97% and the antigen shedding rate is ≤5%; 0.1% BSA is the concentration in the storage buffer (PBS containing 0.1% BSA), which is used to maintain the long-term stability of the reagent. The two have different functions and the concentration is set in a stepwise manner to optimize the results. (3) Detection process: Sample processing: Serum samples are inactivated at 56℃ for 30 min to remove complement interference, and then diluted with PBS at 1:10 after cooling; (4) Reaction incubation: The diluted sample is mixed with the detection reagent in equal volume and incubated at 37℃ in the dark for 1 hour; (5) Result interpretation: When the antigen coupled to the surface of AuNPs-SH particles binds to the specific antibody in the sample, a cross-linking network of AuNPs-SH particles-antigen-antibody-antigen-AuNPs-SH particles is formed. The particle size of the aggregated AuNPs-SH particles increases, the surface plasmon resonance effect changes, and the solution color gradually changes from red to blue-purple. The essence of this color change reaction is the aggregation phenomenon of AuNPs-SH particles caused by the specific binding of antigen and antibody. Semi-quantitative detection is achieved through color change (or spectral data). Combined with dual-wavelength spectral analysis, the absorption peak at 650 nm is enhanced. The ratio of absorbance at 520 nm (red dispersed state) and 650 nm (blue-purple aggregated state) is detected. 650nm / A 520nm≥1.5 is considered positive (1.5-2.0 is weakly positive, >2.0 is strongly positive), which greatly improves the sensitivity and objectivity of the detection and achieves semi-quantitative results. Agglutination status judgment: positive samples are uniform blue-purple agglutinates (without button-shaped precipitates), negative samples are dispersed particles, and are clearly edged, red button-shaped precipitates. (6) The supporting kit includes: the above covalently coupled detection reagents (2mL×1 bottle), PBS buffer (0.1M, pH7.4±0.1, 30mL×1 bottle), positive control (such as syphilis positive serum with TPPA titer of 1:320, 0.5mL×1 bottle), negative control (healthy human serum, 0.5mL×1 bottle), 96-well U-shaped reaction plate and instructions for use, forming a standardized detection system.

[0006] The innovation of this invention lies in the establishment of a universal passive agglutination detection technology system for pathogenic microorganism antigens, specifically including: (1) a pioneering covalent coupling process: using 50nm AuNPs-SH as a carrier, the antigens are directionally coupled to pathogenic microorganism antigens at a mass ratio of 1:50 through thiol-amino covalent bonds (bond energy 30-50kcal / mol), reducing the antigen detachment rate from 20-30% to ≤5%, and maintaining ≥97% activity after 3 months of storage at 4℃, breaking through the stability bottleneck of traditional physical adsorption. (2) a precise blocking technology: the 0.5% BSA blocking process reduces the non-specific adsorption rate from 15-25% to <5% compared to the traditional 1% BSA scheme, and reduces background interference by more than 40%, taking into account both blocking efficiency and antigen activity. (3) a dual-wavelength spectral semi-quantitative criterion: establishing A 650nm / A 520nm The spectral quantitative standard with a value of ≥1.5 (ROC-AUC = 0.98) improved the detection rate of low-titer samples (1:10-1:40) by 26.6% compared with traditional visual interpretation, and reduced the intra-assay coefficient of variation (CV) to 4.8%, filling the gap in objective quantification. (4) Serum optimization processing: The 56℃ inactivation process for pathogen serum (such as syphilis serum) + 1:10 dilution reduced the false positive rate of complement interference from 10% to 0.8%, and the specificity reached 99.2% (sensitivity: 95% CI: 98.5-99.8%), forming a dedicated technical pathway.

