Polypeptide and application thereof in preparation of kit for detecting candida albicans

Abstract

The invention belongs to the field of biological detection, and particularly relates to a polypeptide and application thereof in preparation of a kit for detecting candida albicans. The amino acid sequence of the polypeptide is shown as SEQ ID NO: 1. A kit for detecting candida albicans comprises an enzyme-linked reaction plate coated with the polypeptide. Compared with the current clinical conventional fungal infection detection, the polypeptide and the Elisa Candida albicans detection kit developed based on the polypeptide provided by the invention not only can effectively improve the sensitivity of Candida albicans infection, but also have high Candida albicans specificity.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection, and particularly relates to a polypeptide and its application in the preparation of a kit for detecting Candida albicans. Background Art

[0002] Clinically, the diagnostic methods for Candida albicans mainly include microbial culture and β - D - glucan (β - BDG) detection method. However, the microbial culture method usually takes 3 - 5 days, and has low sensitivity and high false - negative rate for invasive Candida albicans infection. And β - BDG is a pan - fungal diagnostic marker, and this detection method cannot determine the specific fungal species, with poor diagnostic specificity. At the same time, this method is easily interfered by some clinical treatments, such as blood products, etc., resulting in a high false - positive rate. For some patients with difficult and complicated diseases, microbial metagenomic detection is sometimes used clinically. However, since Candida albicans is a common colonizing bacterium in humans, only detecting a small number of DNA copies of Candida albicans cannot accurately determine whether it is colonization or invasive infection. Therefore, there is currently no comprehensive and excellent diagnostic method for invasive Candida albicans infection.

[0003] Currently, the diagnosis of invasive candidiasis lacks accurate and effective biomarkers. A major challenge is to distinguish between commensal colonization and invasive infection, because host - pathogen interactions are common in both states. Serum diagnostic antigens that can distinguish these situations with high sensitivity and specificity in Candida albicans infection remain a complex task. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a polypeptide and its application in the preparation of a kit for detecting Candida albicans.

[0005] To achieve the above - mentioned invention purpose, the present invention provides the following technical solutions:

[0006] A polypeptide, the amino acid sequence corresponding to the polypeptide is as shown in SEQ ID NO: 1.

[0007] The above - mentioned polypeptide is used for capturing anti - Candida albicans antibody.

[0008] The above - mentioned polypeptide is used in the preparation of a kit for detecting Candida albicans.

[0009] Preferably, the kit includes an Elisa kit, an immunomicrosphere / magnetic bead kit, and an immunochromatography kit.

[0010] A kit for detecting Candida albicans, the kit includes an enzyme - linked reaction plate or the reagents for preparing the enzyme - linked reaction plate, and the above - mentioned polypeptide is coated on the enzyme - linked reaction plate.

[0011] In the above-mentioned kit, the preparation method of the enzyme-linked reaction plate comprises the following steps:

[0012] a. Mix the above-mentioned polypeptide with the coating buffer, add it to the enzyme-labeled plate and incubate at a constant temperature;

[0013] b. Discard the supernatant in the enzyme-labeled plate after incubation in a, and add the blocking solution for blocking operation;

[0014] c. Discard the supernatant in the enzyme-labeled plate after blocking in b, and wash the remaining blocking solution to obtain the enzyme-linked reaction plate.

[0015] Preferably, the coating buffer is carbonate coating buffer.

[0016] Preferably, the coating concentration of the polypeptide is 0.5 μg / 100 μL to 1.5 μg / 100 μL.

[0017] In the above-mentioned kit, the kit further comprises an enzyme-labeled secondary antibody, Elisa color development-related reagents, and a positive standard or a standard curve.

[0018] Preferably, the positive standard is positive serum or an antibody against the above-mentioned polypeptide.

[0019] Beneficial effects:

[0020] In the present invention, the Candidalysin peptide is identified as a serum-dominant antigen, and its C-terminal region is the main immunogenic site. Based on this, the present invention provides an indirect ELISA kit, which is a method for detecting the level of anti-candidalysin IgG antibody in serum. In the training and validation cohorts, the performance of serum anti-Candidalysin IgG was good, and the AUC values were all above 0.83.

