CN116284420A

CN116284420A - Specific fungus detection kit

Info

Publication number: CN116284420A
Application number: CN202310371620.5A
Authority: CN
Inventors: 彭忠真; 任磊
Original assignee: Lingao Shuangwan Biotechnology Co ltd
Current assignee: Lingao Shuangwan Biotechnology Co ltd
Priority date: 2023-04-10
Filing date: 2023-04-10
Publication date: 2023-06-23

Abstract

The invention relates to a specific fungus detection kit. The enolase of candida albicans standard strain ATCC10231 is obtained through separation and purification, and the enolase is used as an immunogen, and the candida albicans enolase monoclonal antibody 3G19 is prepared and obtained through a mouse hybridoma technology. The monoclonal antibody has better affinity target capability and binding property, and the detection of various strains proves that the monoclonal antibody has excellent detection specificity and can be used for preparing a candida albicans kit.

Description

Specific fungus detection kit

Technical Field

The application relates to the field of biological detection, in particular to fungus detection, and more particularly relates to a specific fungus detection kit.

Background

In recent years, with changes in human environment, diseases and the like, the incidence of fungus infection has been on the rise year by year, and candidiasis is mainly an acute, subacute or chronic infection caused by candida albicans, and is the most common mycosis. The clinical symptoms are complicated, urgent and slow, and most children are suffering from acute secondary infection. The disease often invades the skin, mucous membranes, and may also cause visceral or systemic infections. All visceral infections are often secondary to a variety of chronic wasting diseases and have long-term use of broad-spectrum antibiotics, corticosteroids and chemo-and radiotherapy-inducing factors. In recent years, with the application of large-dose antibiotics, hormones and immunosuppressants, and the development of organ transplantation, the incidence of the diseases is gradually increased, and the life is endangered with serious consequences.

Candida albicans is one of the important pathogenic bacteria causing fungal infections, and in addition, it is also an important food-borne pathogenic bacteria. Candida albicans (candida albicans) are widely existing in nature and also exist in the oral cavity, the upper respiratory tract, the intestinal tract and the vagina of normal people, and are usually small in number in normal organisms, do not cause diseases and are conditional pathogenicity. When the body resistance is reduced or the flora is deregulated, candida albicans will multiply and change the growth form to invade human cells, causing diseases. Candida albicans cells are oval in shape, and pathogenic strain cells are pseudohyphae. After the bacteria are incubated on blood agar or Sha Baoshi agar for 2-3 d at 37 ℃ or room temperature, gray cheese-like colonies are generated, and the surface layer is seen to be oval budding cells by smear microscopy, and the bottom layer has more pseudohyphae. If inoculated on 4% maize agar, pseudomycelium, blastospores and thick film spores can be seen after incubation for 3-5 d at room temperature. Candida albicans has weak resistance to heat and dies after being heated to 60 ℃ for 1 h. But has strong resistance to drying, sunlight, ultraviolet rays, chemical agents and the like. Therefore, the research of the candida albicans detection method is of great significance in the field of medical clinical examination and food safety detection.

The candida albicans detection method mainly comprises three methods of direct microscopic examination, truffle culture and histopathology in clinic. Direct microscopy is the simplest and most rapid method, but checking positive does not determine the type of fungus, and checking negative does not exclude mycoses. The fungus culture multipurpose salPaul culture medium, which can separate strain, is an important method for identifying white fungus. With the development of detection technologies such as molecular biology and immunology, more and more test methods are used for diagnosing and detecting candida albicans infection.

PCR diagnosis is a common diagnostic method, and since this method can detect extremely trace amounts of fungal DNA within several hours, early rapid diagnosis of Candida albicans infection is possible. The specific universal primer PCR diagnosis method is to design a section of fungus kingdom conserved sequence which is selected by taking hot start PCR as a detection means as a specific universal primer, and the specific universal primer PCR diagnosis method is high in sensitivity, strong in specificity and good in representativeness. Can be used for detecting candida albicans. However, the use of the strain has limitations and cannot replace the culture of fungi.