[0007] The present invention achieves the following significant effects: (1) Improved stability: Antigen shedding rate ≤5%, no significant activity decay after 3 months of liquid storage at 4℃, and the intra-batch CV of Example 1 is 4.8%, which is far superior to traditional technology. (2) Enhanced specificity: Non-specific adsorption rate <5%, rheumatoid factor cross-reactivity rate significantly reduced, complement interference false positive rate reduced to 0.8%, and specificity reaches 99.2%. (3) Breakthrough in semi-quantitative capability: Dual-wavelength spectral criteria achieve objective quantification, and the detection rate of low-titer samples is increased by 26.6%, meeting the precise needs of efficacy monitoring. (4) Strong universality: The core process (covalent coupling, blocking, semi-quantitative criteria) has been verified to be extended to the detection of other pathogens such as Mycoplasma pneumoniae and respiratory syncytial virus, with shedding rates ≤5% and low-titer detection rates increased by 25%-30%, possessing broad clinical application value.

[0008] This invention comprehensively improves the stability, specificity, and quantitative capability of passive agglutination detection of pathogenic microorganism antigens through material optimization, process innovation, and standard establishment, providing a standardized and industrializable technical solution for clinical infection screening and efficacy monitoring. Attached Figure Description

[0009] Figure 1 This is a structural diagram of a passive agglutination method detection instrument;

[0010] Figure 2 This is a comparison diagram of positive and negative results for the method of this invention. Detailed Implementation

[0011] This invention uses Treponema pallidum antigen detection as an example, and details the technical implementation steps by combining material optimization, process parameter verification, and performance evaluation. Its core process can be extended to the detection of antigens from other pathogenic microorganisms, as detailed below:

[0012] Example 1:

[0013] Preparation and Detection Method of Treponema pallidum Antigen Detection Reagent Based on AuNPs-SH

[0014] 1. Preparation of reagents

[0015] 1.1 Materials and Instruments Preparation: 50nm AuNPs-SH particles (Shanghai Maclean, batch number M20230615, concentration 0.1mg / mL, PDI < 0.1); Treponema pallidum-specific antigen (Beijing Keyue Zhongkai's SY01N309 (S309#), purity ≥ 95%, single band on SDS-PAGE, concentration 1mg / mL); 0.1M PBS buffer (pH 7.4±0.1, containing 0.1% BSA); 0.5% BSA blocking solution (prepared based on 0.1M PBS buffer (pH 7.4±0.1) containing 0.1% BSA); high-speed refrigerated centrifuge (Eppendorf 5425R, maximum 15000×g); dynamic light scattering instrument (Malvern ZS90, 25℃). 96-well U-shaped microplate (suitable for passive agglutination reaction); 37℃ constant temperature incubator (protected from light to ensure stable reaction temperature).

[0016] 1.2 Coupling process optimization (including carrier modification and reaction condition optimization) Optimize carrier surface modification to enhance the binding basis with antigen (1) Increase the density of active groups on the carrier surface For AuNPs-SH, increase the number of surface -SH groups by extending the thiolation modification time or increasing the concentration of the modifier (e.g., by treating with excess mercaptosilane reagent, the surface -SH density of the particles can be increased by 20%-50%), providing more binding sites for the -NH2 of the antigen and reducing weak binding caused by insufficient sites. (2) Introduce bifunctional crosslinking agents Add bifunctional crosslinking agents (such as SPDP (N-succinimide-3-(2-pyridinedithio)propionate) and SMCC (succinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid ester)) between AuNPs-SH particles and antigen. One end of the crosslinking agent is covalently bound to the -SH of AuNPs, and the other end is connected to the -NH2 of the antigen through an amide bond, forming a "carrier-crosslinking agent-antigen" bridging structure. This structure is more robust than the direct thiol-amino reaction, and its resistance to dissociation is increased by more than 30%.

[0017] Optimize coupling reaction conditions and enhance covalent binding efficiency (1) Precisely control reaction parameters pH value: On the basis of maintaining PBS buffer pH 7.4, it can be finely adjusted according to the isoelectric point of the antigen (e.g., acidic conditions can enhance the reactivity of -NH2) to avoid the decrease in binding stability due to pH fluctuations. (2) Reaction time and temperature: Extend the incubation time in the 37℃ water bath to 2.5-3 hours (the original plan was 2 hours), or use gradient heating (first pre-react at 30℃ for 30 minutes, then increase to 37℃) to promote the full formation of covalent bonds; at the same time, optimize the magnetic stirring speed (e.g., from a low speed of 100 rpm to 150 rpm) to ensure uniform contact between the antigen and the carrier. (3) Optimize the ratio of antigen to carrier On the basis of the original 1:50 mass ratio, screen the optimal ratio through gradient experiments (e.g., 1:40, 1:50, 1:60) to avoid "multi-layer weak binding" (the outer layer of antigen is easy to fall off) caused by excessive antigen, or insufficient binding caused by excessive carrier. Prioritize the use of a slightly excessive amount of carrier to ensure that each antigen molecule can form a stable covalent bond with the carrier. The antigen-carrier mass ratio is screened as shown in Table 1.