[0021] Compared with the current clinically conventional detection methods for fungal infections (microbial culture and BDG detection). The anti-Candidalysin IgG serum antibody ELISA detection method provided by the present invention can not only effectively improve the sensitivity of Candida albicans infection, but also has high specificity for Candida albicans. This detection can specifically detect invasive infections caused by Candida albicans. Description of the drawings

[0022] Figure 1 : An Elisa flow chart for detecting Candida albicans.

[0023] Figure 2 : Signal-to-noise ratio (P / N) detection results.

[0024] Figure 3: Elisa standard curve.

[0025] Figure 4 : Sensitivity and specificity experiments, left: training cohort; right: validation cohort. Detailed implementation

[0026] The present invention provides a polypeptide, characterized in that the amino acid sequence corresponding to the polypeptide is shown in SEQ ID NO: 1.

[0027] Application of the above polypeptide in capturing anti-Candida albicans antibodies.

[0028] Application of the above polypeptide in preparing a kit for detecting Candida albicans.

[0029] Preferably, the kit includes an Elisa kit, an immunomicrosphere / magnetic bead kit, and an immunochromatography kit. More preferably, the kit is an Elisa kit.

[0030] A kit for detecting Candida albicans, the kit includes an enzyme-linked reaction plate or reagents for preparing an enzyme-linked reaction plate, and the above polypeptide is coated on the enzyme-linked reaction plate.

[0031] In the above kit for detecting Candida albicans, the preparation method of the enzyme-linked reaction plate includes the following steps:

[0032] a. Mix the above polypeptide with a coating buffer, add it to an enzyme-labeled plate, and incubate at a constant temperature;

[0033] Preferably, the coating buffer is a carbonate coating buffer.

[0034] Preferably, the coating concentration of the polypeptide is 0.5 μg / 100 μL to 1.5 μg / 100 μL. More preferably, the coating concentration is 1 μg / 100 μL.

[0035] b. Discard the supernatant in the enzyme-labeled plate after incubation in step a, and add a blocking solution for blocking operation;

[0036] Preferably, the blocking solution is selected from BSA solution, fetal bovine serum, and skim milk. More preferably, the blocking solution is skim milk.

[0037] Furthermore, the skim milk is a 1% - 5% (w / v) skim milk PBS solution and contains 0.02% - 0.08% Tween 20. Preferably, the skim milk is a 3% (w / v) skim milk PBS solution and contains 0.05% Tween 20

[0038] c. Discard the supernatant of the enzyme-labeled plate after blocking in step b, and wash the residual blocking solution to obtain the enzyme-linked reaction plate. Preferably, wash three times with PBST to remove the residual blocking solution.

[0039] In the above kit, the kit further includes an enzyme-labeled secondary antibody, Elisa color development related reagents, and a positive standard or a standard curve.

[0040] Preferably, the positive standard is positive serum or an antibody against the above polypeptide.

[0041] Preferably, the enzyme-labeled secondary antibody is an HRP-conjugated antibody.

[0042] Furthermore, when using the above kit to detect Candida albicans, the above enzyme-labeled secondary antibody is diluted with a blocking solution during use, and the dilution ratio is 1:2000 - 10000, and the preferred dilution ratio is 1:5000.

[0043] In the present invention, unless otherwise specified, all raw material components are commercially available products well-known to those skilled in the art.

[0044] Next, the technical solutions in the present invention will be clearly and completely described in conjunction with the embodiments in the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of them. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present invention without making creative efforts belong to the scope of protection of the present invention.

[0045] Example 1

[0046] Obtaining of polypeptide:

[0047] According to the amino acid sequence shown in SEQ ID NO: 1: SIIGIIMGILGNIPQVIQIIMSIVKAFKGNK, the polypeptide (Candidalysin polypeptide molecule) was chemically synthesized by Beijing Zexiyuan Biotechnology Co., Ltd. (LifeTein).

[0048] Example 2

[0049] Preparation of enzyme-linked reaction plate:

[0050] 1. Coating antigen: Add 100 μL of coating buffer (0.015M Na 2 CO 3 , 0.035M NaHCO 3 , pH 9.6) and 1 μg of Candidalysin polypeptide molecule (5 μg / μL in ddH 2 O) into each well of the microplate. Incubate overnight at 4°C with gentle horizontal rotation on a shaker.

