Method for detecting specific anti-neutrophil cytoplasm antibody in sputum sample

Abstract

The invention discloses a method for detecting a specific anti-neutrophil cytoplasm antibody in a sputum sample, and belongs to the technical field of antibody detection methods. The method comprises the following steps: 1, treating sputum with a PBS solution to obtain a supernatant, 2, treating the residual mucus part in the sputum with DTT, respectively detecting specific ANCA in the samples treated in the two steps, and adding part of detection results to obtain a detection result of the specific anti-neutrophil cytoplasm antibody in the sputum sample. According to the method, the sputum is pretreated by using a two-step method, the advantages of PBS and DTT for treating the sputum are considered, the situation that the detection level of the ANCA antibody is reduced due to mucus adhesion after PBS treatment is singly used is avoided, meanwhile, the situation that the detection level is reduced due to damage to the free ANCA antibody after DTT treatment is singly used is avoided, meanwhile, the dilution ratio of a sample is reduced, and the detection efficiency is improved. And a tedious step of extracting an immune complex through co-immunoprecipitation is omitted, so that the cost is saved.

Description

Technical Field

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

[0002] Antineutrophil cytoplasmic antibodies (ANCA) may be involved in the pathogenesis of ANCA-associated vasculitis in airway immunity, especially eosinophilic granulomatosis with polyangiitis (EGPA), which is characterized by airway involvement. Respiratory samples can better reflect the state of the respiratory system. 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 sputum specimen-specific ANCA detection method.

[0003] ANCA is associated with a necrotizing vasculitis that affects small and medium-sized blood vessels throughout the body. Common specific antigens of ANCA include MPO and PR3. At present, the research on the pathogenic mechanism and clinical application of ANCA in ANCA-associated vasculitis mainly focuses on peripheral blood ANCA. However, for patients with lung involvement as the main clinical manifestation, there is an inconsistency between the immune inflammatory state of the respiratory system and the immune inflammatory state of the circulation, especially EGPA. The serum ANCA positivity rate of EGPA patients is only 30-40%, while 79% of EGPA with negative serum ANCA are sputum ANCA positive, and the sputum ANCA concentration is related to the level of airway inflammation. In order to facilitate the exploration of the role of local respiratory specific ANCA in the pathogenesis of EGPA, it is crucial to find reasonable and effective sputum processing and ANCA detection methods.

[0004] In the process of specific ANCA detection of sputum specimens, due to the presence of a large amount of mucin in the sputum, various cells and soluble media are adhered into a jelly state. Improper handling of mucin will inevitably lead to detection errors and instability of the test results. Dithiothreitol (DTT) is a commonly used mucolytic agent, and its mechanism of action is to destroy the disulfide bonds in mucin. In previous studies, DTT was often used to directly treat sputum, obtain sputum supernatant, and detect its liquid protein level. However, DTT has no specificity in destroying disulfide bonds in proteins, and may also affect the disulfide bonds in the antibodies to be detected, thereby changing the test 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 direct treatment of sputum specimens with DTT will seriously affect the accuracy of specific ANCA detection of 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 purpose 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 PBS and DTT in treating sputum, avoids the reduction in ANCA antibody detection level caused by mucus adhesion after treatment with PBS alone, and avoids the destruction of free ANCA antibodies after treatment with DTT alone, resulting in a reduction in detection level. The establishment of the two-step method for pre-treating sputum in the present invention provides a more reliable sputum specimen processing method for exploring the pathogenesis of EGPA airway lesions, finding therapeutic targets, monitoring therapeutic effects, etc.

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

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

[0008] First, a PBS solution is added to the sputum sample, vortexed, shaken, and the supernatant is collected after the first centrifugation as the PBS supernatant;

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

[0010] The specific anti-neutrophil cytoplasmic antibodies in the PBS supernatant and the DTT supernatant are detected 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 anti-neutrophil cytoplasmic antibodies are MPO-ANCA and PR3-ANCA.

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

[0013] Optionally, the vortex time is 15 seconds and the oscillation time is 15 minutes.

