CN113791216A

CN113791216A - An ELISA detection method for detecting human serum Candida glabrata enolase IgG antibody

Info

Publication number: CN113791216A
Application number: CN202111015550.7A
Authority: CN
Inventors: 陈兴
Original assignee: Shijiazhuang Heya Biotechnology Co ltd
Current assignee: Shijiazhuang Heya Biotechnology Co ltd
Priority date: 2021-08-31
Filing date: 2021-08-31
Publication date: 2021-12-14

Abstract

An ELISA detection method for detecting human serum Candida glabrata enolase IgG antibody, comprising the following steps: equilibrating each reagent to a room temperature of 20-25° C.; using 100 μL of sample diluent as a blank control, adding a blank coating an enzyme-linked plate Take 100 μL of negative serum as the negative control, add the negative control hole of the coated enzyme-linked plate; take 100 μL of the positive serum as the positive control, add the positive control hole of the coated enzyme-linked plate; add 100 μL of the serum to be tested at a 1:500 dilution to the detection Well; incubate at 37°C for 60min; wash; add 100 μL of enzyme-labeled antibody to the well of the coated enzyme-linked plate, place at 37°C for 30min; wash; develop color; stop the reaction; The light absorption value at the wavelength of 450nm, after judging the validity of the test, calculate the S/N value. The invention uses Candida glabrata enolase as an antigen to establish an indirect ELISA detection technology of human serum Candida glabrata enolase IgG antibody, the detection specificity reaches 100%, and the ELISA detection kit used has high sensitivity and The batch-to-batch stability was good.

Description

ELISA detection method for detecting human serum candida glabrata enolase IgG antibody

Technical Field

The invention relates to an ELISA detection method for detecting a human serum Candida glabrata enolase IgG antibody, belonging to the technical field of biology.

Background

Candida is a normal parasitic flora of human body, but when certain factors destroy the balance between the immune system of a host and candida, the candida can invade host tissues and blood, grow and reproduce in the host tissues to cause pathological changes such as tissue damage, organ dysfunction and inflammatory response, and the infection is Invasive Candidiasis (IC). The current clinical medicine is increasingly challenged by IC, and IC morbidity continues to rise in many areas, with mortality rates as high as 40% even if patients receive antifungal therapy. In most clinical cases, candida albicans is the most common pathogen causing IC. In recent years, IC-causing cases of other non-candida albicans species have become more prevalent due to prophylactic or empirical first line antifungal use. The candida glabrata is not sensitive to the antifungal drug fluconazole commonly used in clinic, and easily forms protective biofilms in various cannulas, so that the trend of increasing the separation rate is more prominent.

For patients with IC, early clinical diagnosis and initiation of antifungal therapy is critical to improving patient survival. To date, blood culture or fluid culture in sterile sites has been considered the gold standard for diagnosis of invasive candidiasis. However, blood culture sensitivity is low (< 50%), and detection takes a long time, up to about 8 days. In recent years, new technologies such as beta-glucan detection and PCR have been developed in molecular biology for IC diagnosis, but they have their own disadvantages and cannot fully satisfy clinical needs. For example, although the beta-glucan detection has already begun to be applied clinically, the method is greatly interfered by food, medicines and other pathogenic infections, and has a prominent false positive problem; the PCR detection has the potential of quickly diagnosing IC, the existing problem is that the standardization of the detection is difficult to finish, the extremely high sensitivity of the PCR detection reduces the specificity of the detection, and the problems that candida has large interference on the experimental result and false positive exists widely in the environment and cannot be overcome in a short time.

The serological method using candida specific antigen or antibody as the detection object becomes a hotspot in the research field of laboratory rapid diagnosis IC by virtue of the advantages of sensitivity, simplicity, convenience and easiness in development. In recent years, a series of target molecules with serological diagnostic value are identified by technologies such as 2D immunoelectrophoresis, and among them, enolase (Eno) is considered to have the most potential by many researchers. In vitro studies on candida albicans have found that Eno has two significant characteristics particularly suitable for application to serological diagnostic studies: the content is rich, and in vitro experiments show that the Eno content in the total protein of the candida albicans yeast phase and the mycelium phase can reach 0.7 percent and 2 percent respectively; secondly, Eno can stimulate the body to generate high-titer antibodies, which are main immunodominant antigens of Candida albicans.

