Test strip and kit for detecting helicobacter pylori antigen in excrement and preparation method of test strip and kit

Abstract

The invention belongs to the technical field of helicobacter pylori antigen detection, and particularly relates to a test strip and kit for helicobacter pylori antigen detection in excrement and a preparation method thereof.The test strip comprises a sample pad, a combination pad and a nitrocellulose membrane, the sample pad is treated with a sample pad pretreatment solution, the combination pad is treated with a combination pad pretreatment solution, and the nitrocellulose membrane is prepared from a nitrocellulose membrane. A colloidal gold labeled binding antibody is coated on the binding pad, the binding antibody is an anti-helicobacter pylori monoclonal antibody, a quality control line and a detection line are arranged on the nitrocellulose membrane, the quality control line is coated with a goat anti-mouse IgG antibody and a rabbit anti-chicken IgY antibody, the detection line is coated with an anti-helicobacter pylori monoclonal antibody, and the kit comprises a test strip, according to the kit and the test strip, the sensitivity and the specificity of detecting helicobacter pylori in excrement can be improved, and compared with the prior art, the kit and the test strip have the advantages that the detection range is wider, the detection is rapid and accurate, the operation is simple and convenient, and the kit and the test strip are safe and reliable.

Description

Technical Field

[0001] The invention belongs to the technical field of Helicobacter pylori antigen detection, and particularly relates to a test strip, a kit and a preparation method thereof for detecting Helicobacter pylori antigen in feces. Background Art

[0002] Helicobacter pylori (HP) is a Gram-negative, microaerophilic bacterium that parasitizes the stomach and duodenum. Its infection is very common, with an infection rate of over 50% in the global natural population. Helicobacter pylori infection is closely related to the occurrence of chronic gastritis, peptic ulcer, gastric mucosa-associated lymphoid tissue lymphoma and gastric cancer. The World Health Organization's International Agency for Research on Cancer has classified it as a Class I carcinogen. According to statistics from the National Cancer Center in March 2023, gastric cancer is one of the most common malignant tumors in my country, with non-cardia gastric cancer accounting for 85% to 90% of the total, of which 90% is related to HP infection. At present, the HP infection rate and gastric cancer incidence rate in the Chinese population are still high. Screening and diagnosis of HP in asymptomatic and sick (postoperative) populations are extremely important for the prevention and control of HP infection.

[0003] Diagnostic methods for Helicobacter pylori infection are divided into two categories based on whether the sample is invasive or not: invasive and noninvasive. Invasive methods require gastric mucosal tissue to be obtained through gastroscopy before testing. These methods are invasive and include histological examinations (such as hematoxylin and eosin staining, Warthin-Starry silver staining, modified Giemsa staining, toluidine blue staining, acridine orange staining, and immunohistochemistry), bacterial culture, rapid urease test (RUT), and Helicobacter pylori nucleic acid testing. Noninvasive methods primarily include serological (antibody) testing, stool Helicobacter pylori antigen testing or genetic testing, and urea breath test (UBT).

[0004] The "Consensus on Screening and Management of Helicobacter pylori Infection in Physical Examination Populations" provides a systematic guidance framework for health examination institutions and clinical gastroenterologists on the screening, diagnosis, treatment, and management of Helicobacter pylori (H. pylori) infection. The consensus emphasizes that non-invasive methods for diagnosing H. pylori infection are preferred for asymptomatic individuals undergoing physical examinations. The urea breath test is the most recommended method (evidence level: 1; recommendation strength: A). The monoclonal stool antigen test can be used as an alternative, as the latter is more convenient for sampling at home and then sending for testing (evidence level: 2; recommendation strength: B).

[0005] The monoclonal fecal antigen test has been clinically verified to have good sensitivity and specificity, and its accuracy is similar to that of UBT. The fecal antigen test does not require fasting, is easy to collect, safe and simple, and has rapid detection. The sampling kit can be stored for a long time at ambient temperature and is easy to transport. At the same time, it can be sampled and sent for testing at home, which is more convenient. It has advantages in the detection of people who do not cooperate well with UBT (such as some elderly people, children, etc.), and is suitable for all people (normal adults, pregnant women, the elderly, etc.). Self-testing monoclonal fecal antigen tests can be used for epidemiological surveys, thereby saving manpower and space.

