Colloidal gold immunochromatography test strip for saliva sample detection and preparation method thereof

Abstract

The invention discloses a colloidal gold immunochromatography test strip for saliva sample detection and a preparation method thereof.The test strip comprises a supporting layer, and a sample pad, a combination pad, an NC membrane and a water absorption layer are sequentially arranged on the supporting layer from the sample introduction end; at least partial areas of the sample pad and the combination pad are obtained by drying after being pretreated by saliva sample treatment liquid, and the saliva sample treatment liquid comprises the following components: a zwitterionic anti-adsorption buffer system, an antibody conformation relaxation agent, a chelating agent type colloid redispersant, hydrophilic small molecular protein and a cyclodextrin type hydrophobic shielding agent. According to the method, systematic improvement is performed from the biochemical performance of the sample and the state of the antigen target, so that the defects that the target is covered by the colloidal environment of saliva and the target is lost due to aggregation of the target antigen are effectively overcome, and the detection sensitivity is greatly improved.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, specifically to a colloidal gold immunochromatographic test strip for saliva sample detection and its preparation method. Background Technology

[0002] Compared to routine test samples such as serum, plasma, or urine, saliva is a highly heterogeneous and complex biological fluid, and its composition and physicochemical properties pose significant challenges to immunochromatographic detection. In addition to water, saliva contains a large amount of mucins (such as MUC5B and MUC7), glycoproteins, peptides, enzymes, and inorganic salts. Although its overall protein concentration is lower than that of serum, the proportion of high-molecular-weight viscous components is significantly higher. These viscous components easily form a three-dimensional colloidal network structure, which can affect the analysis of target analytes for the following reasons: target analytes are embedded or adsorbed by mucins, forming a physical shield; under the influence of mucins, target analytes undergo non-specific association with high-abundance proteins or glycoproteins, leading to conformational changes; target analytes exist in the form of complexes or aggregates, reducing the effective free concentration.

[0003] Therefore, it is difficult to fully expose the immunorecognition epitopes of the target analyte, and even if the antibody itself has high affinity, it is difficult to achieve effective binding, which directly restricts the sensitivity and accuracy of immunochromatographic detection.

[0004] The structural design of existing lateral flow immunochromatographic test strips is typically optimized for low-viscosity, low-structure sample systems such as blood and urine. The fundamental premise is that the target analyte can migrate in a relatively free molecular state under capillary drive. However, this premise often fails under saliva sample conditions. Specifically, the sample migration between the sample pad and the nitrocellulose membrane is uneven, with significant retention. Even if low-abundance analytes can form complexes with labeled antibodies, they are unlikely to accumulate to the visible threshold in the detection line area, resulting in insufficient sensitivity.

[0005] To address the aforementioned issues, existing technologies typically employ the following approaches for improvement: enhancing the signal intensity of the marker, such as increasing the colloidal gold particle size, using enhancers to enhance color development, or introducing fluorescent probes; increasing the amount of antibody or improving antibody affinity; or introducing external instruments to assist in enrichment or signal reading.

[0006] However, the above methods still cannot meet the requirements for processing saliva samples. This is mainly because: increasing the signal intensity of the marker does not solve the problem of the target analyte being embedded or masked. Instead, it is easy to cause background increase due to non-specific adsorption in saliva; simply increasing the amount of antibody is difficult to overcome the bottleneck of limited immunobinding kinetics and is likely to aggravate non-specific binding of the test strip; instrument-assisted enrichment (such as magnetic separation, electrophoresis or centrifugation) can improve sensitivity, but it significantly increases the complexity of operation, which deviates from the original intention of immunochromatography to be "ready to use and test", and is difficult to apply to home or field testing scenarios.

[0007] Furthermore, existing sample preparation solutions are limited in that they are mostly developed for blood / serum / urine systems, lacking specificity for mucin networks, IgA complexes, and salivary colloidal structures. This often results in slow chromatography, high background, signal heterogeneity, and poor repeatability in saliva testing. Operationally, many existing "effective sample preparation" methods require multiple pretreatment steps such as centrifugation and incubation, making them unsuitable for lateral chromatography test strips or home testing. Summary of the Invention

[0008] To address the aforementioned technical problems, this invention proposes a colloidal gold immunochromatographic test strip for saliva sample detection. By multidimensionally regulating the depolymerization state, conformational accessibility, and dispersion stability of the target analyte, it transforms the analyte from an "undetectable state" to a "highly efficient capture state" in the immunochromatographic system, thereby significantly improving detection sensitivity.

