A method for reducing the amount of rinsing solution used in solid-phase immunoassay.

Abstract

This invention belongs to the field of molecular biology detection technology and is mainly used to solve the problem of large amounts of washing solution consumption caused by frequent rinsing in solid-phase immunoassay methods. Specifically, based on the characteristic that the artificial introduction of upstream reagents in solid-phase immunoassay methods will not affect downstream steps, the liquid in the upstream rinsing step is recovered and used for rinsing downstream steps. To avoid cross-binding of upstream analyte-specific reagents between different samples, the recovered washing solution is used for downstream rinsing using an instantaneous rinsing method to minimize the time spent in the process. When the entire batch of samples is testing for the same analyte, the recovered washing solution can be used for immersion washing in each rinsing step.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology detection technology, specifically relating to a method for reducing the consumption of rinsing solution during solid-phase immunological detection. Background Technology

[0002] Currently, biomolecular detection of protein properties is mainly based on immunological principles, such as Western blotting hybridization, enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, and immunofluorescence. The common reaction principle of these technologies is based on the specific binding relationship between antigens and antibodies; that is, specific antibody molecules bind to corresponding specific molecules in the analyzed sample, thereby achieving the labeling purpose.

[0003] Solid-phase immunoassay refers to methods where the analyte is immobilized in a solid medium and then detected using immunological principles (such as Western blotting, immunohistochemistry, and enzyme-linked immunosorbent assay). In these methods, to achieve a visual conversion of the initial labeling step, other labeling steps are often used to amplify or visualize the preceding labeling signal. Subsequent labeling steps are also often based primarily on immunological binding principles and combined with chemical or physical techniques.

[0004] In the multi-step labeling reaction process described above, a rinsing step is usually required to avoid interference from previous step substances on subsequent labeling or signal conversion. This involves using a rinsing buffer to remove molecules (such as primary and secondary antibodies) that did not bind to the analyte after the previous reaction. Each rinse requires a significant amount of rinsing buffer, and typically, a single step's rinsing process needs to be repeated 2-3 times. A large amount of rinsing buffer is consumed after the overall detection is completed.

[0005] In automating the aforementioned detection process at high throughput, the large consumption of rinsing fluid places a significant burden on system maintenance and increases the pressure on waste fluid treatment. Some automated equipment uses a micro-flushing method to reduce waste fluid consumption, but this may sacrifice detection quality or require additional steps. A large-volume rinsing method is a direct approach that ensures detection quality, but its high fluid consumption must be overcome. Summary of the Invention

[0006] One characteristic of solid-phase immunoassay is that reagents in subsequent detection steps have specific binding properties to reagents in adjacent preceding steps, while reagents in intervening steps have no recognition relationship and do not interfere with each other. This invention provides a method for reducing the amount of rinsing solution used in solid-phase immunoassay to overcome the problem of high liquid consumption in large-volume rinsing methods.

[0007] To address the aforementioned issues, a method is proposed to reduce the amount of rinsing solution used in solid-phase immunoassay. This method involves recovering the rinsing solution used in the upstream rinsing step and reusing it in the downstream rinsing step, which requires the same basic rinsing solution.

[0008] To avoid cross-binding of potentially residual analytical-specific reagents in the rinsing solution between different samples, all rinsing steps using recycled rinsing buffer are performed using an instantaneous rinsing method, after which the rinsing solution is removed; immersion rinsing with time retention is not used. When all samples are detecting the same molecular target, either instantaneous rinsing or immersion rinsing with time retention can be used, after which the rinsing solution is removed.

[0009] The following modes are used for flushing fluid recovery:

[0010] 1) Rinse fluid recovery in continuous repetitive mode:

[0011] After completing rinsing step 1, the rinsing solution is recovered;

[0012] The recovered rinsing solution is used to rinse step 2, and then recovered again after completion;

[0013] The recycled rinsing solution is used to rinse step 3, and then recycled again after completion;

[0014] Repeat the process until all tests are completed. This rinsing method can significantly reduce the amount of rinsing fluid consumed.

[0015] 2) Flush fluid recovery in a segmented, repetitive mode:

[0016] After completing rinsing step 1, the rinsing solution is recovered;

[0017] The recovered rinsing solution is used to rinse step 2, and the rinsing solution is recovered again after the step is completed.