The antibody detection method in immunology is to measure the antibody through antigen detection, such as complement fixation test, immune diffusion test, direct or indirect agglutination test, etc., and the method has better sensitivity and low cost. The monoclonal immunohistochemical technology is to detect fungi in tissue with high sensitivity and specificity, and the monoclonal antibodies applied at present are anti-candida albicans, anti-trichobacterium rubrum and anti-aspergillus, so that the combination of the immunohistochemical and culture method can more clearly know whether the fungi in the tissue is saprophytic pollution or pathogenic bacteria, and can prove the existence of mixed infection. CN110204616a discloses an anti-candida albicans enolase specific monoclonal antibody, and the target of the monoclonal antibody is enolase (eno 1), also called 2-phosphate-D-glycerate hydrolase. The enzyme catalyzes the conversion of phosphoglycerate to phosphoenolpyruvic acid, and is the rate-limiting enzyme in the glycolysis process of candida albicans. The enzyme has been considered an ancient, conserved, single-function protein, however as molecular biology studies on immunogenic proteins have progressed, it has been found that it is the most important target molecule for serological detection of candida albicans, e.g. enolase-induced disease can be observed in patients with invasive candida infection. At present, an ELISA method is established by utilizing the matching of an anti-enolase monoclonal antibody and a sheep anti-enolase polyclonal antibody, and enolase protein in candida albicans culture supernatant can be detected. However, at present, the types of alternative anti-candida albicans enolase specific monoclonal antibodies are not enough, and particularly, the types of antibodies and kits thereof suitable for large-scale marketable detection suitable for marketable application are not enough.

Disclosure of Invention

In one aspect of the invention, a monoclonal antibody specific for anti-candida albicans enolase is provided.

Furthermore, the light chain variable region sequence of the candida albicans enolase monoclonal antibody 3G19 provided by the invention is shown as SEQ ID NO:1, the heavy chain variable region sequence of which is shown in SEQ ID NO: 2.

Furthermore, the light chain and/or heavy chain variable region sequences of the candida albicans enolase monoclonal antibody 3G19 of the present invention can be further conservatively substituted.

The term "conservative substitution" also includes amino acid substitutions having a similar hydropathic index or score. Each amino acid is assigned a hydrophilicity index based on its hydrophobicity and charge characteristics: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9) and arginine (-4.5). Amino acid substitutions with a hydropathic index within +/-0.2 are preferred when making changes based on the hydropathic index. More preferably, the substitution includes amino acids having a hydropathic index within +/-0.1, more preferably within about +/-0.05.

Furthermore, the light chain and/or heavy chain variable region sequences of the candida albicans enolase monoclonal antibody 3G19 of the present invention may be further substituted with conservative amino acids. It includes substitution of similar amino acids based on hydrophilic preparation, and in particular the biologically functionally equivalent proteins or peptides produced therefrom are contemplated for use in immune embodiments, as is currently the case. For example, the greatest local average hydrophilicity of a protein, which depends on the hydrophilicity of adjacent amino acids, is related to its immunogenicity and antigenicity. As detailed in U.S. Pat. No. 4,554,101, the following hydrophilic values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0+/-0.1); glutamic acid (+3.0+/-0.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 +/-0.1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). In varying based on similar hydrophilicity values, preferably, the substituted amino acids have hydrophilicity values within about +/-0.2 of each other, more preferably within about +/-0.1, even more preferably within +/-0.05.

Further, the monoclonal antibodies of the invention also include any substitution, change, modification, substitution, deletion, or addition of one (or more) amino acid of the sequence while still maintaining the biological activity of the corresponding antibody, more preferably at least substantially the same activity.