[0009] The colloidal gold immunochromatographic test strip for saliva sample detection of the present invention includes a support layer, on which a sample pad, a conjugate pad, an NC membrane and an absorbent layer are sequentially disposed from the sample injection end. At least a portion of the sample pad and the conjugate pad are obtained by pretreatment with saliva sample processing solution and then drying. The saliva sample processing solution contains the following components: a zwitterionic anti-adsorption buffer system, antibody conformation relaxant, chelating agent-type colloidal redispersant, hydrophilic small molecule protein and cyclodextrin-type hydrophobic shielding agent.

[0010] This application utilizes a zwitterionic anti-adsorption buffer system to significantly reduce the loss of target antigens due to non-specific adsorption on the porous material surface of the test strip, ensuring maximum target migration to the detection area. The addition of hydrophilic small-molecule proteins further releases target antigens bound to IgA, amylase, and mucin in saliva, increasing the content of free targets. Cyclodextrin-based hydrophobic shielding agents also increase the proportion of effective monomeric targets by inhibiting antigen-antigen self-aggregation. By using the chelating agent EDTA to reduce the network strength of the colloidal system formed by saliva itself, targets can more easily enter a free migration state, releasing antigens from saliva and effectively improving the detection sensitivity of the target antigen. Simultaneously, this application utilizes an antibody conformation relaxant to fully expose the epitopes of the target that have been masked, thus maximizing the detection of target sites without increasing the target content. Therefore, this application weakens the non-covalent interactions between the target analyte and the saliva polymer components, inhibiting the re-aggregation or non-specific adsorption of the target analyte during chromatography. Simultaneously, it reduces the colloidal stability of the saliva system, transforming the target analyte into a freely diffused state, and modulates the conformational state of the target analyte to improve epitope accessibility. Consequently, the identifiable concentration and recognition sites of the target antigen in the original saliva sample are significantly increased compared to existing technologies. The components in the saliva sample processing solution of the test strip in this application work synergistically, no longer adjusting for a single factor, thus truly increasing the effective number of the target and the number of effective sites, thereby greatly improving the sensitivity of saliva sample detection. By treating the sample pad of the test strip with the saliva sample processing solution, this application allows for complete satisfaction of measurement requirements even in the simple operating environment of ordinary test strips, greatly reducing operational difficulty and meeting the technical requirements of POCT.

[0011] Furthermore, the zwitterionic anti-adsorption buffer system is selected from at least one of the HEPES-NaOH buffer system, MOPS, and CAPS.

[0012] Furthermore, the antibody conformation relaxant is selected from at least one of urea, guanidine hydrochloride, and guanidine thiocyanate. This application does not "cleave" the protein, but rather allows it to move from a "closed state" to a "semi-relaxed state," thereby fully exposing the target epitopes that are easily obscured and enhancing antibody affinity.

[0013] Furthermore, the hydrophilic small molecule protein is at least one of casein polypeptide or polylysine.

[0014] Furthermore, the cyclodextrin-based hydrophobic shielding agent is at least one of β-cyclodextrin or hydroxypropyl-β-cyclodextrin.

[0015] Furthermore, the saliva sample processing solution contains the following components: 5-150mM HEPES-NaOH buffer system, 0.1M-2M urea, 0.03%-0.2% (w / w) casein polypeptide, and 1mM-20mM hydroxypropyl-β-cyclodextrin.

[0016] Furthermore, the saliva sample processing solution contains the following components: the concentration of the HEPES-NaOH buffer system is 10-100mM, the concentration of the urea is 0.1M-1M, the weight percentage of the casein polypeptide is 0.03%-0.1%, and the content of the hydroxypropyl-β-cyclodextrin is 1mM-10mM.