[0018] The rinsing solution is used to rinse step n until it is used in rinsing step n. After the rinsing solution is completed, it is not recycled.

[0019] Complete rinsing step n+1 with fresh rinsing solution, and then recover the rinsing solution.

[0020] The rinsing solution recovered in rinsing step n+1 is used to rinse step n+2, and the rinsing solution is recovered after the step is completed.

[0021] The rinsing solution recovered in rinsing step 2n-1 is used to rinse step 2n, and the rinsing solution is not recovered after the rinsing step is completed.

[0022] A recycling cycle consists of n rinsing steps. The recycling cycle is repeated until all tests are completed, where n is an integer greater than or equal to 2.

[0023] 2.1) Two-step segmented repetition mode:

[0024] After completing rinsing step 1, the rinsing solution is recovered;

[0025] The recycled rinsing solution is used to rinse step 2, and is not recycled after completion.

[0026] Complete rinsing step 3 with fresh rinsing solution, and then recycle it.

[0027] The recycled rinsing solution is used to rinse step 4, and is not recycled after completion.

[0028] Repeat until all tests are completed. This rinsing method can reduce rinsing fluid consumption by 50%.

[0029] 2.2) Three-step segmented repetition pattern:

[0030] After completing rinsing step 1, the rinsing solution is recovered;

[0031] The recovered rinsing solution is used to rinse step 2, and then recycled after completion;

[0032] The rinsing solution recovered in rinsing step 2 is used to rinse step 3, and is not recovered after completion;

[0033] Complete rinsing step 4 with fresh rinsing solution, and then recycle it.

[0034] The rinsing solution recovered in rinsing step 4 is used to rinse step 5, and then recycled after completion;

[0035] The rinsing solution recovered in rinsing step 5 is used to rinse step 6, and is not recovered after completion.

[0036] Repeat until all tests are completed. This rinsing method can reduce rinsing fluid consumption by 66%, and so on.

[0037] 3) Segmented random mode of flushing fluid recovery:

[0038] After completing rinsing steps 1 to n with a new rinsing solution, the rinsing solution from m of the rinsing steps is recovered. The recovered rinsing solution from the m steps is then used for rinsing steps n+1 to n+L. If the recovered rinsing solution is insufficient, it is supplemented with a new rinsing solution. Here, n is an integer greater than or equal to 2, m is a positive integer less than or equal to n, and L is an integer greater than or equal to m.

[0039] 3.1) n, m, and L are the same positive integers:

[0040] After completing rinsing step 1, the rinsing solution is recovered;

[0041] Use fresh rinsing solution to rinse step 2, and then recover the rinsing solution after completion;

[0042] Continue until fresh flushing solution is used for flushing step n, after which the flushing solution is recycled;

[0043] Then, use the rinsing solution recovered from any of the rinsing steps 1 to n to complete rinsing step n+1. After completion, the rinsing solution is not recovered.

[0044] Use the rinsing solution recovered from any of the remaining rinsing steps 1 to n to rinse step n+2. After completion, the rinsing solution is not recovered.

[0045] The remaining recycled rinsing solution is used for rinsing step 2n until it is used. After that, the rinsing solution is not recycled.

[0046] A recycling cycle consists of n rinsing steps. The recycling cycle is repeated until all tests are completed, where n is an integer greater than or equal to 2.

[0047] In the above method, the rinsing steps include rinsing after antigen retrieval, rinsing after primary antibody incubation, rinsing after secondary antibody incubation, and rinsing after DAB color development.

[0048] In the above rinsing steps, each slide is rinsed with 3-10 mL of rinsing solution, preferably 5 mL, and the rinsing is repeated 2-5 times, preferably 3 times, using the minimum amount of rinsing solution while ensuring the rinsing effect.

[0049] Compared with existing technologies, this invention, based on the characteristic that the artificial introduction of upstream reagents in solid-phase immunoassay methods will not affect downstream steps, recovers the liquid from the upstream rinsing step for rinsing the downstream step. The method described in this invention can significantly reduce the consumption of rinsing fluid in a large-volume rinsing mode, effectively solving the problem of large rinsing fluid consumption caused by frequent rinsing in solid-phase immunoassay methods. Furthermore, it can adopt a continuous or segmented rinsing fluid recovery mode according to actual needs, making it suitable for use in high-throughput, multi-step automated detection equipment, thereby improving equipment performance and user experience. Attached Figure Description