[0018] Subsequent stabilization treatment reduces antigen shedding and signal attenuation. (1) Enhanced blocking and secondary cross-linking: After antigen coupling is completed, in addition to blocking the free sites with 0.5% BSA, a low concentration of glutaraldehyde (0.1%) or polyethylene glycol (PEG, molecular weight 2000) can be added to perform "secondary fixation" on the bound antigen: glutaraldehyde can cross-link adjacent antigen molecules to form a network structure, and PEG can coat the surface of the complex, reducing the damage of the binding bond to the aqueous environment. 0.5% BSA is used to block the free sites of nanoparticles, and 0.1% BSA is used to preserve the buffer to maintain reagent stability. The two have different functions, and the concentration is set in a stepwise optimization manner. The BSA concentration selection verification results are shown in Table 2. (2) Optimize the preservation system: Add a small amount of stabilizer, such as glycerol (final concentration 5%-10%) or trehalose (final concentration 2%), to the preservation buffer (PBS containing 0.1% BSA) to protect the spatial structure of the antigen-carrier complex by forming hydrogen bonds, thereby reducing the probability of dissociation when stored at 4°C; at the same time, strictly control the ionic strength of the buffer (e.g., 0.1M PBS) to avoid particle aggregation and antigen shedding caused by high salt. (3) Optimize the particle size and dispersibility of the carrier: Based on the selection of 50nm AuNPs-SH particles, ensure that the particles are evenly dispersed (monitor the particle size distribution through a dynamic light scattering instrument, and control the PDI value below 0.1) to avoid local antigen shedding caused by particle aggregation; if other carriers are used, microspheres with porous surface structures (e.g., porous silica spheres) are preferred, as the antigen can be embedded in the pores, resulting in a larger binding area and stronger stability.

[0019] Table 1. Quality Ratio Screening (Antigen: AuNPs-SH)

[0020]

[0021] Table 2. Final BSA concentration (%) [Closed environment test, 0.1% is the storage buffer control]

[0022]

[0023] Note: 0.1% BSA is the concentration in the storage buffer, used to compare the blocking effect; 0.5% is the optimal blocking concentration.

[0024] 1.3 Blocking and Purification Steps

[0025] Specific preparation steps: (1) Pre-dispersion: Take 10 mL of 0.1 mg / mL AuNPs-SH, add 10 mL of PBS to dilute to 0.05 mg / mL, stir magnetically (150 rpm) for 10 min, and confirm the particle size is 50 ± 2 nm and PDI < 0.1 by dynamic light scattering; (2) Covalent coupling: Take 20 μL of antigen: AuNPs-SH at a mass ratio of 1:50. 1 mg / mL antigen solution (20 μg) was added dropwise to the solution in step (1) at a rate of 1 drop / second. The solution was then heated in a water bath at 37°C, protected from light, and stirred at 100 rpm for 2 hours to form a thiol-amino covalent bond. (3) Blocking: 1 mL of 0.5% BSA solution was added to a final concentration of 0.25%, and the concentration was adjusted to 0.5%. (4) Purification: The solution was centrifuged at 12000×g for 15 min at 4°C, and the supernatant was discarded. The precipitate was resuspended in PBS containing 0.1% BSA to 10 mL, and the centrifugation was repeated twice. (5) Final volume preservation: The final concentration was adjusted to 0.02 mg / mL with PBS containing 0.1% BSA. The solution was stored at 4°C protected from light and had a shelf life of 3 months.