[0006] Currently, the main kits registered in China for detecting Helicobacter pylori in stool include enzyme-linked immunosorbent assay (ELISA), latex assay, and colloidal gold assay. ELISA is cumbersome, time-consuming, and requires paired equipment for testing. The latex assay also has limited specificity, inferior to the colloidal gold assay. The colloidal gold assay is highly favored for its high sensitivity and specificity, rapidity, accuracy, ease of use, and safety. For rapid diagnosis, the colloidal gold assay remains the preferred method. However, the sensitivity and specificity of currently available kits are insufficient. Summary of the Invention

[0007] In order to solve the problems in the prior art, the present invention provides a test strip, a kit and a preparation method thereof for detecting Helicobacter pylori antigen in feces, so as to achieve the purpose of detecting Helicobacter pylori in feces with high sensitivity and specificity.

[0008] The present invention solves the technical problem by adopting the following technical solutions:

[0009] The present invention aims to provide a test strip for detecting Helicobacter pylori antigen in feces, comprising a sample pad, a conjugation pad and a nitrocellulose membrane, wherein the sample pad is treated with a sample pad pretreatment liquid, the conjugation pad is treated with a conjugation pad pretreatment liquid, the conjugation pad is coated with a colloidal gold-labeled binding antibody, the binding antibody is an anti-Helicobacter pylori monoclonal antibody, the nitrocellulose membrane is provided with a quality control line and a detection line, the quality control line is coated with a sheep anti-mouse IgG antibody and a rabbit anti-chicken IgY antibody, and the detection line is coated with an anti-Helicobacter pylori monoclonal antibody.

[0010] Furthermore, the sample pad treatment solution contains 0.31% boric acid, 1.91% sodium tetraborate, 3% trehalose, 0.2% Tween-20 and 1% BSA, with water as the solvent.

[0011] Furthermore, the conjugate pad pretreatment solution contains 0.31% boric acid, 1.91% sodium tetraborate, 3% trehalose, 0.2% Tween-20, 1% BSA and 0.1% PEG20000, with water as the solvent.

[0012] A method for preparing a test strip for detecting Helicobacter pylori antigen in feces, comprising:

[0013] Preparing a pretreated sample pad: soaking the sample pad in a sample pad pretreatment solution and then drying;

[0014] Preparation of the conjugate pad: soaking the conjugate pad in a conjugate pad pretreatment solution and then drying it, spraying the conjugate antibody labeled with colloidal gold on the pretreated conjugate pad and drying it;

[0015] Preparation of nitrocellulose membrane: streak anti-Helicobacter pylori monoclonal antibody onto the detection area of ​​nitrocellulose membrane, mix goat anti-mouse IgG antibody and rabbit anti-chicken IgY antibody and streak onto the quality control area of ​​nitrocellulose membrane, and then dry to prepare the detection line and coating line.

[0016] Furthermore, the sample pad used is a glass cellulose membrane with a size of 17 to 42 mm. The sample pad is soaked in a sample pad pretreatment solution for 20 to 30 minutes and then dried at 37 to 40° C. for 3 to 5 hours to obtain a pretreated sample pad.

[0017] Furthermore, the preparation method of the colloidal gold-labeled binding antibody includes: adding colloidal gold to a stirring container, adding 0.1M potassium carbonate at a ratio of 1:100 to 1:200, and stirring for 10 minutes; adding an anti-Helicobacter pylori monoclonal antibody at a concentration of 10 to 30 μg / mL, and stirring for 30 minutes; adding BSA to a final concentration of 1% to 1.2%, and stirring for 30 minutes; centrifuging at 12000 rpm and 4°C for 30 minutes; after the centrifugation is completed, discarding the supernatant, and resuspending the precipitate with a gold label diluent at a ratio of 1:10 to obtain the colloidal gold-labeled binding antibody, and storing at 4°C for future use.

[0018] Furthermore, the gold label diluent contains 0.31% boric acid, 1.91% sodium tetraborate, 3% trehalose, 0.2% Tween-20, 0.05% casein, 0.1% PEG20000, and water is used as the solvent.

