An enzyme conjugate diluent for detecting legionella pneumophila IgM antibody, a detection reagent

Abstract

The present application relates to the technical field of biology, and in particular to an enzyme conjugate diluent and detection reagent for detecting IgM antibody of Legionella pneumophila. The enzyme conjugate diluent comprises 0.01-0.1M buffer, 2-5M chloride salt and the following components with mass volume percentage: 0.5-5% protective agent, 0.01-0.05% CaCl2, 0.1-0.5% preservative and 0.01-0.05% pigment; the chloride salt is NaCl and / or KCl. The enzyme conjugate diluent provided by the present application effectively enhances the antigen-antibody reaction rate by adding chloride salt with appropriate concentration, so that the detection value reaches the saturation state, the influence of random error and systematic error is reduced, thereby effectively improving the precision of detection, and the detection time is shortened.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to an enzyme conjugate diluent and detection reagent for detecting Legionella pneumophila IgM antibody. BACKGROUND

[0002] Legionella pneumophila is an obligate intracellular parasite that is ubiquitous in natural freshwater and artificial water environments, mainly parasitizing in the cells of prokaryotes such as amoeba and tetrahymena. Currently, 15 serotypes have been found, and can be transmitted to humans in the form of aerosols through the respiratory tract via water bodies closely contacted by humans such as drinking water systems, air conditioning cooling water, shower nozzle water, etc.

[0003] The main symptoms of Legionella infection in clinical practice are pneumonia and Pontiac fever. Pneumonia is often severe, accompanied by multiple system damage, and nearly 50% of hospitalized patients need to be admitted to the ICU, with a mortality rate of 5%-30%. Pontiac fever is relatively mild, mainly fever, headache and muscle pain, etc., without lung inflammation, and is similar to influenza symptoms. Legionella pneumophila is ineffective for empirical treatment of beta-lactam. Legionella pneumophila detection methods include: serum specific antibody detection, urine antigen detection, nucleic acid detection, isolation and culture, and lower respiratory tract specimen antigen detection. Among them, isolation and culture positive, urine antigen positive, 4-fold or more changes in acute and convalescent phase double serum antibodies can be used as the basis for etiological diagnosis, and nucleic acid or single blood IgM antibody positive diagnosis has important reference significance.

[0004] When using a detection system to detect samples, random errors and systematic errors are inevitably present, and if the antigen and antibody reaction is not sufficient, the precision will be poor due to these interference factors, SUMMARY

[0005] Therefore, the present application provides an enzyme conjugate diluent and detection reagent for detecting Legionella pneumophila IgM antibody. The enzyme conjugate diluent significantly improves the biological reaction rate of Legionella pneumophila antigen and specific antibody, and improves the precision of detection.

[0006] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions:

[0007] An enzyme conjugate diluent comprises the following components:

[0008] 0.05M buffer, 1.3% protective agent, 0.01% CaCl2, 0.3% preservative, 0.01% pigment and 2-5M chloride salt; the chloride salt is NaCl and / or KCl.

[0009] In some embodiments, the concentration of the chloride salt is preferably 3-5 M; in some specific embodiments, it can be 3 M, 4 M or 5 M.

[0010] In some embodiments, the buffer is any one of Tris-HCl buffer, PBS buffer, MOPS buffer, Bis-Tris buffer, PB buffer and HEPES buffer.

[0011] In some embodiments, the protective agent comprises at least one of BSA, casein and ADP; in some specific embodiments, the protective agent consists of BSA, casein and ADP; and the mass ratio of the BSA, casein and ADP is 1:0.2:0.1.

[0012] The present application does not have special requirements for the type of preservative, and the type commonly used in the art can be used. In some embodiments, the preservative is selected from at least one of Bro, P300, MIT and NaN3. In some specific embodiments, the preservative consists of Bro and P300.

[0013] The present application does not have special requirements for the type of pigment, and the type commonly used in the art can be used. In some embodiments, the pigment comprises at least one of carmine, sunset yellow, fruit green and lemon yellow.

[0014] In the present application, the pH of the enzyme conjugate diluent is 7.4-7.5, and can be 7.4 or 7.45.

