Interferon-gamma detection kit and detection method thereof

Abstract

The invention relates to the technical field of gene detection, in particular to an interferon-gamma detection kit and a detection method thereof. According to the specific technical scheme, the preparation method of the luminous microsphere-IFN-gamma coated antibody complex comprises the following steps: cleaning luminous microspheres with an activation buffer solution, uniformly mixing with the activation buffer solution, sequentially adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxythiosuccinimide, oscillating, uniformly mixing, reacting in a dark place, filtering, washing, and drying to obtain the luminous microsphere-IFN-gamma coated antibody complex. After the reaction is finished, centrifuging to remove supernatant; then sequentially adding an activation buffer solution and deionized water for cleaning, adding a coupling buffer solution into the cleaned luminous microspheres, and ultrasonically mixing uniformly; adding an IFN-gamma coated antibody, uniformly mixing, sealing, and reacting in a dark place; after the reaction is finished, adding a rapid confining liquid, uniformly mixing, and reacting in a dark place; after the reaction is finished, centrifuging to remove supernatant; then adding a washing buffer solution for washing, and centrifuging to remove supernatant; and adding a preservation buffer solution, and uniformly mixing for later use. The detection method disclosed by the invention has high specificity and anti-interference performance, shows potential advantages in the aspects of simplicity and convenience in operation and detection speed, and is expected to be used as a reliable alternative method for detecting the IFN-gamma antigen in a serum sample.

Description

Technical Field

[0001] The present invention relates to the technical field of gene detection, and in particular to an interferon-γ detection kit and a detection method thereof. Background Art

[0002] Interferon-γ (IFN-γ) is an important cytokine, mainly produced by activated T cells and NK cells, and has biological effects such as antiviral and immunomodulatory. The detection of IFN-γ is of great significance for the diagnosis and treatment monitoring of various diseases. In the prior art, the detection methods of IFN-γ include ELISA, etc., but these methods have the disadvantages of complex operation and long detection time. Therefore, it is crucial to provide an efficient and convenient IFN-γ detection method. Summary of the invention

[0003] In view of the deficiencies in the prior art, the present invention provides an interferon-γ detection kit and a detection method thereof, which are simple to operate, rapid in detection and high in sensitivity.

[0004] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0005] The present invention discloses a method for preparing a luminescent microsphere-IFN-γ coated antibody complex, the steps of which are as follows:

[0006] (1) After washing the luminescent microspheres with an activation buffer, the luminescent microspheres were mixed with the activation buffer, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysulfosuccinimide were added in sequence. After oscillation and mixing, the mixture was reacted in the dark. After the reaction was completed, the supernatant was removed by centrifugation;

[0007] (2) In step (1), activation buffer and deionized water are added in sequence for washing, coupling buffer is added to the washed luminescent microspheres, and ultrasonic mixing is performed; IFN-γ coated antibody is added, the microspheres are sealed after mixing, and the reaction is protected from light; after the reaction is completed, rapid blocking solution is added, the microspheres are mixed after mixing, and the reaction is protected from light; after the reaction is completed, the supernatant is removed by centrifugation; then washing buffer is added for washing, and the supernatant is removed by centrifugation; and storage buffer is added for mixing and standby use.

[0008] Preferably, the mass ratio of the IFN-γ coated antibody to the luminescent microspheres is 1 to 2:1 to 5, the concentration of the luminescent microspheres is 1 to 10 mg / mL, the concentration of the IFN-γ coated antibody is 0.5 mg / mL, the activation buffer is 0.1 mol / L MES buffer, the concentrations of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysulfosuccinimide are 10 mg / mL respectively; and the coupling buffer is 0.1 mol / L borate buffer.

[0009] Preferably, the rapid blocking solution is bovine serum albumin BSA, the washing buffer is 0.1 mol / L Na2CO3 or NaHCO3, and the storage buffer is 0.1 mol / L PBS buffer containing 0.05% Proclin-300.

[0010] Correspondingly, a luminescent microsphere-IFN-γ coated antibody complex prepared by the above preparation method.

[0011] Correspondingly, a kit is prepared using the luminescent microsphere-IFN-γ coated antibody complex prepared by the above preparation method.

[0012] Correspondingly, a kit for preparing the luminescent microsphere-IFN-γ coated antibody complex prepared by the above preparation method is provided.

[0013] Correspondingly, a luminescent microsphere-IFN-γ coated antibody complex prepared by the above preparation method or a kit prepared by the above preparation method is used in detecting interferon-γ.