[0051] 2. Closed orifice plate: Aspirate the supernatant in the orifice plate. Wash the orifice plate once with sterile water, add 200 μL of blocking solution (3% non-fat milk w / v in PBS and 0.05% Tween-20) to each well, incubate at room temperature for 1 hour, and gently rotate horizontally.

[0052] 3. Wash the orifice plate 3 times with PBST buffer for 5 minutes each time.

[0053] Example 3

[0054] Preparation of enzyme-linked reaction plate:

[0055] Coating antigen: Add 100 μL of coating buffer (0.015 M Na 2 CO 3 , 0.035 M NaHCO 3 , pH 9.6) and 0.5 μg of Candidalysin polypeptide molecule (5 μg / μL in ddH 2 O) to each well of the microplate. Incubate overnight at 4 °C with gentle horizontal rotation on a shaker.

[0056] The remaining steps are the same as in Example 2.

[0057] Example 4

[0058] Signal-to-noise ratio (P / N) detection:

[0059] Take the enzyme-linked reaction plates in Examples 2 and 3 for Elisa experiment (the process is as Figure 1 shown).

[0060] 1. Add positive serum samples to the orifice plate and incubate at room temperature for 3 hours.

[0061] 2. Wash the orifice plate 3 times with PBST buffer for 5 minutes each time.

[0062] 3. Add HRP-conjugated antibody against human IgG (diluted 1:5000 with blocking solution, goat anti-human IgG H&L (HRP), abcam, ab6858), and incubate at room temperature for 1 hour.

[0063] 4. Wash the orifice plate 3 times with PBST buffer for 5 minutes each time.

[0064] 5. Color development: Remove the supernatant in the orifice plate, pat dry the orifice plate. Add 100 μL of TMB substrate solution (Thermo Scientific Pierce 1-Step Ultra TMB ELISA substrate solution, catalog number 34029), and incubate in the dark for 5 minutes. Add 100 μL of 2 M sulfuric acid solution to each well to terminate the reaction.

[0065] 6. Plate reading record: Place the microplate on the BioTeK microplate reader, measure the absorbance values at 450 nm and 570 nm and record them.

[0066] Compare the P / N value (OD450 of the positive sample divided by OD450 of the negative control sample). The results are as Figure 2 shown (S1 and S2 are two positive serum test samples diluted at different ratios). Compared with the coating amount of 0.5 μg, a coating amount of 1 μg can obtain a better signal-to-noise ratio.

[0067] Example 5

[0068] Construction of the standard curve:

[0069] Take the ELISA plate of Example 2, and refer to the experimental method in Example 4 to construct a standard curve using the standard product. The standard product is an antibody against murine Candidalysin with a concentration of 0.957 mg / ml (prepared by Nanjing Genscript Corporation), and the secondary antibody is an HRP-conjugated goat anti-mouse IgG antibody (prepared by Nanjing Genscript Corporation). The standard product is diluted with the blocking solution at 1:32K, 1:64K, 1:128K, 1:256K, 1:512K, 1:1024K, and 1:2048K.

[0070] Use the sigmoidal, 4PL, X as log(concentration) module in Graphpad software to construct the standard curve, with the OD450nm value in the ELISA test on the y-axis. The test samples pass through the standard curve, as Figure 3 and shown in Table 1:

[0071] Table 1

[0072]

[0073] Example 6

[0074] Sensitivity and specificity experiments:

[0075] A total of 270 cases of children's sera were used for the detection experiment, and each sample was detected in 3 parallel wells.

[0076] There were 58 cases in the healthy group. Healthy group: Physical examination children without acute infection, serious diseases, or underlying diseases.

[0077] 43 cases were positive for Candida albicans infection, including 23 cases positive in blood culture, 10 cases positive in urine culture, and 11 cases positive in fecal culture. Positive for Candida albicans infection: Positive in blood culture. Positive in feces and urine cultures for two consecutive times.

[0078] There were 24 cases of suspected Candida albicans infection and 47 cases of other infections. Suspected Candida albicans infection: The sputum specimen culture was positive. Other infection control group: Bacterial or viral infection was proved by antigen detection or culture method.