[0019] Furthermore, the conjugate pad used is a glass cellulose membrane with a specification of 8 mm. The conjugate pad is soaked in a conjugate pad pretreatment solution for 20 to 30 minutes and then dried at 37 to 40°C for 3 to 5 hours to prepare a pretreated conjugate pad. Then, the colloidal gold-labeled binding antibody is sprayed on the pretreated conjugate pad at 8 to 9 μL / cm and placed in a drying oven at 40°C for 2 hours to dry.

[0020] Furthermore, the method for preparing the nitrocellulose membrane includes: the specification of the nitrocellulose membrane is 25 mm, the anti-Helicobacter pylori monoclonal antibody with a concentration of 1.0-2.0 mg / mL is streaked onto the detection area of ​​the nitrocellulose membrane, and the streaking volume is 1 μL / cm, and the goat anti-mouse IgG antibody with a concentration of 1.0-2.0 mg / mL and the rabbit anti-chicken IgY antibody with a concentration of 0.2-0.4 mg / mL are mixed and then streaked onto the quality control area of ​​the nitrocellulose membrane, and the streaking volume is 1 μL / cm, and placed in a 40°C drying oven for 2 hours to dry, thereby preparing the detection line and quality control line, respectively.

[0021] A kit for detecting Helicobacter pylori antigen in feces comprises the test strip or the test strip prepared by the preparation method, a plastic card housing the test strip and a sample diluent; the test strip also comprises a plastic plate and absorbent paper.

[0022] Compared with the prior art, the beneficial technical effects of the present invention are:

[0023] The kit and test strip of the present invention can improve the sensitivity and specificity of detecting Helicobacter pylori in feces, and have a higher detection range than the existing technology. It can detect Helicobacter pylori antigen concentrations of 0 to 2000 ng / mL, and the detection strain concentration is less than 10 7 There is no cross reaction when the CFU / mL is low, and the detection is fast, accurate, easy to operate, safe and reliable.

[0024] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention, it can be implemented in accordance with the contents of the specification. In order to make the above contents of the present invention and its objectives, features and advantages more obvious and easy to understand, the specific implementation methods of the present invention are specifically listed below. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of the composition of the detection reagent card in the present invention.

[0026] Figure 2 Schematic diagram of the detection results of the detection reagent card in the present invention.

[0027] 1-Plastic cartridge, 2-Plastic plate, 3-Sample pad, 4-Binding pad, 5-Quality control line, 6-Test line, 7-Nitrocellulose membrane, 8-Absorbent paper. DETAILED DESCRIPTION

[0028] The technical solutions of the present invention are further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely exemplary illustrations and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are encompassed within the scope of protection that the present invention is intended to protect.

[0029] In addition, unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0030] The kit includes a test reagent card and sample diluent, such as Figure 1 As shown, the detection reagent card includes a test strip and a plastic card shell 1, the test strip includes a rubber plate 2, a sample pad 3, a conjugate pad 4, a nitrocellulose membrane 7 and a blotting paper 8, and a quality control line 5 and a detection line 6 are provided on the nitrocellulose membrane 7.

[0031] Example 1

[0032] 1. Prepare the sample pad: Cut the glass cellulose membrane into 17*300mm, add 1.5mL of sample pad pretreatment solution to each sample pad, soak for 20min, and place in a 40℃ oven for 4h.

[0033] 2. Prepare the conjugate pad: Cut the glass cellulose membrane into 8*300mm, add 1.0mL of conjugate pad pretreatment solution to each conjugate pad, soak for 20 minutes, and place in a 40℃ oven for 4 hours.

[0034] Prepare gold-labeled antibody: Add 10 mL of colloidal gold to a glass beaker. Place the beaker on a magnetic stirrer and add 100 μL of 0.1 M potassium carbonate. Stir for 10 minutes. Add anti-Helicobacter pylori monoclonal antibody at 10 μg / mL and stir for 30 minutes. Add BSA to a final concentration of 1% and stir for 30 minutes. Centrifuge at 12,000 rpm / min and 4°C for 30 minutes. Discard the supernatant and resuspend the pellet in gold conjugate diluent at a 1:10 ratio to obtain the gold-labeled antibody (i.e., the colloidal gold-labeled binding antibody).