[0017] Furthermore, the concentration of the HEPES-NaOH buffer system is 5 mM, the concentration of the urea is 0.3 M-0.6 M, the weight percentage of the casein polypeptide is 0.03%-0.06%, and the content of the hydroxypropyl-β-cyclodextrin is 1 mM-6 mM.

[0018] Furthermore, the target to be tested is at least one of HCG or PEP.

[0019] Furthermore, the detection limit for PEP is ≥5 ng / mL, and the detection limit for HCG is ≥0.25 mIU / mL.

[0020] The preparation method of the colloidal gold immunochromatographic test strip for saliva sample detection of the present invention includes the following steps:

[0021] Pre-fabricated colloidal gold-antibody labeling; The steps of pretreatment with saliva sample processing solution are as follows: immerse the sample pad and / or conjugate pad to be treated in the saliva sample processing solution, and then dry them. Preparation of gold-labeled pads: The label was resuspended in the purified gold-labeled antibody solution, fixed on a polyester fiber membrane by quantitative spraying at 2 μL / cm, and then dried; Preparation of the detection line: Another monoclonal antibody against the target analyte is sprayed onto a nitrocellulose membrane; Preparation of quality control lines: Secondary antibody containing anti-labeled antibody was sprayed onto a nitrocellulose membrane, fixed onto the nitrocellulose membrane by quantitative streak at 1 μL / cm, and then dried; Assembly steps: On the back plate, arrange the structural layers in the following order, overlapping each other at the ends that are close to each other: nitrocellulose membrane, gold label pad, sample pad and absorbent pad.

[0022] The present invention has the following beneficial effects: ① The test strip of the present invention can increase the proportion of targets that effectively participate in the immune response in a unit volume of sample; increase the apparent binding rate of targets and antibodies; and reduce the loss rate of targets at the front end of chromatography, that is, increase the functional concentration without changing the true concentration of the sample.

[0023] ② This application makes systematic improvements based on the biochemical properties of the sample itself and the state of the antigen target. It has lower requirements for the content of the analyte and lower detection limit. It effectively overcomes the loss of the target caused by the colloidal environment of saliva covering the target and the aggregation of the target antigen itself, and greatly improves the detection sensitivity (the detection limit for PEP can be reduced to as low as 5 ng / mL and the detection limit for HCG can be reduced to as low as 0.25 mIU / mL).

[0024] ③ This application directly treats the sample pad and conjugate pad of the paper strip with saliva sample processing solution. During the test, the enrichment effect of the detection site of the target can be completed simultaneously during the diffusion of the sample on the test strip. This does not require any additional user operation steps, does not rely on any external equipment, and does not increase the detection time, thus providing greater feasibility for the commercialization of POCT. Detailed Implementation

[0025] This invention provides a colloidal gold immunochromatographic test strip for saliva sample detection, comprising a support layer, on which a sample pad, a conjugate pad, an NC membrane, and an absorbent layer are sequentially disposed from the sample injection end. The edges of the structural layers can overlap (e.g., the overlap length can be 1-2 mm). At least a portion of the sample pad and conjugate pad are obtained by pretreatment with a saliva sample processing solution followed by drying. The saliva sample processing solution contains the following components: a zwitterionic anti-adsorption buffer system, antibody conformation relaxant, chelating agent-type colloidal redispersant, hydrophilic small molecule protein, and cyclodextrin-type hydrophobic shielding agent. This application, by treating the sample pad and conjugate pad of the test strip with a unique saliva sample processing solution, transforms the target analyte from a complex, aggregated, or embedded state into a freely diffusible free state after the saliva sample passes through the sample pad, thus fully exposing the immune binding sites. Therefore, no pretreatment steps for saliva samples are required. Instead, the target sample is fully exposed during the original test strip detection process, and the influence of interfering substances is minimized, thus greatly improving the detection sensitivity of the sample.