[0015] The present application also provides the use of the enzyme conjugate diluent in the preparation of a detection reagent for Legionella pneumophila IgM antibody.

[0016] The present application also provides a detection reagent for Legionella pneumophila IgM antibody, which comprises the enzyme conjugate diluent, the enzyme-labeled Legionella pneumophila mouse-derived antibody, the magnetic particle suspension, the Legionella pneumophila antigen solution, the sample diluent and the positive and negative controls.

[0017] The magnetic bead suspension comprises magnetic beads, mouse anti-human IgM and magnetic bead buffer, and the magnetic bead buffer comprises the following components:

[0018] 0.02 M PBS + 1% casein + 0.1% ADP + 5% glycerol + 0.3% P300;

[0019] The Legionella pneumophila antigen solution comprises Legionella pneumophila antigen and antigen diluent, and the antigen diluent comprises the following components:

[0020] 0.02 M PBS + 3% BSA + 1.5% sucrose + 0.1% ADP + 0.2% Bro + 0.1% P300;

[0021] The sample diluent comprises the following components:

[0022] 0.05M Tris-HCl + 1% BSA + 1% EDTA-2Na + 20,000 units / L urokinase + 0.1% T20

[0023] + 0.5% CHAPS + 0.2% sodium azide (10%) + 0.1% P300 + 0.002% sunset yellow + 0.0019% lemon yellow.

[0024] The detection reagent provided by the present application also comprises a positive control and / or a negative control.

[0025] The positive control comprises human Legionella pneumophila IgM antibody positive serum or plasma and a positive control diluent; the positive control diluent comprises the following components:

[0026] 0.05M Tris-HCl + 1% BSA + 1% negative serum + 1% EDTA-2Na + 0.2% sodium azide + 0.1% P300 + 0.006% carmine.

[0027] The negative control comprises 0.05M Tris-HCl + 1% BSA + 1% EDTA-2Na + 0.2% sodium azide (10%) + 0.1% P300 + 0.005% fruit green.

[0028] In the detection reagent for Legionella pneumophila IgM antibody, enzyme conjugate diluent, magnetic particle suspension, Legionella pneumophila antigen solution, sample diluent, negative control and positive control provided by the present application, % represents the mass-volume ratio concentration of each component.

[0029] The detection reagent described in the present application uses the principle of capture method for detection, uses anti-human IgM antibody to coat magnetic particles, prepares an antigen solution by using Legionella pneumophila antigen, labels Legionella pneumophila antibody with horseradish peroxidase to prepare an enzyme conjugate solution, forms a solid-phase secondary antibody-IgM antibody-antigen-enzyme-labeled antibody complex through immune reaction, the complex catalyzes the emission of light photons from the luminescent substrate, and the luminescent intensity is proportional to the content of Legionella pneumophila IgM antibody. In the first step, 10 μL of sample, 20 μL of magnetic beads and 100 μL of sample diluent are sequentially added, and the reaction is carried out at 37°C for 15 min; in the second step, 50 μL of antigen solution and 50 μL of enzyme conjugate solution (enzyme conjugate diluent + enzyme-labeled antibody) are sequentially added, and the reaction is carried out at 37°C for 17 min.

[0030] Experiments show that the enzyme conjugate diluent provided by the application effectively enhances the antigen-antibody reaction rate by adding a suitable concentration of chloride salt, so that the detection value reaches a saturation state, the influence of random error and systematic error is reduced, thereby effectively improving the precision of detection, and the detection time is shortened. DETAILED DESCRIPTION

[0031] The application provides an enzyme conjugate diluent and detection reagent for detecting Legionella pneumophila IgM antibody. Those skilled in the art can improve the process parameters according to the content herein. It should be particularly pointed out that all similar replacements and changes are obvious to those skilled in the art, and they are all regarded as included in the application. The method and application of the application have been described by the preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application herein without departing from the content, spirit and scope of the application, to realize and apply the technical content of the application.

[0032] The test materials used in the application are all ordinary commercially available products, which can be purchased in the market.