[0035] The conjugate pad was coated with gold-labeled antibody, i.e., gold spraying: the gold-labeled antibody was sprayed on the pretreated conjugate pad at 8 μL / cm and dried in a drying oven at 40°C for 2 h.

[0036] 3. Prepare nitrocellulose membrane: Cut nitrocellulose membrane into 25 x 300 mm pieces. Prepare 1.0 mg / mL anti-Helicobacter pylori monoclonal antibody as the test line coating antibody. Mix 2.0 mg / mL goat anti-mouse IgG antibody and 0.3 mg / mL rabbit anti-chicken IgY antibody as the control line coating antibody. Streak the membrane at a rate of 1 μL / cm. Dry in a 40°C oven for 2 h.

[0037] 4. Assemble the sample pad, conjugate pad, nitrocellulose membrane and absorbent paper prepared above. Figure 1 As shown in the figure, they are overlapped on the plastic plate in sequence, placed in the plastic card shell, and covered with the plastic card shell to complete the production of the detection reagent card.

[0038] Example 2

[0039] 1. Prepare the sample pad: Cut the glass cellulose membrane into 17*300mm, add 1.5mL of sample pad pretreatment solution to each sample pad, soak for 30min, and place in a 40℃ oven for 3h.

[0040] 2. Prepare the conjugate pad: Cut the glass cellulose membrane into 8*300mm, add 1.0mL of conjugate pad pretreatment solution to each conjugate pad, soak for 30 minutes, and place in a 40℃ oven for 3 hours.

[0041] Prepare gold-labeled antibody: Add 10 mL of colloidal gold to a glass beaker. Place the beaker on a magnetic stirrer and add 150 μL of 0.1 M potassium carbonate. Stir for 10 minutes. Add anti-Helicobacter pylori monoclonal antibody at 20 μg / mL and stir for 30 minutes. Add BSA to a final concentration of 1.1% and stir for 30 minutes. Centrifuge at 12,000 rpm / min at 4°C for 30 minutes. Discard the supernatant and resuspend the pellet in gold conjugate diluent at a 1:10 ratio to obtain the gold-labeled antibody (i.e., the colloidal gold-labeled binding antibody).

[0042] The conjugate pad was coated with gold-labeled antibody, i.e., gold spraying: the gold-labeled antibody was sprayed on the pretreated conjugate pad at 8.5 μL / cm and dried in a drying oven at 40°C for 2 h.

[0043] 3. Prepare nitrocellulose membrane: Cut the nitrocellulose membrane into 25 x 300 mm pieces. Prepare 1.5 mg / mL of anti-Helicobacter pylori monoclonal antibody as the test line coating antibody. Mix 2.0 mg / mL of goat anti-mouse IgG antibody and 0.4 mg / mL of rabbit anti-chicken IgY antibody as the control line coating antibody. Streak the membrane at a rate of 1 μL / cm. Dry in a 40°C oven for 2 h.

[0044] 4. Assemble the sample pad, conjugate pad, nitrocellulose membrane and absorbent paper prepared above. Figure 1 As shown in the figure, they are overlapped on the plastic plate in sequence, placed in the plastic card shell, and covered with the plastic card shell to complete the production of the detection reagent card.

[0045] Example 3

[0046] 1. Prepare the sample pad: Cut the glass cellulose membrane into 42*300mm, add 4.5mL of sample pad pretreatment solution to each sample pad, soak for 30min, and place in a 37℃ oven for 5h.

[0047] 2. Prepare the conjugate pad: Cut the glass cellulose membrane into 8*300mm, add 1.0mL of conjugate pad pretreatment solution to each conjugate pad, soak for 30 minutes, and place in a 37℃ oven for 5 hours.

[0048] Prepare gold-labeled antibody: Add 10 mL of colloidal gold to a glass beaker. Place the beaker on a magnetic stirrer and add 200 μL of 0.1 M potassium carbonate. Stir for 10 minutes. Add anti-Helicobacter pylori monoclonal antibody at 30 μg / mL and stir for 30 minutes. Add BSA to a final concentration of 1.2% and stir for 30 minutes. Centrifuge at 12,000 rpm / min and 4°C for 30 minutes. Discard the supernatant and resuspend the pellet in gold conjugate diluent at a 1:10 ratio to obtain the gold-labeled antibody (i.e., the colloidal gold-labeled binding antibody).