[0026] The preparation method of the colloidal gold immunochromatographic test strip for saliva sample detection of the present invention includes the following steps: Pre-fabricated colloidal gold-antibody labeling; The steps of pretreatment with saliva sample processing solution are as follows: immerse the sample pad or conjugate pad to be treated in the saliva sample processing solution, and then dry it. Preparation of gold-labeled pads: The label was resuspended in the purified gold-labeled antibody solution, fixed on a polyester fiber membrane by quantitative spraying at 2 μL / cm, and then dried; Preparation of the detection line: Another monoclonal antibody against the target analyte is sprayed onto a nitrocellulose membrane; Preparation of quality control lines: Secondary antibody containing anti-labeled antibody was sprayed onto a nitrocellulose membrane, fixed onto the nitrocellulose membrane by quantitative streak at 1 μL / cm, and then dried; Assembly steps: On the back plate, arrange the structural layers in the following order, overlapping each other at the ends that are close to each other: nitrocellulose membrane, gold label pad, sample pad and absorbent pad.

[0027] In some embodiments, the test strip of the present invention further includes a protective film layer and a protective shell disposed on the outside of the test strip.

[0028] The preferred embodiments of the present invention will be described in detail below with reference to specific examples, so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby making a clearer and more explicit definition of the scope of protection of the present invention.

[0029] Example 1: Preparation of a colloidal gold immunochromatographic test strip for direct determination of saliva 1.1 Preparation of colloidal gold solution: Take 100 ml of 0.01% chloroauric acid aqueous solution and heat to boiling. While stirring, accurately add 0.7 ml of 1% trisodium citrate aqueous solution. The golden yellow chloroauric acid aqueous solution turns purple-red within 2 minutes. Continue boiling for 15 minutes. After cooling, restore to the original volume with distilled water. The gold sol prepared in this way has a highest absorption peak in the visible light region of 535 nm, and the measured value OD535 is about 1.1.

[0030] 1.2 Colloidal Gold-Antibody Labeling and Purification: Adjust the pH of 1 mL of colloidal gold solution to 8.0 using 0.1 Mk2CO3 solution. Then, add 1 mg of the antibody to be labeled dropwise to the colloidal gold solution and mix at low speed at room temperature for 60 minutes. Add 1 mL of blocking agent (100 mM Tris-HCl (pH 8.5), 1% BSA, 5% skim milk, 0.5% PEG8000, 0.1% Proclin 300), and continue mixing by rotation for 30 minutes. Centrifuge at 4℃ / 14,000 rpm for 5 minutes, discard the supernatant, and resuspend the precipitate in reconstitution solution (50 mM Tris-HCl (pH 8.0), 0.1% BSA, 7.5% trehalose, 0.1% Tween 20). Repeat twice. Resuspend the precipitate in 1 mL of reconstitution solution and store at 4℃ for later use.

[0031] 1.3 Processing of various chromatography-related membranes: Glass fiber membranes were immersed in sample processing solution (the saliva sample processing solution of this invention or conventional saliva sample processing solution: 50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1% Triton X-100, 0.5% sodium caseinate, 0.5% N-acetyl-L-cysteine), and then dried at 37°C for 12 hours to prepare sample pads. The labeled and purified gold-labeled antibody solution was resuspended and fixed onto a polyester fiber membrane using a quantitative spraying method at 2 μL / cm, and then dried at 37°C for 12 hours. Detection line (T line): another monoclonal antibody (1 mg / mL) targeting the analyte was sprayed onto the membrane. Control line (C line): a secondary antibody against the labeled antibody (e.g., goat anti-mouse IgG, 1 mg / mL) was sprayed onto the membrane and fixed onto a nitrocellulose membrane (NC membrane) using a quantitative streaking method at 1 μL / cm, and then dried at 37°C for 12 hours.

[0032] 1.4 Attachment of the Chromatography Membrane and Cutting and Assembly of the Test Strips: On the PVC backing board, attach the dried components in sequence with overlap: NC membrane → gold label pad → sample pad → absorbent pad. Each component should overlap by approximately 1-2 mm to ensure smooth chromatography without gaps. Cut the assembled plate into test strips of uniform width (4 mm) using a strip cutter. Stack the cartridge, saliva swab, and test strips in sequence, press them together with a cartridge press, and then close the cartridge cap.