[0050] Figure 1 Analysis of the relationships between steps in solid-phase immunoassay (reagent interactions, rinsing residues, and their mutual interference);

[0051] Figure 2 hCG detection in placental tissue (①③ irrigation using the invented method, ②④ irrigation using conventional methods; ①② field of view 1 100×, ③④ field of view 2 100×);

[0052] Figure 3 Detection of Ki-67 in tonsil tissue (①③ irrigation by the invented method, ②④ irrigation by conventional method; ①② field of view 1100×, ③④ field of view 2100×);

[0053] Figure 4 Detection of CKpan in skin tissue (①③ irrigation by the invented method, ②④ irrigation by conventional method; ①② field of view 1100×, ③④ field of view 2400×). Detailed Implementation

[0054] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are only for explaining the present invention and are not intended to limit the present invention.

[0055] Example 1

[0056] Example of solid-phase immunological detection: Immunohistochemistry was used to detect the following tissue sections: one placental tissue section for hCG molecule detection; one tonsil tissue section for Ki-67 molecule detection; and one skin tissue section for CKpan molecule detection.

[0057] The rinsing process during staining is performed in a continuous, repeating pattern. The specific process is as follows:

[0058] 1) Bake placental tissue slices, tonsil tissue slices, and skin tissue slices in a 70℃ oven for 1 hour.

[0059] 2) The tissue sections were dewaxed and hydrated by passing xylene I (3 minutes), xylene II (3 minutes), anhydrous ethanol I (3 minutes), anhydrous ethanol II (3 minutes), 95% ethanol (3 minutes), 85% ethanol (3 minutes), and 75% ethanol (3 minutes).

[0060] 3) Place the slides in EDTA antigen retrieval solution (pH 8.0) and retrieval in a 98°C water bath for 30 minutes.

[0061] 4) Rinsing step 1: Rinse each slide with 5 mL of rinsing buffer, recover the liquid after rinsing, repeat 3 times, use a total of 15 mL for each slide, and use a total of 45 mL of rinsing buffer for 3 slides.

[0062] 5) Primary antibody incubation: Add 150 μL of rabbit anti-human hCG antibody to placental tissue sections, 150 μL of mouse anti-human Ki-67 antibody to tonsil tissue sections, and 150 μL of mouse anti-human CKpan antibody to skin tissue sections. Incubate at room temperature for 45 minutes.

[0063] 6) Rinsing step 2: Rinse each slide with 5 mL of the rinsing buffer recovered in rinsing step 1. After rinsing, the liquid is recovered. Repeat 3 times, for a total of 45 mL of rinsing buffer used for 3 slides.

[0064] 7) Secondary antibody incubation: Add 150 μL of HRP-labeled goat anti-mouse / rabbit IgG polymer antibody to each slide and incubate at room temperature for 25 minutes.

[0065] 8) Rinsing step 3: Rinse each slide with 5 mL of the rinsing buffer recovered in rinsing step 2. After rinsing, the liquid is recovered. Repeat 3 times, for a total of 45 mL of rinsing buffer used for 3 slides.

[0066] 9) DAB color development: Add 150 μL of DAB color development working solution to each slice and react at room temperature for 5 minutes.

[0067] 10) Rinsing step 4: Rinse each slide with 5 mL of the rinsing buffer recovered in rinsing step 3. After rinsing, the liquid is recovered. Repeat 3 times. A total of 45 mL of rinsing buffer is used for 3 slides. All the above rinsing steps are done by instant rinsing. Remove the liquid as soon as possible after rinsing.

[0068] 11) Collect the slides and stain them with hematoxylin staining solution for 3 minutes.

[0069] 12) Rinse with distilled water, then rinse under tap water for 20 minutes.

[0070] 13) The sections were dehydrated and cleared by passing them through 75% ethanol (3 minutes), 85% ethanol (3 minutes), 95% ethanol (3 minutes), anhydrous ethanol I (3 minutes), anhydrous ethanol II (3 minutes), xylene I (3 minutes), and xylene II (3 minutes).

[0071] 14) Central resin sealing.

[0072] 15) Observe under a microscope, showing positive staining of the slides against the background.

[0073] Following this washing method, a total of 45 mL of washing buffer was consumed for the three slides during the washing process after antigen retrieval, primary antibody, secondary antibody, and DAB staining. In comparison, the conventional method consumes 180 mL of washing buffer for the same process. The buffer consumption is 25% of that of the existing method.