[0026] 2. Detection Method

[0027] 2.1 Sample Processing (Inactivation and Dilution) Serum was inactivated by incubating at 56°C for 30 min; after cooling to room temperature, it was diluted 1:10 with PBS (10 μL serum + 90 μL PBS). Positive controls (syphilis-positive serum with a TPPA titer of 1:320, stored in the laboratory) and negative controls (serum from healthy individuals) were prepared in triplicate for each sample.

[0028] 2.2 Reaction Incubation Procedure: Add the following to each well of a 96-well U-shaped plate: 100 μL of diluted sample (or control); 100 μL of reagent from Example 1; mix with a plate shaker for 10 seconds, and incubate at 37°C in the dark for 1 hour.

[0029] 2.3 Result Interpretation Criteria (Qualitative + Semi-Quantitative) Qualitative Interpretation: Positive—uniform aggregation at the bottom of the well, no button-shaped blue-purple precipitate; Negative—clear edges, red button-shaped precipitate. Quantitative: Measure the absorbance of the reaction solution at 520nm and 650nm, and calculate the ratio: A 650nm / A 520nm≥1.5 is considered positive; 1.5-2.0 is weakly positive; >2.0 is strongly positive.

[0030] 3. Performance verification data (sensitivity, specificity, etc.) are shown in Tables 3 and 4.

[0031] 3.1 The kit contains

[0032] Test reagents: prepared in Example 1, 2 mL × 1 bottle; PBS buffer: 0.1 M, pH 7.4 ± 0.1, 30 mL × 1 bottle;

[0033] Positive control: 1:320 syphilis positive serum, 0.5 mL × 1 bottle; Negative control: healthy human serum, 0.5 mL × 1 bottle; 96-well U-shaped reaction plate × 1 piece; Instructions for use (including quality control chart). Precautions: Avoid freezing reagents; a pH deviation of ±0.2 will increase the false positive rate by 5%; different batches of reagents should not be mixed.

[0034] 3.2 Reagent kit performance validation (n=100, sensitivity: 95% CI: 96.5-99.9%)

[0035] Table 3 Performance evaluation indicators of the reagent kit of the present invention

[0036]

[0037] 3.3 Reagent kit storage conditions and stability verification: After 120 days, the shedding rate was 4.9%, meeting the requirement of ≤5%.

[0038] Table 4 Evaluation indicators of storage conditions for the reagent kit of the present invention

[0039]

[0040] In the accompanying drawings of this invention, Figure 1 The diagram shows the structure of a passive agglutination assay instrument, which includes: (1) the instrument body; (2) the sample injection unit; (3) the serum complement inactivation unit; (4) the sample addition unit; (5) the incubation unit; and (6) the colorimetric unit. Figure 2 The following are experimental and positive / negative control images of a 96-well U-shaped plate, including: (1) 96-well U-shaped plate; (2) U-shaped plate test wells; (3) negative result (red button-shaped precipitate); (4) positive result (uniform blue-purple agglomerate A). 650nm / A 520nm ≥1.5); (5) Negative control; (6) Weak positive control (uniform blue-purple agglomerate A) 650nm / A 520nm (1.5-2.0); (7) Strong positive control (uniform blue-purple agglomerate A) 650nm / A 520nm >2.0).

[0041] Example 2:

[0042] Application of the technical solution in the detection of antigens of other pathogenic microorganisms