[0033] The application provides an enzyme conjugate diluent and detection reagent for detecting Legionella pneumophila IgM antibody, wherein the detection reagent comprises an enzyme conjugate, a magnetic particle suspension, a Legionella pneumophila antigen solution, a sample diluent and positive and negative controls.

[0034] The enzyme conjugate diluent is selected from one of the following formulas:

[0035] 0.05M Tris-HCl+1% BSA+0.2% casein+0.01% CaCl2+0.1% ADP+0.2% Bro

[0036] +0.1% P300+0.01% carmine pigment+3M NaCl, pH 7.45;

[0037] 0.05M Tris-HCl+1% BSA+0.2% casein+0.01% CaCl2+0.1% ADP+0.2% Bro

[0038] +0.1% P300+0.01% carmine pigment+4M NaCl, pH 7.45;

[0039] 0.05M Tris-HCl+1% BSA+0.2% casein+0.01% CaCl2+0.1% ADP+0.2% Bro

[0040] +0.1% P300+0.01% carmine pigment+5M NaCl, pH 7.45;

[0041] 0.05M Tris-HCl + 1% BSA + 0.2% casein + 0.01% CaCl2 + 0.1% ADP + 0.2% Bro

[0042] + 0.1% P300 + 0.01% carmine + 2M KCl, pH 7.45.

[0043] 0.05M Tris-HCl + 1% BSA + 0.2% casein + 0.01% CaCl2 + 0.1% ADP + 0.2% Bro

[0044] + 0.1% P300 + 0.01% carmine + 3M KCl, pH 7.45.

[0045] In preparing the above enzyme conjugate diluent, the casein is dissolved first, and then NaCl or KCl is added, otherwise the casein is difficult to dissolve, and there is no special restriction on the order of addition of other components.

[0046] The magnetic bead suspension includes magnetic beads, mouse anti-human IgM, and a magnetic bead buffer, and the magnetic bead buffer includes the following components:

[0047] 0.02M PBS + 1% casein + 0.1% ADP + 5% glycerol + 0.3% P300.

[0048] The Legionella pneumophila antigen solution includes a Legionella pneumophila antigen and an antigen diluent, and the antigen diluent includes the following components:

[0049] 0.02M PBS + 3% BSA + 1.5% sucrose + 0.1% ADP + 0.2% Bro + 0.1% P300.

[0050] The Legionella pneumophila antigen is a Legionella pneumophila lipopolysaccharide antigen. In the specific embodiment of the present application, the preparation method of the Legionella pneumophila lipopolysaccharide antigen is as follows: the Legionella pneumophila antigen stock solution (Legionella pneumophila bacterial solution) and lysozyme are mixed in a volume ratio of 1:1, shaken and mixed uniformly, and after 5 minutes at room temperature, centrifuged at 12000 rpm for 5 minutes, and the supernatant is taken and divided into 500ul per branch to obtain the Legionella pneumophila lipopolysaccharide antigen.

[0051] Legionella pneumophila is a gram-negative bacterium with a three-layer membrane structure. The thin layer of peptidoglycan is much thinner than that of gram-positive bacteria, and the outer membrane contains lipopolysaccharide.

[0052] Lysozyme can effectively hydrolyze the peptidoglycan of bacterial cell wall, and its hydrolysis site is the β-1.4 glycosidic bond between the 1st carbon atom of N-acetylmuramic acid (NAM) and the 4th carbon atom of N-acetylglucosamine (NAG). The peptidoglycan is the main component of bacterial cell wall, which is composed of NAM, NAG and peptide "tail" (4 amino acid residues), NAM and NAG are connected by β-1.4 glycosidic bond, and the peptide "tail" is connected to the 3rd carbon atom of NAM through D-lactoyl carboxyl, the peptide tails are connected by peptide "bridge" (peptide bond or a few amino acids), and NAM, NAG, peptide "tail" and peptide "bridge" together form the multi-layer network structure of peptidoglycan as the skeleton of cell wall. The breakage of any chemical bond in the above structure can cause damage to the bacterial cell wall. The use of lysozyme to treat gram-negative bacteria can destroy the membrane structure to release more lipopolysaccharide from Legionella pneumophila.