[0074] Example 2

[0075] The difference between this embodiment and Embodiment 1 is that the rinsing process in this embodiment is performed in a two-step, segmented, and repeated manner during staining. In rinsing step 3: each slide is rinsed with 5 mL of fresh rinsing buffer, and the liquid is recovered after rinsing, repeated 3 times; in rinsing step 4, the liquid recovered in rinsing step 3 is used.

[0076] Following this washing method, a total of 90 mL of washing buffer was consumed for the three slides during the washing process after antigen retrieval, primary antibody, secondary antibody, and DAB staining. In comparison, the conventional method consumes 180 mL of washing buffer for the same process. The buffer consumption is 50% of that of the existing method.

[0077] Example 3

[0078] The difference between this embodiment and Embodiment 1 is that:

[0079] Rinsing step 2: Rinse each slide with 5 mL of fresh rinsing buffer, recover the liquid after rinsing, repeat 3 times, and use a total of 90 mL of rinsing buffer for 3 slides.

[0080] Rinsing step 3: Rinse each slide with 5 mL of fresh rinsing buffer, recover the liquid after rinsing, repeat 3 times, and use a total of 135 mL of rinsing buffer for 3 slides.

[0081] Rinsing step 4: Rinse each slide with 5 mL of the rinsing buffer recovered in rinsing step 1. After rinsing, the liquid is recovered. Repeat 3 times. A total of 135 mL of rinsing buffer was used for 3 slides. All the above rinsing steps are done by instant rinsing. Remove the liquid as soon as possible after rinsing.

[0082] Example 4

[0083] The difference between this embodiment and embodiment 3 is as follows:

[0084] Rinsing step 4: Rinse each slide with 5 mL of the rinsing buffer recovered in rinsing step 2.

[0085] Example 5

[0086] The difference between this embodiment and embodiment 3 is as follows:

[0087] Rinsing step 4: Rinse each slide with 5 mL of the rinsing buffer recovered in rinsing step 3.

[0088] Example 6

[0089] The difference between this embodiment and Embodiment 1 is that:

[0090] Rinsing step 3: Rinse each slide with 5 mL of fresh rinsing buffer, repeat 3 times, for a total of 90 mL of rinsing buffer used for the 3 slides;

[0091] Rinsing step 4: Rinse each slide with 5 mL of fresh rinsing buffer, repeat 3 times, for a total of 135 mL of rinsing buffer used for the 3 slides.

[0092] Example 7

[0093] The difference between this embodiment and embodiment 6 is that:

[0094] Rinsing step 3: Rinse each slide with 5 mL of the rinsing buffer recovered in rinsing step 2. After rinsing, the liquid is recovered. Repeat 3 times, for a total of 45 mL of rinsing buffer used for 3 slides.

[0095] Rinsing step 4: Rinse each slide with 5 mL of fresh rinsing buffer, recover the liquid after rinsing, repeat 3 times, and use a total of 90 mL of rinsing buffer for 3 slides.

[0096] Example 8

[0097] The difference between this embodiment and embodiment 6 is that:

[0098] Rinsing step 3: Rinse each slide with 5 mL of fresh rinsing buffer, repeat 3 times, for a total of 90 mL of rinsing buffer used for the 3 slides;

[0099] Rinsing step 4: Rinse each slide with 5 mL of the rinsing buffer recovered in rinsing step 2, repeat 3 times, for a total of 90 mL of rinsing buffer used for the 3 slides.

[0100] Example 9

[0101] The difference between this embodiment and Embodiment 1 is that:

[0102] Rinsing step 2: Rinse each slide with 5 mL of fresh rinsing buffer, recover the liquid after rinsing, repeat 3 times, and use a total of 90 mL of rinsing buffer for 3 slides.

[0103] Rinse Step 3: Rinse each slide with 5 mL of the rinsing buffer recovered in Rinse Step 1. After rinsing, the liquid is recovered. Repeat 3 times. A total of 135 mL of rinsing buffer was used for 3 slides.

[0104] Rinsing step 4: Rinse each slide with 5 mL of the rinsing buffer recovered in rinsing step 2. After rinsing, the liquid is recovered. Repeat 3 times. A total of 135 mL of rinsing buffer was used for 3 slides. All the above rinsing steps are done by instant rinsing. Remove the liquid as soon as possible after rinsing.

