A method for detecting specific antineutrophil cytoplasmic antibodies in sputum samples

Abstract

The present invention discloses a method for detecting specific antineutrophil cytoplasmic antibodies in sputum samples, belonging to the technical field of antibody detection methods. The method includes the first step of treating sputum with PBS solution to obtain a supernatant, and the second step of treating the remaining mucus part in the sputum with DTT, and respectively detecting the specific ANCA in the samples treated in the two steps. The sum of part of the detection results is the detection result of the specific antineutrophil cytoplasmic antibody in the sputum sample. The present invention uses a two-step method to pretreat sputum, taking into account the advantages of treating sputum with PBS and DTT, avoiding the reduction of the ANCA antibody detection level caused by mucus adhesion after single use of PBS treatment, and at the same time avoiding the reduction of the detection level caused by the destruction of free ANCA antibodies after single use of DTT treatment. At the same time, the method of the present invention reduces the dilution factor of the sample and omits the cumbersome immunoprecipitation step for extracting immune complexes to save costs.

Description

Technical Field

[0001] The present invention relates to the technical field of antibody detection methods, and particularly to a method for detecting specific antineutrophil cytoplasmic antibodies in sputum samples. Background Art

[0002] Antineutrophil cytoplasmic antibody (ANCA) may be involved in the pathogenesis of ANCA-associated vasculitis in airway immunity, especially eosinophilic granulomatosis with polyangiitis (EGPA) with airway involvement as the main clinical manifestation. Respiratory samples can better reflect the respiratory system status. Compared with lung tissue pathology and bronchoalveolar lavage fluid, sputum specimens are safer, non-invasive, and simpler to collect. However, there is currently no standardized method for detecting specific ANCA in sputum specimens.

[0003] ANCA is associated with a necrotizing vasculitis involving medium- and small-sized blood vessels throughout the body. Common specific antigens of ANCA include MPO and PR3. Currently, research on the pathogenic mechanism and clinical application of ANCA in ANCA-associated vasculitis mainly focuses on peripheral blood ANCA. For those with lung involvement as the main clinical manifestation, there is a discrepancy between the respiratory system immune inflammatory state and the circulating immune inflammatory state, especially in EGPA. The positive rate of serum ANCA in EGPA patients is only 30-40%, while 79% of EGPA patients with negative serum ANCA are positive for sputum ANCA, and the sputum ANCA concentration is correlated with the airway inflammation level. In order to facilitate the exploration of the role of local specific ANCA in the respiratory tract in the pathogenesis of EGPA, it is crucial to find a reasonable and effective sputum treatment and ANCA detection method.

[0004] In the process of specific ANCA detection in sputum specimens, due to the presence of a large amount of mucin in sputum, various cells and soluble mediators are adhered into a jelly-like state. Improper treatment of mucin will inevitably lead to detection errors and instability of detection results. Dithiothreitol (DTT) is a commonly used mucolytic agent, and its mechanism of action is to break the disulfide bonds in mucin. In previous studies, DTT was mostly used to directly treat sputum to obtain sputum supernatant and detect its liquid-phase protein level. However, the destructive effect of DTT on the disulfide bonds in proteins is not specific, and it may also affect the disulfide bonds in the antibodies to be detected, thereby changing the detection results. At the same time, previous studies have confirmed that BPI-ANCA IgA and non-specific ANCA IgG can still be detected in the sputum supernatant treated with DTT. The above shows that directly treating sputum specimens with DTT will seriously affect the accuracy of specific ANCA detection in sputum specimens. At present, there is no perfect sputum pretreatment method for sputum specimens used for specific ANCA detection. Summary of the Invention

[0005] The object of the present invention is to provide a method for detecting specific anti-neutrophil cytoplasmic antibodies in sputum samples to solve the problems existing in the above-mentioned prior art. The present invention takes into account the advantages of treating sputum with PBS and DTT, avoids the reduction of ANCA antibody detection level caused by mucus adhesion after single use of PBS treatment, and at the same time avoids the reduction of detection level caused by the destruction of free ANCA antibodies after single use of DTT treatment. The establishment of the two-step pretreatment method for sputum in the present invention provides a more reliable sputum specimen processing method for exploring the pathogenesis of EGPA airway lesions, finding treatment targets, and monitoring curative effects.