The current theoretical research on serological diagnosis of Candida albicans is relatively sufficient, and a large number of researches show that the Candida albicans Eno antigen antibody detection is a beneficial supplement to the gold standard blood culture method. On this basis, research teams have begun to develop commercial detection reagents for candida albicans Eno. For example, the company Vircell, spain, has initially introduced ELISA reagents that can detect candida albicans Eno antibodies. However, the biggest challenge in the field of IC diagnosis at present is that the research on the detection of non-candida albicans infection is severely insufficient, and the serological diagnosis technology of candida glabrata infection is still blank at present.

Disclosure of Invention

The invention provides an ELISA detection method for detecting human serum candida glabrata enolase IgG antibody, which overcomes the defects of the prior art, establishes an indirect ELISA detection technology for the human serum candida glabrata enolase IgG antibody by using candida glabrata enolase as an antigen, and has the detection specificity reaching 100 percent.

The technical scheme adopted by the invention for solving the technical problems is as follows:

an ELISA detection method for detecting a human serum Candida glabrata enolase IgG antibody is an indirect ELISA detection method, an ELISA kit is used for detecting the human serum Candida glabrata enolase Cg-Eno IgG antibody, and the method specifically comprises the following steps:

a. taking out each reagent of the kit, and balancing to room temperature of 20-25 ℃;

b. sample adding: adding 100 mu L of sample diluent serving as a blank control into blank holes coated with the enzyme linked plate; adding 100 mu L of negative serum as a negative control solution into the coated enzyme linked plate negative control hole; adding 100 mu L of positive serum as a positive control solution into the coated enzyme-linked plate positive control hole; adding 100 mu L of 1:500 diluted serum to be detected into a detection hole;

c. incubating the coated enzyme-linked plate at 37 ℃ for 60 minutes;

d. washing: throwing off samples in the holes, adding 400 mu L of washing solution into the holes coated with the enzyme linked plate, repeatedly washing for 5 times by using the washing solution, and patting dry on absorbent paper;

e. adding 100 mu L of enzyme-labeled antibody into the coated enzyme-linked plate hole, and standing for 30 minutes at 37 ℃;

f. washing: repeating the washing step of step d;

g. color development: adding 50 mu L of substrate solution A and 50 mu L of substrate solution B, shaking up lightly, and reacting for 15 minutes at 37 ℃ in a dark place;

h. adding 50 mu L of final solution into each hole to terminate the reaction, placing an enzyme label plate on an enzyme label instrument, adjusting 0 by using a blank hole, reading the light absorption value under the wavelength of 450nm, judging the effectiveness of the test, and then calculating the S/N value, wherein the S/N value is equal to the OD450 of the sample to be detected/the negative control OD450, and the S/N value is more than or equal to 2.5 and is positive; 2.0< S/N <2.5 is weakly positive; S/N is less than or equal to 2.0, and the negative result is obtained.

The ELISA kit comprises an ELISA plate, an enzyme-labeled antibody, a washing solution, a substrate color development solution, a sample diluent, a stop solution, a positive control solution and a negative control solution, wherein the ELISA plate is coated by a purified Cg-Eno recombinant protein antigen, and the concentration of the Cg-Eno recombinant protein antigen is 1 mug/mL.

According to the ELISA detection method for detecting the human serum Candida glabrata enolase IgG antibody, the coating process of the ELISA plate is as follows: diluting the purified Cg-Eno recombinant protein antigen to 1 mu g/mL by using a carbonate buffer solution with the pH of 9.5 and 0.05mol/L, adding the diluted Cg-Eno recombinant protein antigen to a 96-well coated plate, standing the plate at 100 mu L/well for overnight at 4 ℃, washing the plate for 3 times by using a washing solution, adding 1% BSA (bovine serum albumin) 0.01mol/L PBS with the pH of 7.2, sealing the plate overnight, throwing off the sealing solution, naturally drying the plate for 4-6 hours at room temperature with the humidity of below 40%, drying the plate in vacuum, sealing the plate, and storing the plate at 4 ℃ to obtain the coated enzyme-linked plate.