[0105] Example 10

[0106] The difference between this embodiment and Embodiment 1 is that:

[0107] Rinsing step 2: Rinse each slide with 5 mL of fresh rinsing buffer, recover the liquid after rinsing, repeat 3 times, and use a total of 90 mL of rinsing buffer for 3 slides.

[0108] Rinsing step 3: Each slide was rinsed with 5 mL of the rinsing buffer recovered in rinsing step 2. The liquid was recovered after rinsing. This was repeated 3 times, and a total of 135 mL of rinsing buffer was used for the 3 slides.

[0109] Rinsing step 4: Rinse each slide with 5 mL of the rinsing buffer recovered in rinsing step 1. After rinsing, the liquid is recovered. Repeat 3 times. A total of 135 mL of rinsing buffer was used for 3 slides. All the above rinsing steps are done by instant rinsing. Remove the liquid as soon as possible after rinsing.

[0110] Example 11

[0111] The difference between this embodiment and Embodiment 1 is that:

[0112] Rinsing step 2: Rinse each slide with 5 mL of fresh rinsing buffer, recover the liquid after rinsing, repeat 3 times, and use a total of 90 mL of rinsing buffer for 3 slides.

[0113] Rinse Step 3: Rinse each slide with 5 mL of the rinsing buffer recovered in Rinse Step 1. After rinsing, the liquid is recovered. Repeat 3 times. A total of 135 mL of rinsing buffer was used for 3 slides.

[0114] Rinsing step 4: Rinse each slide with 5 mL of the rinsing buffer recovered in rinsing step 3. After rinsing, the liquid is recovered. Repeat 3 times. A total of 135 mL of rinsing buffer was used for 3 slides. All the above rinsing steps are done by instant rinsing. Remove the liquid as soon as possible after rinsing.

[0115] Example 12

[0116] The difference between this embodiment and Embodiment 1 is that:

[0117] Rinsing step 2: Rinse each slide with 5 mL of fresh rinsing buffer, recover the liquid after rinsing, repeat 3 times, and use a total of 90 mL of rinsing buffer for 3 slides.

[0118] Rinse Step 3: Rinse each slide with 5 mL of the rinsing buffer recovered in Rinse Step 1. After rinsing, the liquid is recovered. Repeat 3 times. A total of 135 mL of rinsing buffer was used for 3 slides.

[0119] Rinsing step 4: Rinse each slide with 5 mL of the rinsing buffer recovered in rinsing step 3. After rinsing, the liquid is recovered. Repeat 3 times. A total of 135 mL of rinsing buffer was used for 3 slides. All the above rinsing steps are done by instant rinsing. Remove the liquid as soon as possible after rinsing.

[0120] Example 13

[0121] The difference between this embodiment and Embodiment 1 is that: in this embodiment, immunohistochemistry is used to detect hCG molecules in a placental tissue sections, where a is greater than or equal to 2. That is, all samples in this embodiment are tested for the same molecular target. The rinsing step adopts either a time-retaining immersion method or an instant rinsing method, and the liquid is removed as soon as possible after rinsing.

[0122] Example 14

[0123] The difference between this embodiment and Embodiment 1 is that: in this embodiment, immunohistochemistry is used to detect ki-67 molecules in b tonsil tissue sections, and b is greater than or equal to 2. That is, all samples in this embodiment are tested for the same molecular target. The rinsing step adopts either immersion rinsing or instant rinsing. The liquid is removed as soon as possible after rinsing.

[0124] Example 15

[0125] The difference between this embodiment and Embodiment 1 is that: in this embodiment, immunohistochemistry is used to detect CKpan molecules in c skin tissue sections, where c is greater than or equal to 2. That is, all samples in this embodiment are tested for the same molecular target. The rinsing step is to use either an immersion rinsing method or an instant rinsing method, and the liquid is removed as soon as possible after rinsing.

[0126] Example 16

[0127] The difference between this embodiment and Embodiment 1 is that in the rinsing step, each slice is rinsed with 3-10 mL of rinsing solution each time, and each rinsing step is repeated 2-5 times.

[0128] Comparative experimental cases

[0129] Immunohistochemistry was used to simultaneously examine two groups of tissue sections. Each group included: one placental tissue section for detecting hCG molecules; one tonsil tissue section for detecting Ki-67 molecules; and one skin tissue section for detecting CKpan molecules.