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

[0015] Optionally, the amount of the DTT solution added 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 performed at 2500 rpm at 4°C for 10 min.

[0018] Optionally, the detection method is an 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 PBS and DTT in treating sputum, avoiding the reduction in ANCA antibody detection level caused by mucus adhesion after single PBS treatment, and avoiding the destruction of free ANCA antibodies after single DTT treatment, resulting in a reduction in detection level. 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 omits the cumbersome immunoprecipitation step of extracting immune complexes to save costs.

[0021] The establishment of the two-step method for pre-treating sputum in the present invention provides a more reliable sputum specimen processing method for exploring the pathogenesis of EGPA airway lesions, finding treatment targets, monitoring therapeutic effects, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1 The specific operation steps of the sputum treatment of the present invention are as follows;

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

[0025] Figure 3 The results of PR3-ANCA (A) and MPO-ANCA (B) tests in sputum of asthma, HASM, EGPA-V and healthy controls;

[0026] Figure 4 The following are microscopic observations of qualified sputum cell smears of EGPA-V (A) and healthy control group (B) (400 times magnification);

[0027] Figure 5 This is the result of the spike recovery experiment;

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

[0029] Figure 7 Are the detection rates of each liquid phase protein in PBS and DTT supernatants; A is a liquid phase protein with high detection in DTT supernatant and low detection in PBS supernatant, B is a liquid phase protein with high detection in both DTT and PBS, C is a liquid phase protein with low detection in both DTT and PBS, and D is a liquid phase protein with low detection in DTT and high detection in PBS. DETAILED DESCRIPTION

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0032] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may 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 associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0033] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0034] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0035] The present invention provides a method for detecting specific anti-neutrophil cytoplasmic antibodies in sputum samples, comprising a first step of treating sputum with a PBS solution to obtain a supernatant, a second step of treating the remaining mucus portion in the sputum with DTT, respectively detecting the specific ANCA in the samples treated in the two steps, and adding the partial detection results to obtain the detection result of the specific anti-neutrophil cytoplasmic antibodies in the sputum sample.

[0036] The specific steps include:

[0037] First, a PBS solution is added to the sputum sample, vortexed, shaken, and the supernatant is collected after the first centrifugation as the PBS supernatant;

[0038] Add DTT solution to the precipitate after centrifugation, vortex and shake again, filter and centrifuge again, collect the supernatant as DTT supernatant;

[0039] The specific anti-neutrophil cytoplasmic antibodies in the PBS supernatant and the DTT supernatant are detected 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.

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

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

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

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

[0044] In a specific embodiment, the amount of the DTT solution added 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 centrifugation again is performed at 2500 rpm at 4°C for 10 min.

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

[0048] Example

[0049] 1. Sample source

[0050] 54 qualified sputum samples were collected 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 vasculitis-type EGPA (EGPA-V), and 17 healthy subjects). All research steps in the examples of the present invention were reviewed and approved by the Ethics Committee of Peking Union Medical College Hospital (approval no. I-22PJ1119).

[0051] 2. Sputum sample processing

[0052] Within two hours, the collected complete sputum samples were transferred to a 50 mL plastic conical tube and phosphate buffered saline (PBS) was added in a volume of 3 times the volume of sputum. After vortexing for 15 seconds and shaking for 15 minutes, centrifugation was performed at 4°C (2800 rpm, 10 minutes). The supernatant components in the conical tube were dried as much as possible and stored at -80°C as PBS supernatant. 0.1 wt% DTT (3 times the volume of sputum) was added to the mucus precipitate in an equal volume to the absorbed PBS supernatant. After vortexing for 15 seconds and shaking for 15 minutes again, the liquefied sputum was filtered into another plastic conical tube using a double layer of cheesecloth. After centrifugation at 4°C (2500 rpm, 10 minutes), the supernatant components were aspirated as DTT supernatant. The concentrations of MPO-ANCA and PR3-ANCA were then detected by ELISA for the treated PBS supernatant and DTT supernatant, respectively. The final concentration of liquid phase proteins (including MPO-ANCA and PR3-ANCA) in each sputum was the sum of the test results of PBS and DTT supernatants. The remaining precipitate was used for sputum quality assessment and cell classification and counting. For specific operation steps, see Figure 1 .