Furthermore, the candida albicans enolase monoclonal antibody 3G19 of the present invention may also be conjugated to a label. The marker may be any entity whose presence may be conveniently detected. For example, the labels May be direct labels, such as those described in detail in U.S. patent No. 5,656,503 to May et al. Direct labeling includes, but is not limited to, entities that can be readily seen by the naked eye in their natural state or by means of filters and/or applied stimuli such as UV light to excite fluorescence. Examples include radioactive compounds, chemiluminescent compounds, electroactive compounds (e.g., redox labels), and fluorescent compounds. Direct particle labels such as dye sols, metal sols (e.g. gold) and colored latex particles are also very suitable and are preferred together with fluorescent compounds. Among these options, colored latex particles and fluorescent compounds are most preferred. Concentration of the label into a small area or volume should produce a readily detectable signal, such as a strongly colored area. Indirect labels, such as enzymes, e.g., alkaline phosphatase and horseradish peroxidase, may also be used, although such indirect labels typically require the addition of one or more developers, e.g., substrates, before a visible signal can be detected.

Coupling of the label to a binding agent such as an antibody of the invention may be by covalent or non-covalent (including hydrophobic) bonding, or by adsorption. Techniques for such coupling are common in the art and can be readily adapted to the particular reagents used.

Such as antibodies of the invention, may also be coated on a solid support. For example, the antibodies may be coated on synthetic plastic materials, magnetic particles, microtiter assay plates, microarray chips, latex beads, filters comprising cellulose or synthetic polymeric materials, glass or plastic slides, dipsticks, capillary filling devices, and the like.

Further, the antibodies of the invention are attached to a solid support to allow for crude separation of the enzyme. The separation technique preferably preserves the activity of the component to be collected to the greatest extent. Various techniques of different efficiency may be used to obtain a coarser separation. The particular technique used depends on the efficiency of the separation, the associated cytotoxicity, ease and speed of performance, and the need for advanced equipment and/or expertise. Methods for separation may include, but are not limited to, magnetic separation, use of antibody-coated magnetic beads, affinity chromatography, and "panning" with antibodies attached to a solid matrix. Techniques to provide accurate isolation include, but are not limited to MACS, dynal magnetic bead selection, and FACS.

Furthermore, the invention also provides a kit for detecting candida albicans, which is characterized by comprising the candida albicans enolase monoclonal antibody 3G19.

Furthermore, the invention also provides a test strip for detecting candida albicans, which is characterized by comprising the candida albicans enolase monoclonal antibody 3G19.

Further, the present invention also provides a specific method for detecting candida albicans, which comprises an ELISA method, a double antibody sandwich method, etc., which comprises using the candida albicans enolase monoclonal antibody 3G19 of the present invention.

Furthermore, the invention also provides application of the candida albicans enolase monoclonal antibody 3G19 in preparing a kit for detecting candida albicans.

Advantageous effects

The enolase of candida albicans standard strain ATCC10231 is obtained through separation and purification, and the enolase is used as an immunogen, and the candida albicans enolase monoclonal antibody 3G19 is prepared and obtained through a mouse hybridoma technology. The monoclonal antibody has better affinity target capability and binding property, and the detection of various strains proves that the monoclonal antibody has excellent detection specificity and can be used for preparing a candida albicans kit.

Drawings

FIG. 1 is a potency assay for candida albicans enolase monoclonal antibody 3G 19;

FIG. 2 is a Western blot detection result diagram of candida albicans enolase monoclonal antibody 3G 19; wherein, the candida albicans suspension in the lane 2 and the recombinant candida albicans enolase in the lane 3 can form a single strip with the candida albicans enolase monoclonal antibody 3G19, and the BSA in the lane 1 does not react with the candida albicans enolase monoclonal antibody 3G19.

Detailed Description

The following detailed description of embodiments of the invention is provided for the purpose of illustration only and is not to be construed as limiting the invention. In addition, all reagents employed in the examples below are commercially available or may be synthesized in accordance with text or known methods, and are readily available to those skilled in the art for reaction conditions not listed, if not explicitly stated.

EXAMPLE 1 preparation of Candida albicans enolase

The candida albicans standard strain ATCC10231 is cultured for 24 hours by shaking with a liquid sandcastle culture medium to obtain a bacterial suspension. Controlling the concentration of bacterial suspension according to a turbidimetry method, wherein the final concentration is 2 multiplied by 10 ⁹ /mL. And adopting a Lyicase enzymolysis wall breaking method to break the wall. Protein solution was precipitated successively with 50% and 75% amine sulfate, and after centrifugation, 50% and 75% amine sulfate solution precipitate was collected and put into distilled water for overnight dialysis.

