CN-122017229-A - Colloidal gold immunochromatography test strip for saliva sample detection and preparation method thereof

Abstract

The application discloses a colloidal gold immunochromatographic test strip for saliva sample detection and a preparation method thereof, wherein the test strip comprises a support layer, a sample pad, a combination pad, an NC film and a water absorption layer are sequentially arranged on the support layer from a sample injection end, at least partial areas of the sample pad and the combination pad are obtained by drying after pretreatment of saliva sample treatment liquid, and the saliva sample treatment liquid comprises the following components: a zwitterionic anti-adsorption buffer system, an antibody conformation relaxant, a chelating agent colloid redispersing agent, a hydrophilic small molecular protein and a cyclodextrin hydrophobic shielding agent. The application starts from the biochemical property of the sample and the state of the antigen target, effectively overcomes the defects of target coverage caused by the colloid environment of saliva and target antigen aggregation, and greatly improves the detection sensitivity.

Inventors

• WANG CHEN
• ZHAN WEI
• HE JINLI
• LI YIZHEN
• ZHANG XUNFU

Assignees

• 芯孚医疗科技(杭州)有限公司

Dates

Publication Date

20260512

Application Date

20260209

Claims (10)

1. 1. A colloidal gold immunochromatographic test strip for saliva sample detection comprises a supporting layer, wherein a sample pad, a binding pad, an NC film and a water-absorbing layer are sequentially arranged on the supporting layer from a sample injection end, and the colloidal gold immunochromatographic test strip is characterized in that at least partial areas of the sample pad and the binding pad are obtained by pretreatment of saliva sample treatment liquid and drying, and the saliva sample treatment liquid comprises a zwitterionic anti-adsorption buffer system, an antibody conformation relaxing agent, a chelating agent colloid redispersing agent, hydrophilic micromolecular proteins and a cyclodextrin hydrophobic shielding agent.
2. 2. The colloidal gold immunochromatographic strip for saliva sample detection according to claim 1, in which the zwitterionic anti-adsorption buffer system is selected from at least one of HEPES-NaOH buffer system, MOPS and CAPS.
3. 3. The colloidal gold immunochromatographic strip for saliva sample detection according to claim 1, in which the antibody conformation relaxant is at least one selected from urea, guanidine hydrochloride and guanidine thiocyanate.
4. 4. The colloidal gold immunochromatographic strip for saliva sample detection according to claim 1, in which the hydrophilic small molecule protein is at least one of casein polypeptide or polylysine.
5. 5. The colloidal gold immunochromatographic strip for saliva sample detection according to claim 1, wherein the cyclodextrin-based hydrophobic shielding agent is at least one of beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin.
6. 6. The colloidal gold immunochromatographic strip for saliva sample detection according to claim 1, wherein the saliva sample treatment liquid comprises the following components of 5-150mM HEPES-NaOH buffer system, 0.1-2M urea, 0.01% -0.2% casein polypeptide by weight and 1mM-20mM hydroxypropyl-beta-cyclodextrin.
7. 7. The colloidal gold immunochromatographic strip for saliva sample detection according to claim 6, in which the saliva sample treatment solution comprises the following components, HEPES-NaOH buffer system at a concentration of 10-100mM, urea at a concentration of 0.1M-1M, casein polypeptide at a weight percentage of 0.03% -0.1%, and hydroxypropyl-beta-cyclodextrin at a content of 1mM-10mM.
8. 8. The colloidal gold immunochromatographic strip for saliva sample detection according to claim 7, in which the concentration of the HEPES-NaOH buffer system is 5mM, the concentration of urea is 0.3M-0.6M, the weight percentage of casein polypeptide is 0.03% -0.06%, and the content of hydroxypropyl-beta-cyclodextrin is 1mM-6mM.
9. 9. The colloidal gold immunochromatographic strip for saliva sample detection according to any one of claims 1 to 8, in which the target to be detected is at least one of HCG or PEP.
10. 10. The method for preparing the colloidal gold immunochromatographic test strip for saliva sample detection according to any one of claims 1 to 9, comprising the steps of: prefabricating colloidal gold-antibody labeling; the step of pretreatment of saliva sample treatment fluid comprises the steps of immersing a sample pad and/or a bonding pad to be treated in the saliva sample treatment fluid, and then drying; Preparing a gold-labeled pad, namely re-suspending the labeled and purified gold-labeled antibody solution, fixing the solution on a polyester fiber membrane in a quantitative spraying mode of 2 mu L/cm, and then drying; preparing a detection line, namely spraying another monoclonal antibody aiming at a target object on a nitrocellulose membrane; preparing a quality control line, namely spraying a secondary antibody of an anti-labeling antibody on a nitrocellulose membrane, fixing the secondary antibody on the nitrocellulose membrane in a quantitative membrane-dividing mode of 1 mu L/cm, and then drying; The assembling step is that on the back plate, the structures are arranged in the following order and are overlapped with each other at the end parts close to each other, namely, the nitrocellulose membrane, the gold mark pad, the sample pad and the water absorption pad.