[0053] 3. Sputum MPO-ANCA and PR3-ANCA testing

[0054] ELISA was used to detect PR3- and MPO-ANCA IgG in sputum PBS and DTT supernatants, respectively. Each sample was tested in duplicate, and the procedure followed the kit instructions.

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

[0056] 4. Specific ANCA test results in sputum

[0057] The specific ANCA test results in sputum showed:

[0058] (1) In the same sputum, the levels of 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 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, the MPO-ANCA in the sputum of the asthma (72.7RU / mL, p=0.003), HASM (79.7RU / mL, p=0.0013) and EGPA-V (75.3RU / 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, respectively, were above this value.

[0060] 5. Cell composition test results

[0061] Figure 4 Figure A and Figure B are qualified sputum from EGPA-V and healthy controls, respectively, showing macrophages (blue arrows), neutrophils (purple arrows), lymphocytes (green arrows), plasma cells (red arrows), and eosinophils (black arrows). Degranulation can be seen in some eosinophils.

[0062] 6. Recovery test results

[0063] The spike recovery experiment was used to evaluate the validity of sputum protein detection. According to the 2002 ERJ working group report, the immunological test is considered valid when the medium recovery rate is greater than 80%. The specific method of the spike recovery experiment is shown in Table 1.

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

[0065]

[0066]

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

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

[0069] Eosinophilic peroxidase (EPX) is a cationic protein with strong adhesion, which may affect the ELISA test results using HRP as a chromogenic substrate. The present invention adds different concentrations of exogenous EPX (specific concentrations: 10000, 2000, 400, 80, 16pg / mL) to two sputum PBS and DTT supernatant samples to evaluate whether high concentrations of EPX will affect the detection of ANCA. The results show that the detection results of PR3-ANCA and MPO-ANCA using the method of treating sputum samples of the present invention are not affected by eosinophilic peroxidase ( Figure 6 ).

[0070] 8. Effects of different sputum processing methods on the detection rate of other fluid-phase proteins

[0071] Figure 7 The detection rates of various liquid-phase proteins based on the detection method of this study were demonstrated, among which 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 had higher detection rates only in DTT supernatant (>50%); TNF-α and ECP had higher detection rates only in PBS supernatant (>50%). Only IL-2, IL-4 and IL-17A had detection rates of less than 50% in both supernatants. This shows that compared with the treatment of sputum with PBS or DTT alone, the treatment method of sputum in this invention can improve the detection rate of other liquid-phase proteins without affecting the detection of cellular components.

[0072] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for detecting specific anti-neutrophil cytoplasmic antibodies in sputum samples, characterized in that: The following steps are involved: First, a PBS solution is added to the sputum sample, vortexed, shaken, and the supernatant is collected after the first centrifugation as the PBS supernatant; Add DTT solution to the precipitate after centrifugation, vortex and shake again, filter and centrifuge again, collect the supernatant as DTT supernatant; The specific anti-neutrophil cytoplasmic antibodies in the PBS supernatant and the DTT supernatant are detected 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.

2. The method according to claim 1, characterized in that The specific anti-neutrophil cytoplasmic antibodies are MPO-ANCA and PR3-ANCA.

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

4. The method according to claim 1, characterized in that: The vortex time was 15 s, and the oscillation time was 15 min.

5. The method according to claim 1, characterized in that The first centrifugation was performed at 2800 rpm at 4°C for 10 min.

6. The method according to claim 1, characterized in that The added amount of the DTT solution was the same as the volume of the PBS supernatant.

7. The method according to claim 1, characterized in that The mass concentration of the DTT solution is 0.1%.

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

9. The method according to claim 1, characterized in that: The detection method is ELISA detection method.

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