The DEAE-Sephadex A-50 column was equilibrated with a Tris-HCl solution having a concentration of 10mmol/L, pH7.8, and the resulting dialyzed protein solution was passed through the column at a flow rate of 50mL/h, and after passing through the peak, the column was washed with 0.2M NaCl, followed by elution peak containing a protein having a relative molecular mass of 47000, and desalted by dialysis with distilled water. The DEAE-Sephadex A-50 column was equilibrated with 10mM Tris-HCl solution at pH8.0, and the peak eluted in the previous step was passed through the column at a flow rate of 18mL/h as in the other steps. ConA-Sepharose-4B column was equilibrated with 10mM Tris-HCl pH7.4, and the above step was passed over the column at a flow rate of 13 mL/h. SDS-PAGE identified a single protein with a single band and a relative molecular weight of 47000 was obtained.

Measurement of candida albicans enolase protease activity: the activity (Ua) of candida albicans enolase was determined by measuring the amount of NADH converted to NAD per unit time during the reaction using a spectrophotometer with a wavelength of 340nm, using saccharomyces cerevisiae enolase as a standard (us=300U/mg, ph7.4, 25 ℃). The reaction mixture was 50mM Tris-HCl pH7.4,5mM MgCl ₂ 2mM EDTA,2mM NADH,1mMADP,10U pyruvate kinase, 10U lactase dehydrogenase and different enolases. The total reaction volume was 1mL and the reaction temperature was 25 ℃. The purified candida albicans enolase enzyme Ua was identified as 2845.3U/mg. The candida albicans enolase (CAEnolase) with better enzyme activity is kept for standby in a refrigerator at 4 ℃.

EXAMPLE 2 preparation of Candida albicans enolase monoclonal antibody

The experimental mouse line BALB/c mice are provided by the animal center of the national academy of military medical science. Primary immunization after 50mg of candida albicans enolase protein purified in example 1 was diluted with physiological saline, mixed with an equal volume of freund's complete adjuvant, stirred and emulsified, 3 BALB/c mice were immunized subcutaneously at two points and the rest were injected intraperitoneally. The same dose of antigen and incomplete adjuvant are uniformly mixed for secondary immunization after 2 weeks, the method is the same as the primary immunization, and the No. 1 mouse with the highest ELISA (enzyme-linked immunosorbent assay) measuring serum titer is selected to have the titer of more than 1:50000 after 7d, and the same dose of antigen is used for intraperitoneal injection for 3 days before fusion, so that the immunization is enhanced for 1 time.

Taking the 1 st mouse after the booster immunization, killing the 1 st mouse after the orbit blood sampling, taking the spleen from the plate after the alcohol sterilization, and transferring the spleen into a 50ml centrifuge tubeDiluting with RPMI 1640-30 ml, and collecting 1×10 ⁸ Individual cells were kept ready for use. Taking SP2/0 cells 1×10 ⁷ And mixing with the spleen cells, centrifuging at 2000r/min for 5min, discarding supernatant, adding 1ml PEG4000 in water bath at 37deg.C for 1min, stirring, and standing in water bath at 37deg.C for 1min. The fusion was terminated by adding 1, 4, 15ml RPM 1640 in 1, 2, 3min, respectively, and centrifuging at 1000r/min for 5min. Removing supernatant, homogenizing, adding HAT culture solution, mixing, dripping into 96-well plate, and adding CO ₂ In an incubator, the liquid is changed regularly, meanwhile, the enzyme protein purified in the embodiment 1 and the BSA control protein are used as coating antigens, the cell culture supernatant is screened by adopting indirect ELISA, the screened 47 positive clones are cloned by a limiting dilution method, and the monoclonal antibody 3G19 with the best specificity is obtained by screening. The hybridoma cell is used for inducing BALB/c mouse ascites monoclonal antibody, and the antibody is purified, and the BCA is adopted to measure the protein concentration to be 2.4mg/mL for later use.