[0043] Using the same process parameters as in Example 1, the passive agglutination quantitative detection of other pathogenic microorganism antigens, such as Mycoplasma pneumoniae antigen, respiratory syncytial virus antigen, and influenza A virus antigen, was performed using 50nm AuNPs-SH: Covalent coupling: antigen to AuNPs-SH mass ratio 1:50, stirred at 37℃ in the dark for 2 hours; Blocking and purification: 0.5% BSA blocking, centrifugation at 12000×g to a final concentration of 0.02mg / mL; Detection procedure: Sample inactivation at 56℃ for 30 minutes → 1:10 dilution → mixing with equal volume of reagent → incubation at 37℃ for 1 hour → A 650nm / A 520nm A value ≥1.5 is considered positive (when detecting respiratory pathogen IgM antibodies, serum samples should be pretreated with donkey anti-human IgG antibody at a final concentration of 2 μg / mL, incubated at 37℃ for 15 min, and then centrifuged to collect the supernatant, which can effectively remove IgG interference; a positive IgM result indicates recent infection). Results showed: Mycoplasma pneumoniae: shedding rate 3.2%; Respiratory syncytial virus: shedding rate 4.1%, activity ≥96% after 3 months of storage at 4℃; non-specific adsorption rate <5%, detection rate of low-titer samples increased by 25%-30% compared to traditional TPPA, intra-assay CV <5%. Validation of the detection of Mycoplasma pneumoniae and respiratory syncytial virus antigens showed that its performance parameters (shedding rate, specificity, detection rate) were consistent with those of Treponema pallidum detection, demonstrating the universality of the technology.

[0044] The above embodiments demonstrate that the present invention, through the combination of 50nm AuNPs-SH covalent coupling process, 0.5% BSA blocking technology, dual-wavelength criteria, and serum optimization treatment, significantly improves the stability, specificity, and semi-quantitative capability of passive agglutination detection of pathogenic microorganism antigens. Moreover, the process is industrializable and applicable to the diagnosis and monitoring of various pathogen infections in clinical settings.

[0045] The scope of protection of this invention includes: a 0.02 mg / mL detection reagent (stable at 4°C for 3 months in the dark) prepared by covalently coupling 50 nm AuNPs-SH with Treponema pallidum-specific antigen at a mass ratio of 1:50 via thiol-amino groups, blocking with 0.5% BSA, and centrifugation purification, and its preparation method (including dilution, coupling, blocking, purification, and storage steps); and the detection method using this reagent (including serum inactivation at 56°C, 1:10 dilution, incubation with the reagent, and qualitative analysis by visual inspection or A...). 650nm / A 520nm≥1.5 spectral quantitative interpretation); detection kits containing the reagent, PBS buffer, BSA solution, positive and negative controls, 96-well U-shaped reaction plate and instructions; and methods and kits that extend the above covalent coupling process, blocking method and detection procedure to the detection of antigens of other pathogens such as Mycoplasma pneumoniae are all within the scope of protection of this invention.

Claims

1. A semi-quantitative detection reagent and method for passive agglutination of pathogenic microbial antigens based on covalent coupling of thiol-modified gold nanoparticles, characterized in that, The core technical features include: (1) a thiol-amino covalent coupling process using 50nm thiol-modified gold nanoparticles and Treponema pallidum antigen; (2) a precise blocking process using 0.5% BSA; and (3) the establishment of an A 650nm / A 520nm ≥1.5 is a positive dual-wavelength spectral semi-quantitative criterion; (4) serum samples are inactivated at 56℃ for 30 min and then diluted 1:

10.

2. The semi-quantitative detection reagent and method for passive agglutination of pathogenic microbial antigens based on covalent coupling of thiol-modified gold nanoparticles according to claim 1, characterized in that, The specific process of thiol-amino covalent coupling between the 50nm thiol-modified gold nanoparticles and Treponema pallidum antigen is as follows: using 50nm thiol-modified gold nanoparticles as a carrier, the antigen and AuNPs-SH are covalently linked by a "thiol-amino" bond at a mass ratio of 1:

50. After centrifugation and purification, a ready-to-use liquid reagent with a concentration of 0.02mg / mL is prepared. The antigen shedding rate is ≤5% after 120 days of ELISA verification (4.9% verified after 120 days). It can be stably stored at 4℃ in the dark for 3 months.

3. The semi-quantitative detection reagent and method for passive agglutination of pathogenic microbial antigens based on covalent coupling of thiol-modified gold nanoparticles according to claim 1, characterized in that, The 0.5% BSA precise blocking process is as follows: blocking is performed using BSA at a final concentration of 0.5%. The blocking solution is prepared using 0.1M PBS buffer (pH 7.4±0.1) containing 0.1% BSA as the base solvent (i.e., the base buffer contains 0.1% BSA, and BSA powder is added to achieve a final concentration of 0.5%). A final concentration of 5-10% glycerol or 2% trehalose is added to the storage buffer as a stabilizer, with an ionic strength equivalent to 0.15M NaCl concentration, to avoid particle aggregation and antigen shedding.