[0033] Example 2: Sensitivity assessment target and sample preparation for direct salivary immunochromatographic assay To better evaluate the effectiveness of this invention in detecting saliva samples, two representative targets in saliva were selected: pepsin (PEP) and human chorionic gonadotropin (HCG). Clinically high-concentration positive saliva samples were used as samples, diluted with negative saliva samples to prepare concentration gradient sensitivity reference standards for performance testing, as detailed in Table 1. Table 1

[0034] Example 3: Comparison of the effects of different anti-adsorption buffer systems and their dosages on saliva sample testing. The concentration gradient HEPES-NaOH buffer system was prepared according to the method in Table 2. Using conventional saliva sample processing solution as a control, saliva samples were prepared according to the method in Example 2. Each saliva sample was tested using the test strips prepared in Example 1. The differences in the effect of test strips prepared using HEPES-NaOH and Tris-HCl (pH 8.0) as buffer systems on the saliva detection effect were compared. The concentrations of the HEPES-NaOH buffer system used were also compared. The specific formulations of the sample processing solutions are shown in Table 2. Table 2

[0035] The sensitivity of saliva samples treated with different sample processing solutions to concentration gradient targets is shown in Table 3. Table 3

[0036] The results showed that using the HEPES-NaOH buffer system as the buffer system for saliva sample processing can, to a certain extent, increase the detection sensitivity of targets in saliva sample detection and lower the detection limits for PEP and HCG. Adding 10 mM HEPES-NaOH buffer system is sufficient to promote the improvement of detection sensitivity. This invention utilizes the zwitterionic system of the HEPES-NaOH buffer system to form a highly hydrated, near-neutral "dynamic water shell" at the solid-liquid interface, simultaneously weakening the three main driving forces of electrostatic adsorption, hydrophobic adsorption, and ion bridge adsorption. This significantly reduces the non-specific loss of antigens on the porous material surface, thereby minimizing antigen loss during the test strip chromatography process and ensuring sufficient migration to the detection area of ​​the test strip.

[0037] In other possible implementations, MOPS or CAPS zwitterionic anti-adsorption buffer systems can be used to replace HEPES.

[0038] Example 4: Comparison of the effect of adding antibody conformation relaxant to saliva sample processing solution on detection sensitivity Saliva sample processing solutions containing different concentration gradients of urea were prepared according to the method in Table 4. Using conventional saliva sample processing solutions as a control, test strips were prepared according to the method in Example 1, and samples were prepared according to the method in Example 2. Each saliva sample was tested, and the differences in the effects of using urea as a de-adhesive and existing saliva sample processing solutions on the saliva detection effect were compared. The specific formulations of each sample processing solution are shown in Table 4. Table 4

[0039] The sensitivity detection results of different formulation sample treatment solutions to concentration gradient targets are shown in Table 5: Table 5

[0040] The results showed that using urea instead of the deadhesive N-acetyl-L-cysteine ​​in the saliva sample processing solution significantly improved the detection sensitivity of the targets HCG and PEP in saliva samples. The detection limit for PEP was further reduced from 25 ng / mL (Table 3) to 20 ng / mL, while the detection limit for HCG was reduced from 2 mIU / mL (Table 3) to 1 mIU / mL. This is because N-acetyl-L-cysteine ​​(NAC) mainly exerts its deadhesive effect by cleaving the disulfide bonds of mucin, and it only targets mucin. Urea, on the other hand, breaks the original hydrogen bond network of proteins, causing the structure of most interfering proteins in saliva to gradually become loose and disordered, leading to the disintegration of their secondary and tertiary structures, thus reducing the interference of HCG and PEP detection. Unlike existing technologies that use N-acetyl-L-cysteine ​​to "cleave" proteins for debinding, urea loosens the molecular conformation of the target protein, moving it from a "closed state" to a "semi-relaxed state." This fully exposes the target, which is easily obscured, thus increasing the detection sensitivity of the analyte. In other possible implementations, urea can be replaced with guanidine hydrochloride or guanidine thiocyanate, or any substance that can loosen the protein structure in a similar way.