According to the ELISA detection method for detecting the human serum Candida glabrata enolase IgG antibody, the preparation process of the purified Cg-Eno recombinant protein antigen comprises the following steps:

a. obtaining recombinant candida glabrata enolase Cg-Eno gene and constructing plasmid: selecting a gene sequence with a sequence number XM-446328.1 registered in Genebank as a Cg-Eno sequence; obtaining a target gene by adopting a whole-gene synthesis mode, and constructing a pET-30a (+) -Cg-Eno recombinant plasmid after the target gene is subjected to enzyme digestion and connection to obtain a recombinant expression vector;

b. expression and purification of recombinant protein: introducing the recombinant expression vector into a host bacterium escherichia coli Rosetta (DE3) to obtain a Cg-eno recombinant strain, and inducing the expression of the recombinant strain by IPTG; collecting recombinant strains, carrying out resuspension on the collected strains by using normal saline, carrying out ultrasonic bacteria breaking, filtering by using a 0.45-micron filter membrane, purifying the obtained Cg-Eno recombinant protein by using a Ni column, and eluting by using imidazole buffer solution with gradient concentration to obtain the purified Cg-Eno recombinant protein antigen.

According to the ELISA detection method for detecting the human serum Candida glabrata enolase IgG antibody, the enzyme-labeled antibody is 1: HRP-goat anti-human IgG antibody at 4000 dilutions.

According to the ELISA detection method for detecting the human serum Candida glabrata enolase IgG antibody, the preparation process of the washing solution is as follows: 0.5g of NaH₂PO₄，6.0g Na₂HPO₄0.5mL of Tween-20, 8.5g of NaCl was dissolved in 500mL of water, and the pH was adjusted to 7.4, thereby obtaining a volume of 1L.

According to the ELISA detection method for detecting the human serum Candida glabrata enolase IgG antibody, the preparation process of the substrate color developing solution is as follows: dissolving 4.2g of citric acid, 13.6g of sodium acetate and 0.5g of carbamide peroxide in 100mL of deionized water, fixing the volume to 1L, and adjusting the pH value to 5.0 to obtain a substrate A solution; dissolving lg TMB in 50mL of dimethyl sulfoxide, adding 100mL of formaldehyde and 16.0g of polyethylene pyrrolidone after dissolving, and diluting to 1L with deionized water after dissolving to obtain a substrate B solution.

According to the ELISA detection method for detecting the human serum Candida glabrata enolase IgG antibody, the preparation process of the sample diluent is as follows: 0.5g of NaH₂PO₄，6.0g Na₂HPO₄8.5g NaCl dissolved in 500mL water, adjusted to pH 7.4, most preferablyThe final volume is 1L, 1g of BSA is added until the BSA is completely dissolved, and a sample diluent is obtained; the stop solution is a 2mol/L sulfuric acid solution.

The ELISA detection method for detecting the human serum Candida glabrata enolase IgG antibody comprises the following steps: collecting serum 10 of clinically confirmed candida glabrata infected patients, mixing the serum uniformly, inactivating the serum for 30min at 56 ℃, and diluting the serum with a sample diluent 1:500 dilution; the negative control solution is: 10 serum samples of clinical candida glabrata infection normal physical examination are collected, mixed evenly, inactivated at 56 ℃ for 30min, and the serum is diluted by a sample diluent 1: and (5) diluting by 500.

According to the ELISA detection method for detecting the human serum Candida glabrata enolase IgG antibody, the standard for judging the test effectiveness is as follows: the positive control serum OD450>0.5, the negative control serum OD450 <0.18, blank wells OD450 0, the experiment was established, otherwise not valid.

The invention has the beneficial effects that: the invention establishes an indirect ELISA detection technology of human serum candida glabrata enolase IgG antibody by taking recombinant candida glabrata enolase protein as an antigen, and the detection specificity reaches 100 percent. The prepared ELISA kit has good cross reactivity with other clinical common candida infection, and has no cross with the serum of a common bacterial infection patient. The kit has high sensitivity and good batch-to-batch stability.