[0053] The sample diluent comprises the following components:

[0054] 0.05M Tris-HCl + 1% BSA + 1% EDTA-2Na + 20,000 units / L urokinase + 0.1% T20

[0055] + 0.5% CHAPS + 0.2% sodium azide (10%) + 0.1% P300 + 0.002% sunset yellow + 0.0019% lemon yellow.

[0056] The detection reagent also comprises a positive control and / or a negative control, wherein:

[0057] The positive control comprises: human Legionella pneumophila IgM antibody positive serum or plasma and positive control diluent; the positive control diluent comprises the following components:

[0058] 0.05M Tris-HCl + 1% BSA + 1% negative serum + 1% EDTA-2Na + 0.2% sodium azide + 0.1% P300 + 0.006% carmine;

[0059] The negative control comprises: 0.05M Tris-HCl + 1% BSA + 1% EDTA-2Na + 0.2% sodium azide (10%) + 0.1% P300 + 0.005% fruit green.

[0060] The application is further described below in conjunction with examples:

[0061] Example 1

[0062] I. Experimental principle: The product uses capture method principle for detection, with anti-human IgM antibody coated magnetic microparticles, Legionella pneumophila antigen preparation antigen solution, horseradish peroxidase labeled Legionella pneumophila antibody preparation enzyme binding, through the immune response to form solid phase secondary antibody-IgM antibody-antigen-enzyme labeled antibody complex, the complex catalyzes the light emitting substrate to emit photons, and the light intensity is proportional to the content of Legionella pneumophila IgM antibody.

[0063] II. Kit composition

[0064] Table 1

[0065]

[0066] III. Preparation of each component

[0067] 1. Magnetic bead preparation (take preparation of 240 ml magnetic microparticle suspension as an example)

[0068] 1.1 Wash before activation: Place 2.4 ml of carboxyl magnetic microparticle working solution provided by the relevant technical personnel on the magnetic rack to separate the supernatant to the supernatant, and discard the supernatant; Accurately measure 24 ml of buffer A into the reaction container, slowly shake the magnetic microparticles in the reaction container by hand, mix on the shaking bed at 200 r / min for 5 min, and repeat the washing for 4 times. Finally, place the reaction container on the magnetic rack to separate the supernatant to the supernatant, and discard the supernatant.

[0069] 1.2 Activation: First, add 4 ml of 20 mg / ml EDC solution (dissolved in buffer B, effective within 5 min), then add 4 ml of 20 mg / ml NHS solution (dissolved in buffer B, effective within 5 min), tightly cover the bottle cap / container, and place it on the shaking bed at room temperature for 1 h.

[0070] 1.3 Wash after activation: Place the reaction container on the magnetic rack to separate the supernatant to the supernatant, and discard the supernatant; Accurately measure 24 ml of buffer B into the reaction container, slowly shake the magnetic microparticles in the reaction container by hand, mix on the shaking bed at 200 r / min for 5 min, and repeat the washing for 2 times. Finally, place the reaction container on the magnetic rack to separate the supernatant to the supernatant, and discard the supernatant.

[0071] 1.4 Coating: First, add the specified amount of buffer B liquid provided by the relevant technical personnel to the reaction container, and slowly shake the magnetic microparticles in the reaction container by hand, then add the specified amount of magnetic microparticle LP-IgM coating antibody, and the total liquid volume is 8 ml. Tightly cover the bottle cap / container, and place it on the shaking bed at room temperature for 2 h.

[0072] 1.5 Sealing: After the reaction is completed, the reaction vessel is placed on the magnetic stand to separate the supernatant until it is clear, and the supernatant is discarded. 24 ml of the sealing solution is accurately measured into the reaction vessel; the magnetic microparticles in the reaction vessel are slowly shaken by hand, and the reaction vessel is placed on a shaker at 200 r / min for 10 min, and the sealing is completed once. The sealing is repeated 4 more times, and finally the reaction vessel is placed on the magnetic stand to separate the supernatant until it is clear, and the supernatant is discarded.