[0006] To achieve the above object, the present invention provides the following scheme:

[0007] The present invention provides a method for detecting specific anti-neutrophil cytoplasmic antibodies in sputum samples, comprising the following steps:

[0008] First, add PBS solution to the sputum sample, vortex and oscillate, and collect the supernatant after the first centrifugation as the PBS supernatant;

[0009] Add DTT solution to the precipitate after centrifugation, vortex and oscillate again, filter and then centrifuge again, and collect the supernatant as the DTT supernatant;

[0010] Detect the specific anti-neutrophil cytoplasmic antibodies in the PBS supernatant and the DTT supernatant respectively, and the sum of the detection results of the specific anti-neutrophil cytoplasmic antibodies in the PBS supernatant and the DTT supernatant is the detection result of the specific anti-neutrophil cytoplasmic antibodies in the sputum sample.

[0011] Optionally, the specific antineutrophil cytoplasmic antibody is MPO-ANCA and PR3-ANCA.

[0012] Optionally, the addition amount of the PBS solution is 3 times the volume of the sputum sample.

[0013] Optionally, the vortex time is 15 s and the oscillation time is 15 min.

[0014] Optionally, the first centrifugation is at 2800 rpm for 10 min at 4°C.

[0015] Optionally, the addition amount of the DTT solution is the same as the volume of the PBS supernatant.

[0016] Optionally, the mass concentration of the DTT solution is 0.1%.

[0017] Optionally, the second centrifugation is at 2500 rpm for 10 min at 4°C.

[0018] Optionally, the detection method is the ELISA detection method.

[0019] The present invention discloses the following technical effects:

[0020] The present invention uses a two-step method to pretreat sputum, taking into account the advantages of treating sputum with PBS and DTT, avoiding the reduction of ANCA antibody detection level caused by mucus adhesion after single use of PBS treatment, and at the same time avoiding the reduction of detection level caused by the destruction of free ANCA antibodies after single use of DTT treatment. At the same time, the method of the present invention reduces the dilution multiple of the sample, helps to improve the detection sensitivity of low-titer ANCA, and saves costs by omitting the cumbersome step of immunoprecipitation to extract immune complexes.

[0021] The establishment of the two-step method for pretreating sputum in the present invention provides a more reliable method for processing sputum specimens for exploring the pathogenesis of EGPA airway lesions, finding treatment targets, and monitoring treatment effects. Description of the Drawings

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0023] Figure 1 It is a specific operation step flow chart for sputum treatment in the present invention;

[0024] Figure 2Detection differences of PR3-ANCA (A) and MPO-ANCA (B) in the PBS and DTT supernatants of the present invention;

[0025] Figure 3 Detection results of PR3-ANCA (A) and MPO-ANCA (B) in the sputum of asthma, HASM, EGPA-V, and healthy control groups;

[0026] Figure 4 Microscopic observation diagrams (400-fold magnification) of qualified sputum cell smears of EGPA-V (A) and healthy control group (B);

[0027] Figure 5 Results of the standard addition recovery experiment;

[0028] Figure 6 Influence of eosinophil peroxidase (EPX) on the detection results of PR3-ANCA (A) and MPO-ANCA (B);

[0029] Figure 7 Detection rates of each liquid-phase protein in the PBS and DTT supernatants; among them, A is the liquid-phase protein with high detection in the DTT supernatant and low detection in the PBS supernatant, B is the liquid-phase protein with high detection in both DTT and PBS, C is the liquid-phase protein with low detection in both DTT and PBS, and D is the liquid-phase protein with low detection in the DTT supernatant and high detection in the PBS supernatant. Detailed implementation manners

[0030] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0031] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0032] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0033] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the specification of the present invention, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and embodiments of the present invention are merely exemplary.

[0034] Regarding the terms "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0035] The present invention provides a method for detecting specific antineutrophil cytoplasmic antibodies in a sputum sample, which includes a first step of treating the sputum with a PBS solution to obtain a supernatant, a second step of further treating the remaining mucus part in the sputum with DTT, separately detecting the specific ANCA in the samples of the two steps, and adding part of the detection results to obtain the detection result of the specific antineutrophil cytoplasmic antibody in the sputum sample.

[0036] Specifically, it includes the following steps:

[0037] First, add a PBS solution to the sputum sample, vortex and oscillate, and collect the supernatant after the first centrifugation as the PBS supernatant.

[0038] Add a DTT solution to the precipitate after centrifugation, vortex and oscillate again, filter and then centrifuge again, and collect the supernatant as the DTT supernatant.