Drawings

FIG. 1 is a SDS-PAGE analysis of nickel agarose affinity chromatography elution fractions of recombinant Cg-Eno proteins of the invention;

FIG. 2 is an SDS-PAGE analysis of the final purification of recombinant Cg-Eno protein;

FIG. 3 is Western Blot analysis of recombinant Cg-Eno protein.

In fig. 1: m, Protein Marker; 1. sampling; 2. flowing out; 3-4, 20mM Imidazole; 5-6, 50mM Imidazole; 7. 500mM Imidazole; in fig. 2: m, Protein Marker; 1. a protein of interest; in fig. 3: m, Prestained Marker; 1. a protein of interest.

Detailed Description

The present invention will be further described with reference to the following examples.

An ELISA detection method for detecting a human serum Candida glabrata enolase IgG antibody is characterized in that an indirect ELISA detection method is used for detecting a human serum Candida glabrata enolase Cg-Eno IgG antibody, and a special ELISA kit is used for detecting the Cg-Eno IgG antibody in the detection process.

The ELISA kit comprises an ELISA plate, an enzyme-labeled antibody, a washing solution, a substrate developing solution, a sample diluent, a stop solution, a positive control solution and a negative control solution, wherein the ELISA plate is coated with a purified Cg-Eno recombinant protein antigen to prepare a coated enzyme-linked plate. The specifications of each reagent are as follows:

the coating process of the ELISA plate comprises the following steps: diluting the purified Cg-Eno recombinant protein antigen to 1 mu g/mL by using a carbonate buffer solution with the pH of 9.5 and 0.05mol/L, adding the diluted Cg-Eno recombinant protein antigen to a 96-well coated plate, standing the plate at 100 mu L/well for overnight at 4 ℃, washing the plate for 3 times by using a washing solution, adding 1% BSA (bovine serum albumin) 0.01mol/L PBS with the pH of 7.2, sealing the plate overnight, throwing off the sealing solution, naturally drying the plate for 4-6 hours at room temperature with the humidity of below 40%, drying the plate in vacuum, sealing the plate, and storing the plate at 4 ℃ to obtain the coated enzyme-linked plate.

The enzyme-labeled antibody is 1: HRP-goat anti-human IgG antibody (ABCam) diluted at 4000 was diluted with the sample diluent.

The preparation process of the washing liquid comprises the following steps: 0.5g of NaH₂PO₄，6.0g Na₂HPO₄0.5mL of Tween-20, 8.5g of NaCl was dissolved in 500mL of water, and the pH was adjusted to 7.4, thereby obtaining a volume of 1L.

The preparation process of the substrate color development liquid comprises the following steps: dissolving 4.2g of citric acid, 13.6g of sodium acetate and 0.5g of carbamide peroxide in 100mL of deionized water, fixing the volume to 1L, and adjusting the pH value to 5.0 to obtain a substrate A solution; dissolving lg TMB in 50mL of dimethyl sulfoxide, adding 100mL of formaldehyde and 16.0g of polyethylene pyrrolidone after dissolving, and diluting to 1L with deionized water after dissolving to obtain a substrate B solution.

The preparation process of the sample diluent comprises the following steps: 0.5g of NaH₂PO₄，6.0g Na₂HPO₄8.5g of NaCl is dissolved in 500mL of water, the pH value is adjusted to 7.4, the volume is finally determined to be 1L, 1g of BSA is added until the BSA is completely dissolved, and a sample diluent is obtained; the stop solutionIs a 2mol/L sulfuric acid solution.

The positive control solution is: collecting serum 10 of clinically confirmed candida glabrata infected patients, mixing the serum uniformly, inactivating the serum for 30min at 56 ℃, and diluting the serum with a sample diluent 1:500 dilution; the negative control solution is: 10 serum samples of clinical candida glabrata infection normal physical examination are collected, mixed evenly, inactivated at 56 ℃ for 30min, and the serum is diluted by a sample diluent 1: and (5) diluting by 500.