[0014] Correspondingly, a method for detecting interferon-γ using the luminescent microsphere-IFN-γ coated antibody complex prepared by the above preparation method or the prepared kit is as follows: adding the luminescent microsphere-IFN-γ coated antibody complex and the biotin-IFN-γ labeled antibody to the IFN-γ antigen standard, incubating at 15 to 42° C. for 5 seconds to 10 minutes, and gently stirring; then, adding streptavidin-coated photosensitive microspheres to the reaction mixture, and incubating at 15 to 42° C. in the dark for another 15 seconds to 10 minutes, and measuring its CE intensity.

[0015] Preferably, the preparation process of the streptavidin-coated photosensitive microspheres is as follows: after centrifuging the suspended photosensitive microspheres to remove the supernatant, add MES solution to wash and then add MES solution, mix the suspended microspheres, add 10 mg / mL of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 10 mg / mL of N-hydroxysulfosuccinimide, mix and centrifuge, resuspend with PBS buffer, add streptavidin, shake and mix overnight, add BSA to block, centrifuge, add TBS-T, shake and centrifuge, and store the suspended microspheres in TBS-T.

[0016] Preferably, the dilution volume ratio of the luminescent microsphere-IFN-γ coated antibody complex is 1:4-29, when measuring the CE intensity, the laser power is 80-120 mW, the irradiation time is 600-1400 ms, the volume ratio of the luminescent microsphere-IFN-γ coated antibody complex to the IFN-γ antigen standard is 1-5:1-5, the volume ratio of the luminescent microsphere-IFN-γ coated antibody complex to the biotin-IFN-γ labeled antibody is 1-2:1-5; the volume ratio of the luminescent microsphere-IFN-γ coated antibody complex to the streptavidin-coated photosensitive microsphere is 1-2:1-4.

[0017] The present invention has the following beneficial effects:

[0018] The present invention prepares a luminescent microsphere-IFN-γ coated antibody complex by direct coupling of luminescent microspheres with IFN-γ coated antibodies. The prepared luminescent microsphere-IFN-γ coated antibody complex is combined with a biotin-IFN-γ labeled antibody and a streptavidin-coated photosensitive microsphere for incubation, and then used for photochemiluminescence immunoassay for IFN-γ antigen detection. The detection results show that the photochemiluminescence method has high specificity and anti-interference performance. At the same time, the photochemiluminescence method not only has similar detection results to the immunofluorescence labeling method, but also shows potential advantages in terms of ease of operation and detection speed, and is expected to be used as a reliable alternative method for IFN-γ antigen detection in serum samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the principle diagram of interferon-γ antigen detection (A: interferon-γ antigen is absent; B: interferon-γ antigen is present);

[0020] Figure 2 This is a study on the optimal mass ratio of luminescent microspheres and IFN-γ coated antibodies;

[0021] Figure 3 This is a study diagram of the optimal volume ratio of the luminescent microsphere-IFN-γ coated antibody complex and the diluent;

[0022] Figure 4 For laser power research;

[0023] Figure 5 For irradiation time study;

[0024] Figure 6 To study the volume ratio of luminescent microsphere-IFN-γ coated antibody complex and standard in IFN-γ-Ag detection;

[0025] Figure 7 To study the volume ratio of luminescent microsphere-IFN-γ coated antibody complex and biotin-IFN-γ labeled antibody in IFN-γ-Ag detection;

[0026] Figure 8 To study the volume ratio of luminescent microspheres-IFN-γ coated antibody complexes and streptavidin coated photosensitive microspheres in IFN-γ-Ag detection;

[0027] Fig. 9 Optimize the incubation temperature;

[0028] Fig.10 Optimize for incubation time;

[0029] Fig.11 is the specificity of the IFN-γ antibody;

[0030] Fig.12 HOOK effect in IFN-γ detection;

[0031] Fig.13 is the linear relationship between IFN-γ-Ag concentration and CL value;

[0032] Fig.14 The anti-interference ability of IFN-γ antibody. DETAILED DESCRIPTION

[0033] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0035] The present invention discloses a method for preparing a luminescent microsphere-IFN-γ coated antibody complex, the steps of which are as follows:

[0036] (1) After washing the luminescent microspheres with an activation buffer, the luminescent microspheres are mixed with the activation buffer, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysulfosuccinimide are added in sequence, and the mixture is shaken and mixed to react in the dark. After the reaction is completed, the supernatant is removed by centrifugation; wherein the mass ratio of the IFN-γ coated antibody to the luminescent microspheres is 1 to 2:1 to 5, the concentration of the luminescent microspheres is 1 to 10 mg / mL, the concentration of the IFN-γ coated antibody is 0.5 mg / mL, the activation buffer is 0.1 mol / L MES buffer, and the concentrations of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysulfosuccinimide are 10 mg / mL respectively;