Description

Colloidal gold immunochromatography test strip for saliva sample detection and preparation method thereof Technical Field The invention relates to the technical field of biological detection, in particular to a colloidal gold immunochromatographic test strip for saliva sample detection and a preparation method thereof. Background Saliva is a highly heterogeneous complex biological fluid, whose composition and physicochemical properties present significant challenges for immunochromatographic assays, as compared to conventional test samples such as serum, plasma, or urine. In addition to moisture, saliva contains a large amount of mucins (such as MUC5B, MUC), glycoproteins, polypeptides, enzymes and inorganic salts, and the total protein concentration is lower than that of serum, but the proportion of high molecular viscosity components is significantly higher. The viscous components are easy to form a three-dimensional colloid network structure, and can influence the analysis process of the target analyte due to the fact that the target analyte is embedded or adsorbed by mucin to form a physical shield, the target analyte and high-abundance proteins or glycoproteins are in nonspecific association under the action of mucin to cause conformational change, and the target analyte exists in a complex or aggregate form to reduce the effective free concentration. Therefore, it is difficult to fully expose the immunorecognition epitope of the target analyte, and even if the affinity of the antibody itself is high, it is difficult to achieve effective binding, directly restricting the sensitivity and accuracy of immunochromatography detection. The structural design of the existing lateral flow immunochromatographic test strip is generally optimized based on a low-viscosity and low-structural sample system such as blood, urine and the like, and the basic premise is that target analytes can migrate in a relatively free molecular state under capillary drive. However, under saliva sample conditions, this premise is often not true, and is particularly characterized by uneven migration of the sample between the sample pad and the nitrocellulose membrane, significant retention, and difficulty in accumulating the low abundance analytes to a visible threshold, i.e., insufficient sensitivity, in the detection line region, even if the analytes can form complexes with the labeled antibodies. In response to the above problems, the prior art has generally been improved by increasing the signal intensity of the label, such as increasing the colloidal gold particle size, enhancing color development with an enhancer, introducing fluorescent probes, increasing the amount of antibody or increasing the affinity of antibody, introducing external instrumentation to aid in enrichment or signal reading. However, the method still cannot meet the requirements of processing saliva samples, mainly because the problem that target analytes are embedded or shielded cannot be solved by improving the signal strength of the markers, but background rise is easily caused by nonspecific adsorption in saliva, the bottleneck of limited immunobinding kinetics is difficult to break through by simply increasing the dosage of antibodies, nonspecific binding of test strips is easily aggravated, and the sensitivity can be improved by instrumental auxiliary enrichment (such as magnetic separation, electrophoresis or centrifugation), but the operation complexity is obviously increased, so that the method is not suitable for home or field detection scenes. Moreover, the limitations of the existing sample pad treatment solutions are mostly developed for blood/serum/urine systems, and lack pertinence to mucin networks, igA complexes, saliva colloid structures. Problems such as slow chromatography, high background, nonuniform signals, poor repeatability and the like often occur in saliva detection. In the operation level, many "effective sample processing" needs multi-step pretreatment operations such as centrifugation, incubation, etc., and thus is not suitable for detection of lateral chromatography test strips or home detection. Disclosure of Invention In order to solve the technical problems, the invention provides a colloidal gold immunochromatographic test strip for saliva sample detection, which enables the colloidal gold immunochromatographic test strip to be converted from an 'undetectable state' to an 'efficient and capturable state' in an immunochromatographic system by regulating and controlling the depolymerization state, conformational accessibility and dispersion stability of a target analyte in a multidimensional manner, so that the detection sensitivity is remarkably improved. The colloidal gold immunochromatographic test strip for saliva sample detection comprises a supporting layer, wherein a sample pad, a binding pad, an NC film and a water absorption layer are sequentially arranged on the supporting layer from a sample injection end, at least pa