EXAMPLE 3 determination of the 3G19 Titer of Candida albicans enolase monoclonal antibody

The 96-well enzyme-linked plate is coated with the enzyme protein purified in example 1, washed 3 times with a washing solution after passing through the solution at 4 ℃, added with a sealing solution at 37 ℃ for 4 hours, washed 3 times, patted dry and stored at 4 ℃. And (3) adding the purified ascites monoclonal antibody to a coated ELISA plate, taking blank mouse serum as a negative control, selecting positive serum as a positive control, incubating at 37 ℃ for 30min, washing the plate for 4 times, beating to dry, adding a 1:5000 diluted ELISA secondary antibody, incubating at 37 ℃ for 30min, washing the plate for 4 times, and beating to dry. DAB color development is carried out at 37 ℃ for 30min, stop solution is added, and OD value is measured at 450 nm. The results are shown in FIG. 1.

From the results shown in FIG. 1, the candida albicans enolase monoclonal antibody 3G19 prepared by the invention has better titer characteristics.

And (3) identifying the subtype of the candida albicans enolase monoclonal antibody 3G19 to be IgG2a by adopting a monoclonal antibody subtype identification kit.

EXAMPLE 4 specific identification of Candida albicans enolase monoclonal antibody 3G19

Mixing Candida albicans suspension, recombinant Candida albicans enolase, product No. pro-2297, prospec and BSA protein with sample buffer at a ratio of 3:1, respectively, and homogenizing at 100Heating in water bath at temperature of 5min. The separation gel and the concentrated gel solution were prepared, after polymerization of the concentrated gel solution, the sample comb was carefully pulled out, and 10. Mu.l of Candida albicans suspension, recombinant Candida albicans enolase, cat No. pro-2297, prospec and BSA protein were added sequentially to the sample wells. And (3) starting electrophoresis under the condition of constant pressure of 80V, and adjusting the voltage to 180V until the sample solution enters the separation gel until the electrophoresis is finished. Immunoblotting detects the reactivity and specificity of antibodies: the gel was removed, the edges of the gel were cut off, and immersed in TF buffer for 30min. 2 thick 7 filter papers of the same size as the gel were further taken, and 1 nitrocellulose membrane (PVDF membrane) was soaked with TF buffer. Transfer was performed with a semi-dry gum transfer machine. Sequentially placing 1 wet thick filter paper, 1 nitrocellulose membrane, electrophoresis gel, 3 wet filter paper, and 0.8mA/cm on the electrode plate ² Nitrocellulose membrane, constant voltage 18V transfer for 30min. Taking out the nitrocellulose membrane, immersing the nitrocellulose membrane into a sealing liquid at 37 ℃ for sealing for 60min, and discarding the liquid. 5ml of a suitable amount of candida albicans enolase monoclonal antibody 3G19 solution diluted 1:3000 was added and reacted overnight at 4 ℃. The nitrocellulose membrane was washed 1 time with TBST buffer and then 3 times with TBST buffer for 8min each. The liquid was discarded, and 10ml of an appropriate amount of HRP-labeled secondary antibody was added thereto, and the reaction was allowed to proceed with shaking at 37℃for 30 minutes. The nitrocellulose membrane was washed 1 time with TBST buffer and then with TTBS buffer for 4 times, 10min each. The liquid was discarded, and the mixed ECL color development solution was added to carry out exposure development in a dark room. Positive results should exhibit significant exposure bands, negative results have no exposure bands. The results are shown in FIG. 2.

As can be seen from FIG. 2, the Candida albicans suspension of lane 2 and the recombinant Candida albicans enolase of lane 3 can form a single band with the Candida albicans enolase monoclonal antibody 3G19, while the BSA of lane 1 does not react with the Candida albicans enolase monoclonal antibody 3G19, which shows that the Candida albicans enolase monoclonal antibody 3G19 has better specificity.