[0079] Detection method control group: There were 98 cases in total, including positive and negative G tests.

[0080] Take the enzyme-linked reaction plate prepared in Example 2 and perform serological experiment detection with reference to the operation steps in Examples 4 and 5.

[0081] In the training set, compare the differences in the serum anti-Candidalysin IgG antibody concentration between the positive group of Candida albicans bacteremia (23 cases) and the control group of children (77 cases, healthy group + disease control group + non-albicans Candida group). The ROC curve analyzes anti-Candidalysin IgG antibody as a diagnostic marker for Candida albicans infection. The results are as Figure 4 shown in the left part of, with an AUC value of 0.885 (95% CI: 0.818 - 0.952). When the cut-off value is 0.1768 ng / mL, the sensitivity reaches 82.6% and the specificity is 83.1%.

[0082] In the validation set, select the Candida albicans infection group (41 cases, blood culture + fecal culture + urine culture, at least 1 positive blood culture, and at least two consecutive positive fecal and urine cultures) and the healthy group (58 cases) for comparison. The ROC curve analyzes anti-Candidalysin IgG antibody as a diagnostic marker for Candida albicans infection. The results are as Figure 3 shown in the right part of, with an AUC value of 0.835 (95% CI: 0.757 - 0.913). When the cut-off value is 0.1175 ng / mL, the sensitivity reaches 82.8% and the specificity is 69.0%.

[0083] It should also be noted that in the experiment, it was found that some samples from the healthy cohort showed levels similar to those of certain positive patients. This indicates that there is a certain overlap between the early stage of infection and active colonization. Anti-Candidalysin IgG has a certain lag in the diagnosis of early Candida albicans infection. However, in the middle and late stages of infection, the increase in anti-Candidalysin IgG level can be used as an indicator of invasive candidiasis and can well distinguish colonization and invasive infection.

[0084] Example 7

[0085] Reliability test:

[0086] The Cohen's kappa coefficient (κ) was used to compare the consistency between the ELISA method of the present invention and the clinically conventional methods for detecting fungal infections (BDG detection and microbial culture method). The results are shown in Table 2 as follows:

[0087] Table 2

[0088]

[0089]

[0090] It shows that the anti-Candidalysin IgG serum antibody ELISA detection method has a high consistency with the culture (kappa = 0.689, p < 0.05), while there is no consistency with the BDG experiment (kappa = 0.085, p < 0.05). This may be because BDG is a pan-marker of fungi and not specific to Candida albicans, resulting in low specificity of detection and low consistency between the two methods.

[0091] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A polypeptide, characterized in that The amino acid sequence corresponding to the polypeptide is shown in SEQ ID NO:

1.

2. Use of the polypeptide as claimed in claim 1 in capturing anti-Candida albicans antibodies.

3. Use of the polypeptide according to claim 1 in preparing a kit for detecting Candida albicans.

4. The use according to claim 3, characterized in that The kits include Elisa kits, immune microsphere / magnetic bead kits and immunochromatography kits.

5. A kit for detecting Candida albicans, characterized in that: The kit comprises an enzyme-linked reaction plate or a reagent for preparing an enzyme-linked reaction plate, and the polypeptide according to claim 1 is coated on the enzyme-linked reaction plate.

6. The kit according to claim 5, characterized in that The preparation method of the enzyme linked reaction plate comprises the following steps: a. Mix the polypeptide according to claim 1 with the coating buffer, add it to the ELISA plate and incubate at a constant temperature; b. Discard the supernatant in the ELISA plate after incubation in step a, and add blocking solution for blocking operation; c. discard the supernatant of the ELISA plate after blocking in b, and wash away the residual blocking solution to obtain the ELISA plate.

7. The kit according to claim 6, characterized in that The coating buffer is a carbonate coating buffer.

8. The kit according to claim 6, characterized in that The coating concentration of the polypeptide is 0.5 μg / 100 μL to 1.5 μg / 100 μL.

9. The kit according to claim 5, characterized in that The kit also includes an enzyme-labeled secondary antibody, Elisa color development-related reagents, and a positive standard or a standard curve.

10. The kit according to claim 9, characterized in that The positive standard is a positive serum or an antibody against the polypeptide according to claim 1.

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