[0037] (2) In step (1), activation buffer and deionized water are added in sequence for washing, coupling buffer is added to the washed luminescent microspheres, and ultrasonic mixing is performed; then IFN-γ coated antibody is added, the microspheres are sealed after mixing, and the reaction is protected from light; after the reaction is completed, a quick blocking solution is added, the microspheres are protected from light after mixing; after the reaction is completed, the supernatant is removed by centrifugation; then washing buffer is added for washing, the supernatant is removed by centrifugation; then storage buffer is added for mixing and reserved. The activation buffer is 0.1 mol / L MES buffer, the coupling buffer is 0.1 mol / L borate buffer, the quick blocking solution is bovine serum albumin BSA, the washing buffer is 0.1 mol / L Na2CO3 or NaHCO3, and the storage buffer is 0.1 mol / L PBS buffer containing 0.05% Proclin-300.

[0038] The present invention also discloses a luminescent microsphere-IFN-γ coated antibody complex prepared by the above preparation method, and a kit is prepared by using the luminescent microsphere-IFN-γ coated antibody complex, or a kit is prepared by including the luminescent microsphere-IFN-γ coated antibody complex. The luminescent microsphere-IFN-γ coated antibody complex or the prepared kit is used to detect interferon-γ.

[0039] The present invention also discloses a detection method for detecting interferon-γ by using a luminescent microsphere-IFN-γ coated antibody complex prepared by the above preparation method or a kit prepared by the above preparation method, and the principle thereof is as follows: Figure 1 As shown, specifically: add luminescent microspheres-IFN-γ coated antibody complexes and biotin-IFN-γ labeled antibodies to the IFN-γ antigen standard, incubate at 15-42°C for 5s-10min, and gently stir; then, add streptavidin-coated photosensitive microspheres to the reaction mixture, and incubate at 15-42°C in the dark for another 15s-10min, and measure the CE intensity. The dilution volume ratio of the luminescent microsphere-IFN-γ coated antibody complex is 1:4-29, when measuring the CE intensity, the laser power is 80-120 mW, the irradiation time is 600-1400 ms, the volume ratio of the luminescent microsphere-IFN-γ coated antibody complex to the IFN-γ antigen standard is 1-5:1-5, the volume ratio of the luminescent microsphere-IFN-γ coated antibody complex to the biotin-IFN-γ labeled antibody is 1-2:1-5; the volume ratio of the luminescent microsphere-IFN-γ coated antibody complex to the streptavidin-coated photosensitive microsphere is 1-2:1-4.

[0040] The preparation process of the streptavidin-coated photosensitive microspheres is as follows: after centrifuging the suspended photosensitive microspheres to remove the supernatant, adding MES solution for washing and then adding MES solution, mixing the suspended microspheres, adding 10 mg / mL of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 10 mg / mL of N-hydroxysulfosuccinimide, centrifuging after mixing, resuspending with PBS buffer, adding streptavidin, shaking and mixing overnight, adding BSA for blocking, centrifuging, adding TBS-T, shaking and centrifuging, and storing the suspended microspheres with TBS-T.

[0041] The present invention will be further described below in conjunction with specific embodiments.

[0042] The luminescent microspheres and photosensitive microspheres used in the following examples were purchased from Suzhou Weidu Biotechnology Co., Ltd.

[0043] Example 1

[0044] Direct coupling of luminescent microspheres with IFN-γ coated antibodies to prepare luminescent microsphere-IFN-γ coated antibody complex (FG-IFN-γ)

[0045] The specific process is as follows: (1) 100 μL of luminescent microspheres (10 mg mL -1 ) was added to a centrifuge tube containing 1 mL of activation buffer, mixed by ultrasonication for 7-8 seconds, centrifuged at 14000 rpm, 15°C for 15 min, and the supernatant was removed. - 1 MES buffer, pH 6.0), ultrasonic for 7-8s, remove and mix thoroughly, add activator 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) 7 μL (10 mg mL -1 ), oscillate and mix, then add 66 μL (10 mg mL -1 ), shake and mix, place in a constant temperature shaker to avoid light, react, 40r / min, 25℃, activate for 15min. After activation, centrifuge at 14000rpm, 15℃ for 15min, remove the supernatant. Add 1mL activation buffer, sonicate for 7-8s, remove and invert to mix. Centrifuge at 14000rpm, 15℃ for 15min, remove the supernatant. Add 1mL deionized water, sonicate for 7-8s, remove and invert to mix. Centrifuge at 14000rpm, 15℃ for 15min, remove the supernatant.