[0039] Separate and detect the specific antineutrophil cytoplasmic antibodies in the PBS supernatant and the DTT supernatant, and the sum of the detection results of the specific antineutrophil cytoplasmic antibodies in the PBS supernatant and the DTT supernatant is the detection result of the specific antineutrophil cytoplasmic antibody in the sputum sample.

[0040] In a specific embodiment, the specific antineutrophil cytoplasmic antibodies are MPO-ANCA and PR3-ANCA.

[0041] In a specific embodiment, the addition amount of the PBS solution is 3 times the volume of the sputum sample.

[0042] In a specific embodiment, the vortex time is 15 s and the oscillation time is 15 min.

[0043] In a specific embodiment, the first centrifugation is at 2800 rpm for 10 min at 4°C.

[0044] In a specific embodiment, the addition amount of the DTT solution is the same as the volume of the PBS supernatant.

[0045] In a specific embodiment, the mass concentration of the DTT solution is 0.1%.

[0046] In a specific embodiment, the re - centrifugation is carried out at 2500 rpm for 10 min at 4°C.

[0047] In a specific embodiment, the detection method is the ELISA detection method.

[0048] Example

[0049] 1. Sample source

[0050] Collect 54 qualified sputum samples from patients who visited Peking Union Medical College Hospital from November 2022 to November 2023 (15 cases of asthma, 13 cases of asthma with hypereosinophilic syndrome (HASM), 9 cases of eosinophilic granulomatosis with polyangiitis - vasculitis type (EGPA - V), and 17 healthy subjects). All research steps in the embodiments of the present invention have been reviewed and approved by the Ethics Committee of Peking Union Medical College Hospital (approval no. I - 22PJ1119).

[0051] 2. Sputum sample processing

[0052] Transfer the collected complete sputum sample to a 50 - mL plastic conical tube within two hours, and add phosphate - buffered saline (PBS) with a volume three times that of the sputum. After vortexing for 15 s and shaking and mixing for 15 min, centrifuge at 4°C (2800 rpm, 10 min). Aspirate as much of the supernatant component in the conical tube as possible and store it at - 80°C as PBS supernatant. Add 0.1 wt% DTT (with a volume three times that of the sputum) with the same volume as the aspirated PBS supernatant to the mucus precipitate. After vortexing for 15 s and shaking and mixing for 15 min again, filter the liquefied sputum through double - layer coarse gauze into another plastic conical tube. After centrifuging at 4°C (2500 rpm, 10 min), aspirate the supernatant component as DTT supernatant. Then, use the ELISA method to detect the concentrations of MPO - ANCA and PR3 - ANCA in the processed PBS supernatant and DTT supernatant respectively. The final concentration of the liquid - phase protein (including MPO - ANCA and PR3 - ANCA) in each sputum is the sum of the detection results of the PBS and DTT supernatants. The remaining precipitate part is used for sputum quality assessment and differential cell count. The specific operation steps are shown in Figure 1 .

[0053] 3. Detection of sputum MPO - ANCA and PR3 - ANCA

[0054] Use ELISA to detect PR3 - and MPO - ANCA IgG in the PBS and DTT supernatants of sputum respectively. Each sample is detected in duplicate, and the procedure follows the kit instructions.

[0055] The detection ranges of MPO-ANCA and PR3-ANCA are 1-200 RU / mL and 2-200 RU / mL respectively. If the detected concentration > 200 RU / mL, the sample should be diluted as necessary before re-detection.

[0056] 4. Detection results of specific ANCA in sputum

[0057] The detection results of specific ANCA in sputum showed that:

[0058] (1) In the same sputum sample, both PR3-ANCA (p<0.0001) and MPO-ANCA (p<0.0001) in the DTT supernatant were significantly higher than those in the PBS supernatant ( Figure 2 );

[0059] (2) Compared with healthy controls, PR3-ANCA in sputum of the asthma (p = 0.0015) and HASM (p = 0.0135) groups were significantly increased ( Figure 3 A). Taking the 90th percentile of the healthy control group as the normal threshold, 60%, 38.5% and 44.4% of the asthma, HASM and EGPA-V groups were higher than this value respectively. Compared with the healthy control group, MPO-ANCA in sputum of the asthma (72.7 RU / mL, p = 0.003), HASM (79.7 RU / mL, p = 0.0013) and EGPA-V (75.3 RU / mL, p = 0.0136) groups were significantly increased ( Figure 3 B). Taking the 90th percentile of the healthy control group as the normal threshold, 66.7%, 76.9% and 77.8% of the asthma, HASM and EGPA-V groups were higher than this value respectively.