[0130] The first group underwent a rinsing process during staining according to the continuous repetition pattern described in this invention, while the second group underwent a rinsing process during staining according to conventional methods. The specific process is as follows:

[0131] 1) Bake placental tissue slices, tonsil tissue slices, and skin tissue slices in a 70℃ oven for 1 hour.

[0132] 2) The tissue sections were dewaxed and hydrated by passing xylene I (3 minutes), xylene II (3 minutes), anhydrous ethanol I (3 minutes), anhydrous ethanol II (3 minutes), 95% ethanol (3 minutes), 85% ethanol (3 minutes), and 75% ethanol (3 minutes).

[0133] 3) Place the slides in EDTA antigen retrieval solution (pH 8.0) and retrieval in a 98°C water bath for 30 minutes.

[0134] 4) Rinsing: Each slide in the first group was rinsed with 5 mL of rinsing buffer, and the liquid was recovered after rinsing. This was repeated 3 times, with a total of 15 mL used per slide and 45 mL used for 3 slides. Each slide in the second group was rinsed with 5 mL of rinsing buffer, and the liquid was discarded after rinsing. This was repeated 3 times, with a total of 15 mL used per slide and 45 mL used for 3 slides.

[0135] 5) Primary antibody incubation: Add 150 μL of rabbit anti-human hCG antibody to placental tissue sections, 150 μL of mouse anti-human Ki-67 antibody to tonsil tissue sections, and 150 μL of mouse anti-human CKpan antibody to skin tissue sections. Incubate at room temperature for 45 minutes.

[0136] 6) Rinsing: Each slide in the first group was rinsed with 5 mL of the rinsing buffer from the previous step. The liquid was recovered after rinsing. This was repeated 3 times, with a total of 45 mL of rinsing buffer used for the 3 slides. Each slide in the second group was rinsed with 5 mL of rinsing buffer. The slides were discarded after rinsing. This was repeated 3 times, with a total of 90 mL of rinsing buffer used for the 3 slides.

[0137] 7) Secondary antibody incubation: Add 150 μL of HRP-labeled goat anti-mouse / rabbit IgG polymer antibody to each slide and incubate at room temperature for 25 minutes.

[0138] 8) Rinsing: Each slide in the first group was rinsed with 5 mL of the rinsing buffer from the previous step. The liquid was recovered after rinsing. This was repeated 3 times, with a total of 45 mL of rinsing buffer used for the 3 slides. Each slide in the second group was rinsed with 5 mL of rinsing buffer. The slides were discarded after rinsing. This was repeated 3 times, with a total of 135 mL of rinsing buffer used for the 3 slides.

[0139] 9) DAB color development: Add 150 μL of DAB color development working solution to each slice and react at room temperature for 5 minutes.

[0140] 10) Rinsing: Each slide in the first group was rinsed with 5 mL of the rinsing buffer from the previous step. The liquid was recovered after rinsing. This was repeated 3 times, with a total of 45 mL of rinsing buffer used for the 3 slides. Each slide in the second group was rinsed with 5 mL of rinsing buffer. The slides were discarded after rinsing. This was repeated 3 times, with a total of 180 mL of rinsing buffer used for the 3 slides.

[0141] 11) Collect the slides and stain them with hematoxylin staining solution for 3 minutes.

[0142] 12) Rinse with distilled water, then rinse under tap water for 20 minutes.

[0143] 13) The sections were dehydrated and cleared by passing them through 75% ethanol (3 minutes), 85% ethanol (3 minutes), 95% ethanol (3 minutes), anhydrous ethanol I (3 minutes), anhydrous ethanol II (3 minutes), xylene I (3 minutes), and xylene II (3 minutes).

[0144] 14) Central resin sealing.

[0145] 15) Under microscopic observation, there was no significant difference between the positive staining and the background in both groups of sections (see...). Figure 2-4 )

[0146] In the first group, following the invented washing method, a total of 45 mL of washing buffer was consumed for the three slides during the washing processes after retrieval, primary antibody, secondary antibody, and DAB staining. In the second group, following the conventional method, a total of 180 mL of washing buffer was consumed for the three slides during the washing processes after retrieval, primary antibody, secondary antibody, and DAB staining. The buffer consumption of the first group was 25% of that of the second group.