[0041] Example 5: The role of different concentrations of hydrophilic small molecule proteins in enhancing sensitivity As shown in Table 6, casein peptides with concentration gradients were prepared as hydrophilic small molecule proteins. Conventional saliva sample processing solution was used as a control. Simultaneously, 0.03% casein peptide (CL-30L) and 0.06% casein peptide (CL-60L) were added to the conventional saliva sample processing solution as controls. Test strips were prepared according to the procedure in Example 1, and samples were prepared according to the method in Example 2. The effects of different concentrations of casein peptides in saliva sample detection were studied. The specific formulations of each sample processing solution are shown in Table 6. Table 6

[0042] The sensitivity detection results of different formulation sample treatment solutions to concentration gradient targets are shown in Table 7. Table 7

[0043] The results showed that adding hydrophilic small molecule protein (casein polypeptide) to the system further improved the detection sensitivity of targets in saliva samples. The synergistic effect of the anti-adsorption buffer system of this invention with casein polypeptide and urea was significantly better than that of adding a single component in the control group. With the addition of urea and the use of a HEPES-NaOH (pH 8.0) buffer system in the saliva sample processing system, this embodiment further reduced the detection limit by adding casein polypeptide (the detection limit for PEP decreased from 20 ng / mL to 15 ng / mL; the detection limit for HCG decreased from 1 mIU / mL to 0.5 mIU / mL).

[0044] However, when the PEP content is relatively low, the detection sensitivity is not necessarily higher with a higher amount of casein peptides. In particular, when the amount of casein peptides added is 0.2%, it cannot be detected when the amount of PEP added is 15 ng / mL and 20 ng / mL (Table 7). This indicates that the amount of casein peptides added needs to be controlled within a certain range.

[0045] In the control sample, although the addition of a certain amount of casein peptides could lower the detection limit to some extent, it was still significantly higher than that of the saliva sample processed using the urea-added and HEPES-NaOH (pH 8.0) buffer system in this application. This application utilizes casein peptides, which, although their binding affinity to the target is weaker than that of IgA, amylase, or mucin, can reduce the binding of the target to IgA, amylase, or mucin due to their larger addition amount. Therefore, without altering the target's structure, the target is displaced from the surface of IgA, amylase, mucin, etc., thereby improving the detection sensitivity of the target.

[0046] In other possible implementations, polylysine may also be used.

[0047] Example 6: The role of different concentrations of cyclodextrin in improving sensitivity Saliva sample processing solutions containing hydroxypropyl-β-cyclodextrin at various concentration gradients were prepared according to Table 8. A conventional saliva sample processing solution was used as a control (CL). Simultaneously, 3 mM (CL-30H) and 6 mM (CL-60H) of hydroxypropyl-β-cyclodextrin were added to the control solution as synchronous controls. Test strips were prepared according to the procedure in Example 1, and the samples from Example 2 were tested.

[0048] Table 8

[0049] The sensitivity detection results of different formulation sample treatment solutions to concentration gradient targets are shown in Table 9: Table 9

[0050] The results show that in the processing solution system of this application, the addition of only 1 mM of βCD can significantly improve the detection sensitivity. This is because many antigens in saliva exist in a semi-folded state with their hydrophobic surfaces exposed. βCD can increase the proportion of effective monomeric targets by inhibiting antigen-antigen self-aggregation, thereby improving the detection sensitivity of targets in saliva samples to a certain extent.

[0051] Although the addition of βCD to the control sample processing solution can also improve sensitivity using cyclodextrin-based systems, the synergistic effect of the anti-adsorption buffer system of this invention with hydroxypropyl-β-cyclodextrin and urea is significantly better than the addition of a single component in the control group. This further illustrates that the components in the formulation of the sample processing solution of this application have a synergistic effect with each other.

[0052] Furthermore, when detecting PEP and HCG, the amount of β-CD added is not necessarily better when the target concentration is relatively low (PEP at 10 ng / mL and 15 ng / mL or lower, and HCG at 0.5 mIU / mL or lower). A β-CD addition of ≤6 mM is sufficient to meet the requirements for high-sensitivity detection.