Before detection, a purified Cg-Eno recombinant protein antigen is prepared, and the preparation process comprises the following steps:

a. obtaining recombinant candida glabrata enolase Cg-Eno gene and constructing plasmid: selecting a gene sequence with the sequence number XM-446328.1 registered in Genebank as a Cg-Eno sequence (the corresponding amino acid sequence accession number is NC-006032.2); obtaining a target gene by adopting a whole-gene synthesis mode, and constructing a pET-30a (+) -Cg-Eno recombinant plasmid after the target gene is subjected to enzyme digestion and connection to obtain a recombinant expression vector; the original expression vector is pET-30a (+) and the restriction enzyme cutting site is Nco I/XhoI.

b. Expression and purification of recombinant protein: introducing the recombinant expression vector into a host bacterium escherichia coli Rosetta (DE3) to obtain a Cg-Eno recombinant strain, inducing the expression of the recombinant strain by IPTG (isopropyl-beta-thiogalactoside) at the induction expression temperature of 30-40 ℃, preferably 37 ℃, for 3-5h, preferably 4h, using an inducer IPTG as the IPTG, and adding the IPTG to the final concentration of 0.1-1.0mM, more preferably 0.5 mM; the recombinant strains are collected, the collected strains are resuspended by normal saline, broken by ultrasonic waves, filtered by a 0.45 mu m filter membrane, the obtained Cg-Eno recombinant protein is purified by a Ni column, and is eluted by imidazole buffer solution with gradient concentration, the SDS-PAGE analysis of the elution component of nickel agarose affinity chromatography is shown in figure 1, and the SDS-PAGE analysis of the obtained purified Cg-Eno recombinant protein antigen is shown in figure 2.

Performing color reaction on the prepared purified Cg-Eno recombinant protein antigen: and (3) taking a rabbit anti-His tag antibody as a primary antibody, and developing with a TMB developing kit according to the Western Blot step to show that an obvious band appears at the corresponding position of the recombinant protein, thereby confirming that the obtained protein is the Cg-Eno protein expressed by recombination, and the sequence is shown in figure 3.

The indirect ELISA detection method comprises the following specific processes:

c. incubating the coated enzyme-linked plate at 37 ℃ for 60 minutes;

f. washing: repeating the washing step of step d;

h. adding 50 mu L of final solution into each hole to terminate the reaction, placing an enzyme label plate on an enzyme label instrument, adjusting zero by using a blank hole, reading the light absorption value under the wavelength of 450nm, and judging the effectiveness of the test: the positive control serum OD450 is greater than 0.5, the negative control serum OD450 is less than 0.18, the blank hole OD450 is 0, the experiment is established, otherwise, the experiment is invalid; calculating an S/N value, wherein the S/N value is equal to the OD450 of the sample to be detected/the negative control OD450, and the S/N is more than or equal to 2.5 and is positive; 2.0< S/N <2.5 is weakly positive; S/N is less than or equal to 2.0, and the negative result is obtained.

The performance of the indirect ELISA kit of the invention is detected.

1. Specificity of

The indirect ELISA kit provided by the invention is used for respectively detecting the serums of 5 candida glabrata infected patients and 5 healthy examinees through an indirect ELISA detection technology, the results are shown in the table 1, the serums of 5 candida glabrata infected patients are positive, the serums of 5 healthy examinees are negative, and the detection specificity is 100%.

TABLE 1 Candida glabrata infection and serum CgEno IgG antibody analysis in healthy examiners

2. Cross reaction

Serum of other clinical common candida and clinical common bacterial infection patients is selected, the kit is used for detecting, and the cross reactivity of the detection reagent is analyzed. Specific strains and detection results are shown in Table 2, and it can be seen that Cg-Eno IgG antibody detection has good cross-reactivity with other clinical common candida infections, and has no cross with serum of common bacterial infection patients.

TABLE 2 detection results of serum CgEno IgG antibody in common clinical strain infection

3. Sensitivity of the probe

3 batches of the Cg-Eno IgG antibody ELISA detection kit are adopted to detect 3 portions of Candida glabrata culture positive serum, and the detected titer is 1: 1600-1: 12800, which shows that the kit of the present invention has high sensitivity and good stability among batches, and the results are shown in Table 3.