Example 5 identification of the affinity and sequence of Candida albicans enolase monoclonal antibody 3G19

SPR technique detects candida albicans enolase monoclonal antibody 3G19 antibody affinity: the anti-mouse IgG secondary antibody is fixed on a CM5 chip, a Biacore T200 is utilized to capture a candida albicans enolase monoclonal antibody 3G19 antibody (mouse antibody), a recombinant candida albicans enolase antigen is used as an analyte, buffer solutions are respectively used for dilution to 0, 7.5, 15, 30 and 60nM concentration gradients, and the binding of different antibodies and the recombinant candida albicans enolase antigen is detected by utilizing single-cycle dynamics. Final data were analyzed by kinetics fit using biacore evaluation software 3.0.3.0 on a 1:1 model. The results are shown in Table 1.

TABLE 1 results of affinity properties of Candida albicans enolase monoclonal antibody 3G19

Antibody SPR	Results
		KD(mol/L)	1.68E-10
Ka(1/(mol*s/L))	3.05E+05
		Kd(1/s)	5.13E-05

As can be seen from Table 1, the candida albicans enolase monoclonal antibody 3G19 has better affinity with target protein and stronger binding stability.

mRNA was extracted from the 3G19 hybridoma cell line according to the instructions of the Tiangen bacterial Total RNA extraction kit (cat. No. DP 430). The first strand was synthesized and amplified by PCR according to the instructions of the Invitrogen III First-Strand Synthesis System for RT-PCR kit. TA Cloning was performed according to pEASY-T1 Cloning Kit (Transgen CT 101) Kit instructions. PCR was performed using M13 universal primers and positive clones were sequenced. And (3) identifying the light chain variable region sequence of the candida albicans enolase monoclonal antibody 3G19 by sequence analysis, wherein the light chain variable region sequence is shown as SEQ ID NO:1, the heavy chain variable region sequence of which is shown in SEQ ID NO: 2.

Example 6 detection test strip and identification prepared by Candida albicans enolase monoclonal antibody 3G19

The colloidal gold is prepared by adopting a sodium citrate reduction method, the monoclonal antibody 3G19 is used for marking the colloidal gold, and a differential centrifugation method is adopted for purifying the colloidal gold probe. And respectively taking a candida albicans enolase polyclonal antibody and a goat anti-mouse IgG antibody as a detection line and a quality control line of the test paper, coating an NC film, and assembling the colloidal gold test paper. Specificity tests were performed using candida glabrata, escherichia coli, staphylococcus aureus, candida albicans, yeast, candida parapsilosis, candida krusei, and streptococcus. The results are shown in Table 2.

TABLE 2 antibody 3G19 test strip specificity test results

"-" indicates no band, and "+" indicates the presence of a positive band.

As can be seen from the results in Table 2, the antibody 3G19 test strip of the invention has very good specificity, does not generate specific bands with other non-candida albicans, but only has strong positive band reaction on candida albicans and enolase recombinant proteins of candida albicans, and shows better specificity.

In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction. While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.

Claims

1. A monoclonal antibody 3G19 specifically targeting candida albicans enolase, which is characterized in that the variable region sequence of the light chain is shown as SEQ ID NO:1, the heavy chain variable region sequence of which is shown in SEQ ID NO: 2.

2. The monoclonal antibody 3G19 specifically targeting candida albicans enolase of claim 1, wherein the subtype of the monoclonal antibody is IgG2a.

3. A kit for specifically detecting candida albicans, comprising the monoclonal antibody 3G19 specifically targeting candida albicans enolase of claim 1, wherein the monoclonal antibody comprises a detectable label; the kit is an ELISA detection kit.

4. Use of monoclonal antibody 3G19 specifically targeting candida albicans enolase as claimed in claim 1 in the preparation of a kit for the detection of candida albicans.

5. The use according to claim 4, characterized in that the kit contains a test strip labeled with the monoclonal antibody 3G19 of claim 1 specifically targeting candida albicans enolase.

6. The method of claim 4, wherein the kit is an ELISA assay kit.