[0053] The cyclodextrin-based hydrophobic modifier selected in this application can encapsulate hydrophobic molecules within its cavity through inclusion action, and form the hydrophilic groups of the cyclodextrin-based hydrophobic modifier on the side away from the antibody, thereby improving the detectability of the target during detection.

[0054] Example 7: The role of different concentrations of chelating agent-type colloidal redispersants in improving sensitivity Sample processing solutions containing different concentration gradients of EDTA disodium salt were prepared. Using conventional saliva sample processing solution as a control, test strips were prepared according to the procedure in Example 1, and samples from Example 2 were tested to determine the effect of different concentrations of zwitterionic anti-adsorption buffer systems. The specific formulations of each sample processing solution are shown in Table 10. Table 10 The sensitivity detection results of different formulation sample treatment solutions to concentration gradient targets are shown in Table 11: Table 11

[0055] In the control solution, the addition of disodium EDTA did not improve the detection sensitivity of PEP. However, in the saliva sample processing system of this application, due to the use of the HEPES-NaOH (pH 8.0) buffer system and the replacement of urea with N-acetyl-L-cysteine, the detection limit for PEP can be further reduced from 15 ng / mL (Table 7) to 10 ng / mL. It can be seen that disodium EDTA can improve the detection sensitivity of the saliva sample processing solution of this invention.

[0056] In HCG detection, this invention utilizes a sample processing solution prepared with a HEPES-NaOH (pH 8.0) buffer system. Furthermore, by replacing urea with N-acetyl-L-cysteine ​​and adding 0.1 mM EDTA disodium salt, the detection limit for HCG can be reduced from 1 mIU / mL to 0.5 mIU / mL. This application utilizes the chelating agent EDTA to reduce the network strength of the colloidal system formed by saliva itself, thus making it easier for the target to enter a free migration state and releasing the antigen in the saliva, effectively improving the detection sensitivity of the target antigen.

[0057] Example 8: Comparison of the effects of the amount of each component added to the treatment solution on improving detection sensitivity To further verify the synergistic effect of each component on the saliva sample processing solution, each sample processing solution was prepared according to Table 12, with the conventional saliva sample processing solution as a control. Urea, disodium EDTA, casein polypeptide and hydroxypropyl-β-cyclodextrin were added sequentially to the conventional saliva sample processing solution. Test strips were prepared according to the procedure of Example 1 and tested according to the samples of Example 2.

[0058] Table 12 The sensitivity detection results of different formulation sample treatment solutions to concentration gradient targets are shown in Table 13. As shown: Table 13

[0059] The results show that the system according to the present invention, with the addition of all strategies for synergistic effect, can significantly improve the detection sensitivity of targets in saliva samples (the detection limit of PEP can be reduced to as low as 5 ng / mL, and the detection limit of HCG can be reduced to as low as 0.25 mIU / mL), with strong effectiveness and wide application range.

[0060] When detecting PEP, using the control sample solution, although it contained the same components as the solution of this application (urea, disodium EDTA, and casein peptides, except for the buffer system), the addition of hydroxypropyl-β-cyclodextrin had no effect on the detection efficiency. This precisely demonstrates that only by using the HEPES-NaOH buffer system of this invention in combination with urea, disodium EDTA, and casein peptides can a synergistic effect be achieved with hydroxypropyl-β-cyclodextrin. However, when detecting HCG, even in the control sample solution system, urea, disodium EDTA, casein peptides, and hydroxypropyl-β-cyclodextrin still produced a significant synergistic effect, although it was significantly lower than the effect of the solution prepared using the HEPES-NaOH buffer system of this application. The results of ALL-L, ALL-M, and ALL-H further show that as the amount of each component added to the solution of this application increases to a moderate level, the detection sensitivity for PEP and HCG no longer increases. In other words, a moderate level of each component addition (50mM HEPES-NaOH buffer system, 0.5mM urea, 0.05% casein polypeptide, and 5mM hydroxypropyl-β-cyclodextrin) is already considered optimal.

[0061] The reagent combination of the sample processing solution of the present invention transforms the target analyte in the saliva sample from a complex, aggregated, or embedded state into a freely diffusing state, and maximizes the exposure of the immune binding site, thereby effectively reducing the detection limit of the target in the saliva sample, resulting in high detection sensitivity and greatly reducing the required detection concentration of the sample.