[0060] 5. Detection results of cell composition

[0061] Figure 4 Figure A and Figure B are qualified sputum of EGPA-V and healthy controls respectively. Macrophages (blue arrows), neutrophils (purple arrows), lymphocytes (green arrows), plasma cells (red arrows), eosinophils (black arrows) are shown in the figures, and degranulation can be seen in some eosinophils.

[0062] 6. Results of the recovery experiment

[0063] The spiked recovery experiment was used to evaluate the effectiveness of sputum protein detection. According to the 2002 ERJ Working Group report, when the medium recovery rate > 80%, the immunological detection is considered effective. The specific method of the spiked recovery experiment is shown in Table 1.

[0064] Table 1 Spiked recovery experiment of PR3-ANCA and MPO-ANCA

[0065]

[0066]

[0067] PR3-ANCA and MPO-ANCA spike recovery experiments were respectively performed on 4 sputum samples. The recovery rate ranges of PR3-ANCA and MPO-ANCA were 70.5 - 94.6% and 86.1 - 122.6% respectively, indicating that the average recovery rates of detecting MPO-ANCA and PR3-ANCA by the sputum treatment method of the present invention were both greater than 80%( Figure 5 ).

[0068] 7. Influence of sputum EPX on the detection of PR3-ANCA and MPO-ANCA

[0069] Eosinophil peroxidase (EPX) is a cationic protein with strong adhesion, which may affect the ELISA detection results using HRP as the chromogenic substrate. In the present invention, exogenous EPX with different concentrations (specific concentrations: 10000, 2000, 400, 80, 16 pg / mL) was respectively added to the PBS and DTT supernatants of two sputum samples to evaluate whether high-concentration EPX would affect the detection of ANCA. The results showed that when using the method of treating sputum samples in the present invention to detect PR3-ANCA and MPO-ANCA, the detection results were not affected by eosinophil peroxidase( Figure 6 ).

[0070] 8. Influence of different sputum treatment methods on the detection rates of other liquid-phase proteins

[0071] Figure 7 The detection rates of various liquid-phase proteins based on the detection method of this study are shown. Among them, the detection rates of MPO-ANCA, EPX, and eotaxin-1 in both supernatants were 100%. The detection rates of Eotaxin-2, IL-5, IL-6, IL-1β, IL-10, IFNγ, IL-13, and IL-23 in both supernatants were also higher than 50%. However, PR3-ANCA, eotaxin-3, and IL-22 only had relatively high detection rates (>50%) in the DTT supernatant; TNF-α and ECP only had relatively high detection rates (>50%) in the PBS supernatant. Only IL-2, IL-4, and IL-17A had detection rates less than 50% in both supernatants. It shows that compared with treating sputum with PBS alone or DTT alone, the method of treating sputum in the present invention can improve the detection rate of other liquid-phase proteins without affecting the detection of cell components.

[0072] The embodiments described above are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for detecting specific antineutrophil cytoplasmic antibodies in sputum samples, characterized in that, It includes the following steps: First, add PBS solution to the sputum sample, vortex and oscillate it. After the first centrifugation, collect the supernatant as the PBS supernatant; Add DTT solution to the precipitate after centrifugation, vortex and oscillate it again. After filtration and then centrifugation again, collect the supernatant as the DTT supernatant; Detect the specific antineutrophil cytoplasmic antibody in the PBS supernatant and the DTT supernatant respectively. The sum of the detection results of the specific antineutrophil cytoplasmic antibody in the PBS supernatant and the DTT supernatant is the detection result of the specific antineutrophil cytoplasmic antibody in the sputum sample; The specific antineutrophil cytoplasmic antibody is MPO-ANCA and PR3-ANCA.

2. The method according to claim 1, characterized in that The addition amount of the PBS solution is 3 times the volume of the sputum sample.

3. The method according to claim 1, wherein The time of vortexing is 15 s, and the time of oscillation is 15 min.

4. The method according to claim 1, wherein The first centrifugation is at 2800 rpm for 10 min at 4°C.

5. The method according to claim 1, wherein The addition amount of the DTT solution is the same as the volume of the PBS supernatant.

6. The method according to claim 1, wherein The mass concentration of the DTT solution is 0.1%.

7. The method according to claim 1, characterized in that, The second centrifugation is at 2500 rpm for 10 min at 4°C.

8. The method according to claim 1, wherein The detection method is the ELISA detection method.

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