[0049] The conjugate pad was coated with gold-labeled antibody, i.e., gold spraying: the gold-labeled antibody was sprayed on the pretreated conjugate pad at 9 μL / cm and dried in a drying oven at 40°C for 2 h.

[0050] 3. Prepare nitrocellulose membrane: Cut nitrocellulose membrane into 25 x 300 mm pieces. Prepare 2.0 mg / mL of anti-Helicobacter pylori monoclonal antibody as the test line coating antibody. Mix 1.0 mg / mL goat anti-mouse IgG antibody and 0.2 mg / mL rabbit anti-chicken IgY antibody as the control line coating antibody. Streak the membrane at a rate of 1 μL / cm. Dry in a 40°C oven for 2 h.

[0051] 4. Assemble the sample pad, conjugate pad, nitrocellulose membrane and absorbent paper prepared above. Figure 1 As shown in the figure, they are overlapped on the plastic plate in sequence, placed in the plastic card shell, and covered with the plastic card shell to complete the production of the detection reagent card.

[0052] Performance testing

[0053] The sensitivity, HOOK effect and specificity of Helicobacter pylori antigen detection kits produced by other companies and the kit of this embodiment were tested using corporate reference products.

[0054] (1) Sensitivity and Hook Effect Test: The Helicobacter pylori (HP) Antigen Detection Kit (Colloidal Gold Method) (Registration Certificate: National Medical Device Registration No. 201163402242) (Comparative Reagent) and the kit in this Example were tested. The analytical sensitivity of the product produced by Yinke is a sensitivity reference material capable of detecting 150 ng of H. pylori antigen / mL of extract, as shown in Table 1 below.

[0055] Table 1

[0056]

[0057] Note: - indicates negative, + indicates positive, ++ indicates moderate positive, and +++ indicates strong positive

[0058] The above results show that under the detection condition of Helicobacter pylori antigen concentration of 0-2000 ng / mL, Example 2 has higher sensitivity and detection range than Examples 1 and 3 and the comparative reagent.

[0059] (2) Specificity test: The Helicobacter pylori antigen detection kit (colloidal gold method) (Registration Certificate: National Medical Device Registration No. 20183401879) (comparison reagent) produced by Hangzhou Anxu Biotechnology Co., Ltd. (hereinafter referred to as Anxu) and the detection kit in this example were tested. The cross reaction of the product produced by Anxu was 10 5There was no cross-reaction in the cross-reaction test with other strains / times, see Table 2 below.

[0060] Table 2

[0061]

[0062]

[0063]

[0064] Note: - indicates negative, + indicates positive.

[0065] As shown in Table 2 above, the cross-specificity of the kits of Examples 1-3 is better than that of the comparative reagents, and the detection of the above strain concentrations is less than 10 7 No cross-reaction occurred at CFU / mL.

[0066] The serial numbers of the above embodiments of the present invention are for description only and do not represent the advantages or disadvantages of the embodiments.

[0067] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation methods. The above-mentioned specific implementation methods are merely illustrative and not restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are protected by the present invention.

Claims

1. A test strip for detecting Helicobacter pylori antigen in feces, characterized in that: The invention comprises a sample pad, a conjugate pad and a nitrocellulose membrane. The sample pad is treated with a sample pad pretreatment liquid, the conjugate pad is treated with a conjugate pad pretreatment liquid, the conjugate pad is coated with a conjugate antibody labeled with colloidal gold, the conjugate antibody is an anti-Helicobacter pylori monoclonal antibody, a quality control line and a detection line are provided on the nitrocellulose membrane, the quality control line is coated with a goat anti-mouse IgG antibody and a rabbit anti-chicken IgY antibody, and the detection line is coated with an anti-Helicobacter pylori monoclonal antibody.

2. A test strip for detecting Helicobacter pylori antigen in feces according to claim 1, characterized in that: The sample pad treatment solution contained 0.31% boric acid, 1.91% sodium tetraborate, 3% trehalose, 0.2% Tween-20 and 1% BSA, with water as the solvent.