[0073] 1.6 Post-preparation storage: 240 ml of the magnetic microparticle sealing solution is accurately measured, and the residual magnetic beads in the reaction vessel are transferred to a storage container using the magnetic microparticle sealing solution multiple times, and the mixture is mixed at 200 r / min for 5 min on a shaker, the bottle cap / container is tightly closed, a label is attached, and then it is stored at 2-8°C. The preparation of the magnetic microparticle suspension is completed.

[0074] 2. Preparation of enzyme conjugate diluent

[0075] 2.1 Formula one (control 1):

[0076] 0.05M Tris-HCl + 1% BSA + 0.2% casein + 0.01% CaCl2+ 0.1% ADP + 0.2% Bro

[0077] + 0.1% P300 + 0.01% carmine pigment, pH 7.45;

[0078] 2.2 Formula two:

[0079] 0.05M Tris-HCl + 1% BSA + 0.2% casein + 0.01% CaCl2+ 0.1% ADP + 0.2% Bro

[0080] + 0.1% P300 + 0.01% carmine pigment + 3M NaCl, pH 7.45 (NaCl is added after casein is dissolved, otherwise casein is difficult to dissolve);

[0081] 2.3 Formula three:

[0082] 0.05M Tris-HCl + 1% BSA + 0.2% casein + 0.01% CaCl2+ 0.1% ADP + 0.2% Bro

[0083] + 0.1% P300 + 0.01% carmine pigment + 4M NaCl, pH 7.45 (NaCl is added after casein is dissolved, otherwise casein is difficult to dissolve);

[0084] 2.4 Formula four:

[0085] 0.05 M Tris-HCl + 1% BSA + 0.2% casein + 0.01% CaCl2+ 0.1% ADP + 0.2% Bro

[0086] + 0.1% P300 + 0.01% carmine + 5 M NaCl, pH 7.45 (NaCl is added after casein is dissolved, otherwise casein is difficult to dissolve);

[0087] 2.5 Formula Five:

[0088] 0.05 M Tris-HCl + 1% BSA + 0.2% casein + 0.01% CaCl2+ 0.1% ADP + 0.2% Bro

[0089] + 0.1% P300 + 0.01% carmine + 2 M KCl, pH 7.45 (KCl is added after casein is dissolved, otherwise casein is difficult to dissolve);

[0090] 2.6 Formula Six:

[0091] 0.05 M Tris-HCl + 1% BSA + 0.2% casein + 0.01% CaCl2+ 0.1% ADP + 0.2% Bro

[0092] + 0.1% P300 + 0.01% carmine + 3 M KCl, pH 7.45 (KCl is added after casein is dissolved, otherwise casein is difficult to dissolve);

[0093] 2.7 Formula Seven (Control 2):

[0094] 0.05 M Tris-HCl + 1% BSA + 0.2% casein + 0.01% CaCl2+ 0.1% ADP + 0.2% Bro

[0095] + 0.1% P300 + 0.01% carmine + 0.1 M Na2CO3, pH 7.45;

[0096] 2.7 Formula Eight (Control 3):

[0097] 0.05 M Tris-HCl + 1% BSA + 0.2% casein + 0.01% CaCl2+ 0.1% ADP + 0.2% Bro

[0098] + 0.1% P300 + 0.01% carmine + 0.5 M Na2CO3, pPH 7.45;

[0099] 2.7 Formula Nine (Control 4):

[0100] 0.05M Tris-HCl + 1% BSA + 0.2% casein + 0.01% CaCl2+ 0.1% ADP + 0.2% Bro

[0101] + 0.1% P300 + 0.01% Carmine + 1M Na2CO3, pH 7.45;

[0102] The horseradish peroxidase-labeled murine monoclonal antibody of Legionella pneumophila was added to the above four enzyme conjugate diluent formulations at 1 / 4000.

[0103] 3. Preparation of antigen solution

[0104] 3.1 Preparation of containers

[0105] The containers required for the preparation of the magnetic microparticle Legionella pneumophila antigen were prepared before preparation.

[0106] 3.2 Buffer preparation:

[0107] 3.2.1 Standard formula and raw material requirements of the buffer:

[0108] Table 2

[0109]

[0110] 3.3 Configuration procedure

[0111] 3.3.1 Determine the volume of PBS to be prepared according to the requirements, and calculate the total mass of the buffer according to the formula remarks.