[0147] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for reducing the amount of rinsing solution used in solid-phase immunoassay, characterized in that: The flushing fluid used in the upstream flushing step is recovered and used in the downstream flushing step that requires the same basic flushing fluid; The rinsing fluid recovery follows a segmented, repetitive pattern: After completing rinsing step 1, the rinsing fluid is recovered; the recovered rinsing fluid is used for rinsing step 2, and then recovered again; this continues until the rinsing fluid recovered from rinsing step n-1 is used for rinsing step n, after which the rinsing fluid is not recovered; rinsing step n+1 is completed with fresh rinsing fluid, and then recovered; the rinsing fluid recovered from rinsing step n+1 is used for rinsing step n+2, and then recovered; this continues until the rinsing fluid recovered from rinsing step 2n-1 is used for rinsing step 2n, after which the rinsing fluid is not recovered; a recovery cycle consists of n rinsing steps, and this cycle continues until all tests are completed, where n is an integer greater than or equal to 2.

2. The method according to claim 1, characterized in that, The rinsing fluid recovery follows a two-step, segmented, repetitive pattern: after completing rinsing step 1, the rinsing fluid is recovered; the recovered rinsing fluid is used for rinsing step 2, and the rinsing fluid is not recovered after rinsing step 2; rinsing step 3 is completed with fresh rinsing fluid, and the rinsing fluid is recovered after rinsing step 3; the rinsing fluid recovered from rinsing step 3 is used for rinsing step 4, and the rinsing fluid is not recovered after rinsing step 4; this cycle continues until all tests are completed.

3. The method according to claim 2, characterized in that, The rinsing steps include rinsing after antigen retrieval, rinsing after primary antibody incubation, rinsing after secondary antibody incubation, and rinsing after DAB staining. The specific rinsing methods are as follows: Rinsing after antigen retrieval: Rinse each slide with 3-10 mL of rinsing buffer, recover the liquid after rinsing, and repeat rinsing 2-5 times; Rinsing after primary antibody incubation: Rinse each slide with 3-10 mL of rinsing buffer recovered after retrieval, do not recover the liquid after rinsing, and repeat rinsing 2-5 times; Rinsing after secondary antibody incubation: Rinse each slide with 3-10 mL of fresh rinsing buffer, recover the liquid after rinsing, and repeat rinsing 2-5 times; Rinsing after DAB staining: Rinse each slide with 3-10 mL of rinsing buffer recovered after secondary antibody incubation, and repeat rinsing 2-5 times.

4. The method according to claim 1, characterized in that, The rinsing fluid recovery follows a three-step, segmented, repetitive pattern: After completing rinsing step 1, the rinsing fluid is recovered; the recovered rinsing fluid is used for rinsing step 2, and then recovered again; the rinsing fluid recovered from rinsing step 2 is used for rinsing step 3, and then not recovered; rinsing step 4 is completed with fresh rinsing fluid, and then recovered; the rinsing fluid recovered from rinsing step 4 is used for rinsing step 5, and then recovered again; the rinsing fluid recovered from rinsing step 5 is used for rinsing step 6, and then not recovered; this cycle continues until all tests are completed.

5. A method for reducing the amount of rinsing solution used in solid-phase immunoassay, characterized in that: The flushing fluid used in the upstream flushing step is recovered and used in the downstream flushing step that requires the same basic flushing fluid; The flushing fluid recovery adopts a segmented random mode: after completing flushing steps 1 to n with new flushing fluid, the flushing fluid from m of the flushing steps is recovered. Then, the recovered flushing fluid from the m steps is used for flushing steps n+1 to n+L respectively. If the recovered flushing fluid is insufficient, it is supplemented with new flushing fluid. Here, n is an integer greater than or equal to 2, m is a positive integer less than or equal to n, and L is an integer greater than or equal to m.

6. The method according to claim 5, characterized in that, The rinsing steps include rinsing after antigen retrieval, rinsing after primary antibody incubation, rinsing after secondary antibody incubation, and rinsing after DAB staining. The specific rinsing methods are as follows: Rinsing after antigen retrieval: Rinse each slide with 3-10 mL of rinsing buffer, recover the liquid after rinsing, and repeat rinsing 2-5 times; Rinsing after primary antibody incubation: Rinse each slide with 3-10 mL of fresh rinsing buffer, recover the liquid after rinsing, and repeat rinsing 2-5 times; Rinsing after secondary antibody incubation: Rinse each slide with 3-10 mL of rinsing buffer recovered after antigen retrieval and / or rinsing buffer recovered after primary antibody incubation, do not recover the liquid after rinsing, and repeat rinsing 2-5 times; Rinsing after DAB staining: Rinse each slide with 3-10 mL of fresh rinsing buffer, and repeat rinsing 2-5 times.