3. A test strip for detecting Helicobacter pylori antigen in feces according to claim 1, characterized in that: The conjugate pad pretreatment solution contains 0.31% boric acid, 1.91% sodium tetraborate, 3% trehalose, 0.2% Tween-20, 1% BSA and 0.1% PEG20000, with water as the solvent.

4. A method for preparing a test strip for detecting Helicobacter pylori antigen in feces according to any one of claims 1 to 3, characterized in that: include: Preparing a pretreated sample pad: soaking the sample pad in a sample pad pretreatment solution and then drying; Preparation of the conjugate pad: soaking the conjugate pad in a conjugate pad pretreatment solution and then drying it, spraying the conjugate antibody labeled with colloidal gold on the pretreated conjugate pad and drying it; Prepare nitrocellulose membrane: streak anti-Helicobacter pylori monoclonal antibody onto the test area of ​​nitrocellulose membrane, mix goat anti-mouse IgG antibody and rabbit anti-chicken IgY antibody and streak onto the quality control area of ​​nitrocellulose membrane, and then dry to prepare test line and quality control line respectively.

5. The method for preparing a test strip for detecting Helicobacter pylori antigen in feces according to claim 4, characterized in that: The sample pad is soaked in the sample pad pretreatment solution for 20 to 30 minutes and then dried at 37 to 40° C. for 3 to 5 hours to obtain a pretreated sample pad.

6. The method for preparing a test strip for detecting Helicobacter pylori antigen in feces according to claim 4, characterized in that: The preparation method of the colloidal gold-labeled binding antibody includes: adding colloidal gold to a stirring container, adding 0.1M potassium carbonate at a ratio of 1:100 to 1:200, and stirring for 10 minutes; adding an anti-Helicobacter pylori monoclonal antibody at a concentration of 10 to 30 μg / mL, and stirring for 30 minutes; adding BSA to a final concentration of 1% to 1.2%, and stirring for 30 minutes; centrifuging at 12000 rpm and 4°C for 30 minutes; after the centrifugation, discarding the supernatant, and resuspending the precipitate with a gold label diluent at a ratio of 1:10 to obtain the colloidal gold-labeled binding antibody.

7. A method for preparing a test strip for detecting Helicobacter pylori antigen in feces according to claim 6, characterized in that: The gold label diluent contains 0.31% boric acid, 1.91% sodium tetraborate, 3% trehalose, 0.2% Tween-20, 0.05% casein, 0.1% PEG20000, and water is used as the solvent.

8. A method for preparing a test strip for detecting Helicobacter pylori antigen in feces according to claim 7, characterized in that: The conjugate pad was soaked in the conjugate pad pretreatment solution for 20 to 30 minutes and then dried at 37 to 40°C for 3 to 5 hours to prepare a pretreated conjugate pad. Then, the colloidal gold-labeled binding antibody was sprayed on the pretreated conjugate pad at a concentration of 8 to 9 μL / cm and dried in a drying oven at 40°C for 2 hours.

9. The method for preparing a test strip for detecting Helicobacter pylori antigen in feces according to claim 4, wherein: The method for preparing a nitrocellulose membrane includes: streaking an anti-Helicobacter pylori monoclonal antibody with a concentration of 1.0 to 2.0 mg / mL onto a detection area of ​​the nitrocellulose membrane with a streaking volume of 1 μL / cm; mixing a goat anti-mouse IgG antibody with a concentration of 1.0 to 2.0 mg / mL and a rabbit anti-chicken IgY antibody with a concentration of 0.2 to 0.4 mg / mL, and then streaking the mixture onto a quality control area of ​​the nitrocellulose membrane with a streaking volume of 1 μL / cm; and then placing the mixture in a drying oven at 40°C for 2 hours to dry, thereby preparing a detection line and a quality control line, respectively.

10. A kit for detecting Helicobacter pylori antigen in feces, characterized in that: The invention comprises the test strip according to any one of claims 1 to 3 or the test strip prepared by the preparation method according to any one of claims 4 to 9, and a plastic card shell for containing the test strip and a sample diluent.

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