4. The semi-quantitative detection reagent and method for passive agglutination of pathogenic microbial antigens based on covalent coupling of thiol-modified gold nanoparticles according to claim 1, characterized in that, When Treponema pallidum antibodies are present in the sample, the antibodies bind to the surface antigen of thiol-modified gold nanoparticles (AuNPs-SH) to form a cross-linked structure, causing the AuNPs-SH particles to aggregate. The solution color changes from red to blue-purple, the absorbance at 520 nm (dispersed state) decreases, and the absorbance at 650 nm (aggregated state) increases. The absorbance at both wavelengths is measured using a UV-Vis spectrophotometer, and A is calculated. 650nm / A 520nm A ratio ≥1.5 is considered positive, while a negative sample ratio <1.5 indicates semi-quantitative detection. Aggregation status is assessed as follows: positive samples show uniform blue-purple aggregates (without button-shaped precipitates), while negative samples show dispersed particles with clear edges and red button-shaped precipitates.

5. The semi-quantitative detection reagent and method for passive agglutination of pathogenic microbial antigens based on covalent coupling of thiol-modified gold nanoparticles according to claim 1, characterized in that, The specific optimization treatment for complement inactivation of syphilis serum is as follows: serum samples are inactivated in a 56°C water bath for 30 min, and then diluted with PBS at a ratio of 1:

10. This treatment can reduce the false positive rate caused by complement interference from 10% to 0.8%, and the detection specificity reaches 99.2% (sensitivity: 95% CI: 98.5-99.8%).

6. The semi-quantitative detection reagent and method for passive agglutination of pathogenic microbial antigens based on covalent coupling of thiol-modified gold nanoparticles according to claim 1, characterized in that, The detection kit based on this reagent includes: the covalently coupled detection reagent, 0.1M PBS buffer (pH 7.4±0.1), 0.5% BSA solution, positive control (syphilis-positive serum with TPPA titer 1:320), negative control (serum from healthy individuals), a 96-well U-shaped microplate, and instructions for use.

7. The semi-quantitative detection reagent and method for passive agglutination of pathogenic microbial antigens based on covalent coupling of thiol-modified gold nanoparticles according to any one of claims 1-6, the scope of protection of which includes: (1) A pathogenic microorganism antigen detection reagent, which is prepared by the following process as defined in claims 1-6: 50nm AuNPs-SH is covalently coupled with pathogenic microorganism antigen at a mass ratio of 1:50 via thiol-amino groups, precisely blocked with 0.5% BSA, and then subjected to A... 650nm / A 520nm ≥1.5 semi-quantitative criteria, and a processing procedure for serum samples to be inactivated at 56℃ for 30 min and then diluted 1:10; (2) a method for detecting pathogenic microorganism antibodies, which is based on the antigen detection reagents defined in claims 1-6, and adopts a sample processing procedure (serum inactivated at 56℃ for 30 min and then diluted 1:10), reaction incubation conditions of incubation at 37℃ in the dark for 1 hour and result interpretation rules (A 650nm / A 520nm ≥1.5 spectral criteria); (3) A detection kit comprising: the covalently coupled detection reagent as described in claims 1-6 as in claim 6, 0.1M PBS buffer (pH 7.4 ± 0.1), a positive control (syphilis-positive serum with a TPPA titer of 1:320), a negative control (serum from healthy individuals), and a 96-well U-shaped reaction plate; (4) The thiol-amino covalent coupling process as defined in claims 1-6, the 0.5% BSA blocking method, and A 650nm / A 520nm Application of ≥1.5 semi-quantitative criteria and serum complement inactivation process in the preparation of antigen detection reagents, detection methods or kits for Mycoplasma pneumoniae antigen, respiratory syncytial virus antigen or influenza A virus.

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