7. The method according to claim 5, characterized in that, The rinsing steps include rinsing after antigen retrieval, rinsing after primary antibody incubation, rinsing after secondary antibody incubation, and rinsing after DAB staining. The specific rinsing methods are as follows: Rinsing after antigen retrieval: Rinse each slide with 3-10 mL of rinsing buffer, recover the liquid after rinsing, and repeat rinsing 2-5 times; Rinsing after primary antibody incubation: Rinse each slide with 3-10 mL of fresh rinsing buffer, recover the liquid after rinsing, and repeat rinsing 2-5 times; Rinsing after secondary antibody incubation: Rinse each slide with 3-10 mL of fresh rinsing buffer, do not recover the liquid after rinsing, and repeat rinsing 2-5 times; Rinsing after DAB staining: Rinse each slide with 3-10 mL of rinsing buffer recovered after antigen retrieval and / or rinsing buffer recovered after primary antibody incubation, and repeat rinsing 2-5 times.

8. The method according to claim 5, characterized in that: After completing rinsing step 1, the rinsing solution is recovered; new rinsing solution is used for rinsing step 2, and the rinsing solution is recovered after completion; this continues until new rinsing solution is used for rinsing step n, and the rinsing solution is recovered after completion; then, rinsing step n+1 is completed using the rinsing solution recovered from any of the rinsing steps 1 to n, and the rinsing solution is not recovered after completion; the remaining rinsing solution recovered from any of the rinsing steps 1 to n is used for rinsing step n+2, and the rinsing solution is not recovered after completion; this continues until the remaining recovered rinsing solution is used for rinsing step 2n, and the rinsing solution is not recovered after completion; a recovery cycle is formed by n rinsing steps, and the above recovery cycle is repeated until all tests are completed, where n is an integer greater than or equal to 2.

9. The method according to claim 8, characterized in that, The rinsing steps include rinsing after antigen retrieval, rinsing after primary antibody incubation, rinsing after secondary antibody incubation, and rinsing after DAB staining. The specific rinsing methods are as follows: Rinsing after antigen retrieval: Rinse each slide with 3-10 mL of rinsing buffer, recover the liquid after rinsing, and repeat rinsing 2-5 times; Rinsing after primary antibody incubation: Rinse each slide with 3-10 mL of fresh rinsing buffer, recover the liquid after rinsing, and repeat rinsing 2-5 times; Rinsing after secondary antibody incubation: Rinse each slide with 3-10 mL of rinsing buffer recovered after antigen retrieval, do not recover the liquid after rinsing, and repeat rinsing 2-5 times; Rinsing after DAB staining: Rinse each slide with 3-10 mL of rinsing buffer recovered after primary antibody incubation, and repeat rinsing 2-5 times.

10. The method according to claim 8, characterized in that, The rinsing steps include rinsing after antigen retrieval, rinsing after primary antibody incubation, rinsing after secondary antibody incubation, and rinsing after DAB staining. The specific rinsing methods are as follows: Rinsing after antigen retrieval: Rinse each slide with 3-10 mL of rinsing buffer, recover the liquid after rinsing, and repeat rinsing 2-5 times; Rinsing after primary antibody incubation: Rinse each slide with 3-10 mL of fresh rinsing buffer, recover the liquid after rinsing, and repeat rinsing 2-5 times; Rinsing after secondary antibody incubation: Rinse each slide with 3-10 mL of rinsing buffer recovered after primary antibody incubation, do not recover the liquid after rinsing, and repeat rinsing 2-5 times; Rinsing after DAB staining: Rinse each slide with 3-10 mL of rinsing buffer recovered after antigen retrieval, do not recover the liquid after rinsing, and repeat rinsing 2-5 times.

11. The method according to claim 3, 6, 7, 9 or 10, characterized in that: During the rinsing step, each slice was rinsed with 5 mL of rinsing buffer.

12. The method according to claim 3, 6, 7, 9 or 10, characterized in that: During the rinsing process, each slice is rinsed three times per rinsing step.

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