[0062] The above embodiments are only for illustrating the technical concept and features of the present invention. Their purpose is to enable those skilled in the art to understand the content of the present invention and implement it. They should not be used to limit the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A colloidal gold immunochromatographic test strip for saliva sample detection, comprising a support layer, wherein a sample pad, a conjugate pad, an NC membrane, and an absorbent layer are sequentially disposed on the support layer starting from the sample injection end, characterized in that, At least a portion of the sample pad and conjugate pad are obtained by pretreatment with saliva sample processing solution followed by drying. The saliva sample processing solution contains the following components: zwitterionic anti-adsorption buffer system, antibody conformation relaxant, chelating agent-type colloidal redispersant, hydrophilic small molecule protein, and cyclodextrin-type hydrophobic shielding agent.

2. The colloidal gold immunochromatographic test strip for saliva sample detection according to claim 1, characterized in that, The zwitterionic anti-adsorption buffer system is selected from at least one of HEPES-NaOH buffer system, MOPS and CAPS.

3. The colloidal gold immunochromatographic test strip for saliva sample detection according to claim 1, characterized in that, The antibody conformation relaxant is selected from at least one of urea, guanidine hydrochloride, and guanidine thiocyanate.

4. The colloidal gold immunochromatographic test strip for saliva sample detection according to claim 1, characterized in that, The hydrophilic small molecule protein is at least one of casein polypeptide or polylysine.

5. The colloidal gold immunochromatographic test strip for saliva sample detection according to claim 1, characterized in that, The cyclodextrin-based hydrophobic shielding agent is at least one of β-cyclodextrin or hydroxypropyl-β-cyclodextrin.

6. The colloidal gold immunochromatographic test strip for saliva sample detection according to claim 1, characterized in that, The saliva sample processing solution contains the following components: 5-150 mM HEPES-NaOH buffer system, 0.1 M-2 M urea, 0.01%-0.2% casein polypeptide by weight, and 1 mM-20 mM hydroxypropyl-β-cyclodextrin.

7. The colloidal gold immunochromatographic test strip for saliva sample detection according to claim 6, characterized in that, The saliva sample processing solution contains the following components: the concentration of the HEPES-NaOH buffer system is 10-100mM, the concentration of the urea is 0.1M-1M, the weight percentage of the casein polypeptide is 0.03%-0.1%, and the content of the hydroxypropyl-β-cyclodextrin is 1mM-10mM.

8. The colloidal gold immunochromatographic test strip for saliva sample detection according to claim 7, characterized in that, The concentration of the HEPES-NaOH buffer system is 5 mM, the concentration of the urea is 0.3 M-0.6 M, the weight percentage of the casein polypeptide is 0.03%-0.06%, and the content of the hydroxypropyl-β-cyclodextrin is 1 mM-6 mM.

9. The colloidal gold immunochromatographic test strip for saliva sample detection according to any one of claims 1-8, characterized in that, The target to be tested is at least one of HCG or PEP.

10. The method for preparing the colloidal gold immunochromatographic test strip for saliva sample detection according to any one of claims 1-9, characterized in that, Includes the following steps: Pre-fabricated colloidal gold-antibody labeling; The steps of pretreatment with saliva sample processing solution are as follows: immerse the sample pad and / or conjugate pad to be treated in the saliva sample processing solution, and then dry them. Preparation of gold-labeled pads: The label was resuspended in the purified gold-labeled antibody solution, fixed on a polyester fiber membrane by quantitative spraying at 2 μL / cm, and then dried; Preparation of the detection line: Another monoclonal antibody against the target analyte is sprayed onto a nitrocellulose membrane; Preparation of quality control lines: Secondary antibody containing anti-labeled antibody was sprayed onto a nitrocellulose membrane, fixed onto the nitrocellulose membrane by quantitative streak at 1 μL / cm, and then dried; Assembly steps: On the back panel, arrange the structures in the following order and overlap each other at the ends that are close to each other: nitrocellulose membrane, gold label pad, sample pad and absorbent pad.

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