TABLE 3 Candida glabrata antibody positive serum titer test Table

Note: determination criteria of the kit: the S/N is more than or equal to 2.5 and is positive; 2.0< S/N <2.5 is weakly positive; S/N is less than or equal to 2.0, and the negative result is obtained.

Sequence listing

<110> Shijiazhuang and Subsbiotech GmbH

<120> ELISA detection method for detecting human serum Candida glabrata enolase IgG antibody

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<170> SIPOSequenceListing 1.0

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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ccatggggat gtcagtcact aaagtccacg ccagaacagt ctatgactcc agaggtaacc 60

ctacagttga agtagaggtc tacaccaagg acggtatgtt cagggctatt gtcccatccg 120

gtgcctcaac gggttctcat gaagccctcg agcttcgtga cggtgacaag tccaagtggg 180

aaggtaaagg tgtcaccaag gctgtctcca atgtcaacga tatcattgga ccaaagctga 240

ttgaatccaa gctggatgtc aaggaccaaa aagccatcga tgagttcatg atcaagcttg 300

acggtacaaa gaacaagagt aagttgggtg ccaacgcaat cttgggtgtt tctttggctg 360

tcgctagggc cggtgccgct gccaaagggg tgccactgta ccaacacatc gctgagttgg 420

cggacatgaa gaaagaacca tacgtcattc cttgtccttt cttcaacgtc ttgaacggtg 480

gtgtccatgc tggtggtaac ctggctttgc aagaattttt aattgctcct gttggcgctg 540

agtccttcca tgaggctttg agactaggtt ccgaagtcta ccacaagttg aaggccttgg 600

ctaagaagag aatcggctct tctgctggca atgtcggtga tgagggtggt atcgccccat 660

ctctatctac tccattcgaa gctttggatt tgatctacga cgctatcaaa gaagctggtc 720

acgaaggtaa ggtgaagatt gccatggacc ccgcttcttc ggaatttttc caaggtgaca 780

agtatgactt ggactttaag aacccacatc cagatgctaa gaacaagttg tcaggtgctc 840

aactcggtga ctactacaag accattttgg aaaaatatcc aattgtgtct ttggaagatc 900

catttgctga agacgactgg gaggcttgga ctaacttctt ccctaaggca ggtgttcaaa 960

tcatcgctga tgacttaact gtcaccaacc ctgaaagaat ccaaactgcc attgacaaga 1020

agactgctga ctgtttgttg ttgaaggtta accaaattgg ttctttgaca gaatcaatta 1080

actctgctaa gctggcttac ggtgccggct ggggtgtcca ggtttcccat agatctggtg 1140

aaactgaaga cactttcatt gctgatcttg ttgttggttt gagaactggt caaattaaat 1200

ctggttctct ggctagatca gaaagattgg caaagtggaa ccaaattttg agaattgaag 1260

aggaattggg tgcttcaaac acaatttttg caggtgccaa attccataag ggacaattgt 1320

tgtaactcga g 1331

Claims

1. An ELISA detection method for detecting human serum Candida glabrata enolase IgG antibody is characterized in that: the detection method is an indirect ELISA detection method, and is used for detecting the human serum Candida glabrata enolase Cg-Eno IgG antibody by using an ELISA kit, and the detection method specifically comprises the following steps:

c. incubating the coated enzyme-linked plate at 37 ℃ for 60 minutes;

f. washing: repeating the washing step of step d;

h. adding 50 mu L of final solution into each hole to terminate the reaction, placing an enzyme label plate on an enzyme label instrument, zeroing by using a blank hole, reading a light absorption value under the wavelength of 450nm, judging the effectiveness of the test, and calculating an S/N value, wherein the S/N value is equal to OD450 of a sample to be detected/negative control OD450, and the S/N value is more than or equal to 2.5 and is positive; 2.0< S/N <2.5 is weakly positive; S/N is less than or equal to 2.0, and the negative result is obtained.