[0112] Calculate the amount of each raw material according to the standard formula;

[0113] 3.3.2 Add the required amount of purified water to the container, then add the required amount of NaCl, NaH2PO4·2H2O, and Na2HPO4·12H2O, and stir well to dissolve;

[0114] 3.3.3 Measure the pH value of the solution and record the pH value of the solution (PH = 7.6 ± 0.1);

[0115] 3.4 Preparation of lysozyme solution:

[0116] 3.4.1 Preparation procedure: weigh 10 mg of lysozyme and add 1 ml of PBS to dissolve, prepare a 10 mg / ml lysozyme solution, mix well, and reserve.

[0117] 3.5 Antigen treatment process:

[0118] 3.5.1 The antigen stock solution (Legionella pneumophila bacteria solution) and lysozyme were mixed at a volume ratio of 1:1, shaken to mix, and then incubated at room temperature for 5 minutes. The mixture was then centrifuged at 12,000 rpm for 5 minutes, and the supernatant was aliquoted at 500 μL per sample and used as the antigen.

[0119] Table 3

[0120]

[0121] The antigen was diluted to a titer of 1 / 1000 and added to the antigen diluent to prepare the antigen solution.

[0122] The sample diluent, negative control, and positive control were prepared according to the formulation in Table 1. The positive control was prepared by adding human Legionella pneumophila IgM antibody positive serum or plasma to the positive control diluent at a titer of 1 / 100.

[0123] IV. Full-automatic instrument used in the experiment

[0124] AutoLumo A2000 Plus

[0125] V. Test procedure

[0126] After the test sample (negative control, positive control, or sample to be tested) was prepared and properly placed, the start button was clicked to start the Cutoff value setting procedure or the sample testing procedure, and the instrument performed the following operations:

[0127] (1) The sample holder was transported to the sample loading position, and the reaction container was loaded into the sample loading position.

[0128] (2) When the Cutoff value setting procedure was performed, 100 μL of the negative control and the positive control and 20 μL of the magnetic particle suspension were dispensed.

[0129] (3) When the sample testing procedure was performed, 10 μL of the sample, 20 μL of the magnetic particle suspension, and 100 μL of the sample diluent were dispensed.

[0130] (4) The reaction solution was mixed and incubated at 37°C for 15 minutes.

[0131] (5) After incubation, the reaction solution was washed and separated using the washing solution.

[0132] (6) 50 μL of the enzyme conjugate and 50 μL of the antigen solution were dispensed.

[0133] (7) The reaction solution was mixed and incubated at 37°C for 17 minutes.

[0134] (8) After incubation, the reaction solution was washed and separated using the washing solution.

[0135] (9) Complete 50 μL of substrate A solution and 50 μL of substrate B solution.

[0136] (10) Mix the reaction solution and detect the luminescence intensity.

[0137] Six, experimental results

[0138] Table 4: Effect of adding different concentrations of NaCl in enzyme conjugate diluent on precision of kit

[0139]

[0140] Note: The solubility of NaCl in water solution at 2-8℃ is 6M.

[0141] Result analysis: As the molar concentration of sodium chloride increases, the luminescence value of the sample also increases, and reaches a plateau at 4M, and the precision is also the best, which shows that sodium chloride can significantly enhance the antigen-antibody reaction rate and improve the precision of detection.

[0142] Table 5: Effect of adding different concentrations of KCl in enzyme conjugate diluent on precision of kit

[0143]

[0144]

[0145] Note: The solubility of KCl in water solution at 2-8℃ is 4M; and 2M / control, 3M / control and 4M / control are the average values of the corresponding five groups of data divided by the average value of the control.

[0146] Result analysis: As the molar concentration of potassium chloride increases, the luminescence value of the sample also increases, and reaches a plateau at 4M, and the precision is also the best, which shows that potassium chloride can significantly enhance the antigen-antibody reaction rate and improve the precision.