2. The ELISA detection method for detecting human serum Candida glabrata enolase IgG antibody according to claim 1, characterized in that: the ELISA kit comprises an ELISA plate, an enzyme-labeled antibody, a washing solution, a substrate developing solution, a sample diluent, a stop solution, a positive control solution and a negative control solution, wherein the ELISA plate is coated by a purified Cg-Eno recombinant protein antigen, and the concentration of the Cg-Eno recombinant protein antigen is 1 mu g/mL.

3. The ELISA detection method for detecting human serum Candida glabrata enolase IgG antibody according to claim 2, characterized in that: the coating process of the ELISA plate is as follows: diluting the purified Cg-Eno recombinant protein antigen to 1 mu g/mL by using a carbonate buffer solution with the pH of 9.5 and 0.05mol/L, adding the diluted Cg-Eno recombinant protein antigen to a 96-well coated plate, standing the plate at 100 mu L/well for overnight at 4 ℃, washing the plate for 3 times by using a washing solution, adding 1% BSA (bovine serum albumin) 0.01mol/L PBS with the pH of 7.2, sealing the plate overnight, throwing off the sealing solution, naturally drying the plate for 4-6 hours at room temperature with the humidity of below 40%, drying the plate in vacuum, sealing the plate, and storing the plate at 4 ℃ to obtain the coated enzyme-linked plate.

4. The ELISA detection method for detecting human serum Candida glabrata enolase IgG antibody according to claim 3, characterized in that: the preparation process of the purified Cg-Eno recombinant protein antigen comprises the following steps:

5. The ELISA detection method for detecting human serum Candida glabrata enolase IgG antibody according to claim 4, characterized in that: the enzyme-labeled antibody is 1: HRP-goat anti-human IgG antibody at 4000 dilutions.

6. The ELISA detection method for detecting human serum Candida glabrata enolase IgG antibody according to claim 5, characterized in that: the preparation process of the washing liquid comprises the following steps: 0.5g of NaH₂PO₄，6.0g Na₂HPO₄0.5mL of Tween-20, 8.5g of NaCl was dissolved in 500mL of water, and the pH was adjusted to 7.4, thereby obtaining a volume of 1L.

7. The ELISA detection method for detecting human serum Candida glabrata enolase IgG antibody according to claim 6, characterized in that: the preparation process of the substrate color development liquid comprises the following steps: dissolving 4.2g of citric acid, 13.6g of sodium acetate and 0.5g of carbamide peroxide in 100mL of deionized water, fixing the volume to 1L, and adjusting the pH value to 5.0 to obtain a substrate A solution; dissolving lg TMB in 50mL of dimethyl sulfoxide, adding 100mL of formaldehyde and 16.0g of polyethylene pyrrolidone after dissolving, and diluting to 1L with deionized water after dissolving to obtain a substrate B solution.

8. The ELISA detection method for detecting human serum Candida glabrata enolase IgG antibody according to claim 7, characterized in that: the preparation process of the sample diluent comprises the following steps: 0.5g of NaH₂PO₄，6.0g Na₂HPO₄8.5g of NaCl is dissolved in 500mL of water, the pH value is adjusted to 7.4, the volume is finally determined to be 1L, 1g of BSA is added until the BSA is completely dissolved, and a sample diluent is obtained; the stop solution is a 2mol/L sulfuric acid solution.

9. The ELISA detection method for detecting human serum Candida glabrata enolase IgG antibody according to claim 8, characterized in that: the positive control solution is: collecting serum 10 of clinically confirmed candida glabrata infected patients, mixing the serum uniformly, inactivating the serum for 30min at 56 ℃, and diluting the serum with a sample diluent 1:500 dilution; the negative control solution is: 10 serum samples of clinical candida glabrata infection normal physical examination are collected, mixed evenly, inactivated at 56 ℃ for 30min, and the serum is diluted by a sample diluent 1: and (5) diluting by 500.

10. The ELISA detection method for detecting human serum Candida glabrata enolase IgG antibody according to claim 9, characterized in that: the criteria for determining the effectiveness of the test are: the positive control serum OD450>0.5, the negative control serum OD450 <0.18, blank wells OD450 0, the experiment was established, otherwise not valid.