[0147] Table 6: Effect of adding different concentrations of Na2CO3 in enzyme conjugate diluent on precision of kit

[0148]

[0149] Note: The solubility of Na2CO3 in water solution at 2-8℃ is 1M;

[0150] Result analysis: The addition of sodium carbonate in enzyme conjugate diluent has little effect on the luminescence value of antigen-antibody, and cannot improve the precision.

[0151] From the above data analysis, it can be found that the addition of 2-5M chloride salt (KCl or NaCl) in enzyme conjugate diluent can improve the lipopolysaccharide antigen-antibody reactivity of Legionella pneumophila, thereby improving the precision.

[0152] The above merely preferred embodiments of the present application, it should be noted that for those of ordinary skill in the art, without departing from the principles of the present application, can also make a number of improvements and refinements, these improvements and refinements should also be considered within the scope of the present application.

Claims

1. Use of an enzyme conjugate diluent in the preparation of a detection reagent for Legionella pneumophila IgM antibody; The enzyme conjugate diluent comprises 0.01-0.1M buffer, 3-5M chloride salt and the following components in mass volume percentage: 0.5-5% protective agent, 0.01-0.05% CaCl2, 0.1-0.5% preservative and 0.01-0.05% pigment; The chloride salt is NaCl and / or KCl; The buffer is Tris-HCl buffer; The protective agent consists of BSA, casein and ADP.

2. A detection reagent for IgM antibody to Legionella pneumophila, characterized by comprising, The enzyme conjugate diluent, enzyme-labeled mouse-derived Legionella pneumophila antibody, magnetic particle suspension, Legionella pneumophila antigen solution and sample diluent are included; The enzyme conjugate diluent comprises 0.01-0.1M buffer, 3-5M chloride salt and the following components in mass volume percentage: 0.5-5% protective agent, 0.01-0.05% CaCl2, 0.1-0.5% preservative and 0.01-0.05% pigment; The chloride salt is NaCl and / or KCl; The buffer is Tris-HCl buffer; The protective agent consists of BSA, casein, ADP.

3. The detection reagent according to claim 2, characterized in that, The preservative is selected from at least one of Bro, P300, MIT, NaN3; The pigment comprises at least one of carmine pigment, sunset yellow, fruit green and lemon yellow pigment.

4. The detection reagent according to claim 2, characterized by The enzyme conjugate diluent, enzyme-labeled mouse-derived Legionella pneumophila antibody, magnetic particle suspension, Legionella pneumophila antigen solution and sample diluent are included; The enzyme conjugate diluent comprises 0.01-0.1M buffer, 3-5M chloride salt and the following components in mass volume percentage: 0.5-5% protective agent, 0.01-0.05% CaCl2, 0.1-0.5% preservative and 0.01-0.05% pigment; 5. The detection reagent according to claim 2, characterized by The chloride salt is NaCl and / or KCl. The magnetic particle suspension comprises magnetic beads, mouse anti-human IgM and magnetic bead buffer, wherein the magnetic bead buffer comprises the following components: 0.02M PBS+1% casein+0.1% ADP+5% glycerol+0.3% P300; The Legionella pneumophila antigen solution comprises Legionella pneumophila antigen and antigen diluent, wherein the antigen diluent comprises the following components: 0.02M PBS+3% BSA+1.5% sucrose+0.1% ADP+0.2% Bro+0.1% P300; The sample diluent comprises the following components: 0.05M Tris-HCl+1% BSA+1% EDTA-2Na+20,000 units / L urokinase+0.1% T20+0.5% CHAPS+0.2% sodium azide (10%)+0.1% P300+0.002% sunset yellow pigment+0.0019% lemon yellow pigment.

6. The detection reagent according to claim 2, characterized by A positive control and / or a negative control are further included; The positive control comprises: human Legionella pneumophila IgM antibody positive serum or plasma and positive control diluent; the positive control diluent is composed of the following components: 0.05M Tris-HCl+1% BSA+1% negative serum+1% EDTA-2Na+0.2% sodium azide+0.1% P300+0.006% carmine pigment; The negative control consisted of: 0.05 M Tris-HCl + 1% BSA + 1% EDTA-2Na + 0.2% sodium azide (10%) + 0.1% P300 + 0.005% fruit green.

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