[0046] (2) Add 0.2 mL of coupling buffer (0.1 mol·L) to the washed luminescent microspheres. -1Borate buffer, pH 8.0), ultrasonically mix for 5 seconds, then add 0.8 mL of coupling buffer, ultrasonically mix for 7-8 seconds, remove and invert to mix. Carefully add 10 μL of 0.5 mg·mL -1 Interferon-γ (IFN-γ) coated antibody (Wuhan Cloud Clone Technology Co., Ltd., No. PSA049Hu01) and oscillated to mix, oscillated to mix, sealed with sealing film, placed in a constant temperature oscillating box to avoid light reaction, 40r / min, 25℃, coupling for 2h. After the coupling is completed, 0.1mL of rapid blocking solution (bovine serum albumin BSA) is added, oscillated to mix, blocked in the dark, 40r / min, 25℃ for 2h or overnight blocking for 12-16h. After the blocking is completed, centrifuge to remove the supernatant, first add 0.2mL of washing buffer (0.1mol·L -1 Na2CO3 or NaHCO3, pH 10.0), ultrasonically mix for 5 seconds, then add 0.8mL washing buffer, ultrasonically mix for 7-8 seconds, remove and invert to mix. Centrifuge to remove the supernatant, and repeat washing 3 times. Finally, add 0.2mL storage buffer (0.1mol·L -1 PBS buffer, containing 0.05% Proclin-300, pH 7.4), ultrasonically mix for 5 seconds, then add 0.8 mL of storage buffer, ultrasonically mix for 7-8 seconds, take out and mix by inversion, and store at 2-8°C for use.

[0047] Example 2

[0048] Study on detection of IFN-γ antigen (IFN-γ-Ag) based on photoinduced chemiluminescence immunoassay

[0049] The specific process is as follows: 10 μL of IFN-γ antigen standard was added to a 96-well plate. Then 20 μL of luminescent microspheres-IFN-γ coated antibody complex and 20 μL of biotin-IFN-γ labeled antibody (Wuhan Cloud Clone Technology Co., Ltd., No. PSA049Hu01) were added. The plate was incubated at 37 ° C for 6 minutes and gently stirred on a shaking platform. Finally, 40 μL of streptavidin-coated photosensitive microspheres were added to the reaction mixture in all wells and incubated for another 6 minutes at 37 ° C in the dark. The CL intensity was then measured on a LICA reader.

[0050] The preparation process of photosensitive microspheres coated with streptavidin is as follows: fully mix the suspended photosensitive microspheres with a vortex mixer, take the suspension (i.e., photosensitive microspheres) into a centrifuge tube, centrifuge at 16000rpm for 30min, discard the supernatant, add 1mL of pH6.2, 0.05moL / L MES solution, fully mix the suspended microspheres with a vortex mixer, centrifuge at 16000rpm for 30min, discard the supernatant, repeat twice, add 1mL of pH6.2, 0.05moL / L MES solution, fully mix the suspended microspheres with a vortex mixer, add 25μL of 10mg / mL Sulfo-NHS and 40μL of 10mg / mL EDC, use a vortex shaker to fully mix the suspended microspheres for 30 minutes, centrifuge twice, resuspend in 1 mL of PBS buffer at pH 7.4, add 0.5 mg of streptavidin, shake and mix overnight, add 200 μL of 5% BSA, pH 7.2, block for 2 hours, centrifuge, add 1 mL of TBS-T (25 mM Tris-HCl, pH 7.2, 0.15 M NaCl, 0.05% Tween), shake and centrifuge, repeat 5 times, suspend the microspheres in TBS-T, and store at 2-8°C.

[0051] Example 3

[0052] The following experiments were conducted on the basis of Example 1 and Example 2.

[0053] 1. Optimization of the amount of conjugation between luminescent microspheres and IFN-γ coated antibodies

[0054] The effect of different mass ratios on the chemiluminescence (CL) signal intensity was investigated by systematically changing the coupling volume of IFN-γ coated antibody and luminescent microspheres. -1 ) coated antibody and 1 mg mL -1 The luminescent microspheres (20 μL) were coupled to obtain a series of conjugates with mass ratios of 2:1, 1:1, 2:3, 1:2, 2:5 and 1:3. Subsequently, a fixed concentration of IFN-γ-Ag standard (300 pg mL -1 , 10μL) was added to a 96-well plate, followed by the addition of luminescent microspheres-IFN-γ coated antibody complexes (20μL) and biotin-IFN-γ labeled antibodies (20μL) obtained by coupling at different mass ratios. After incubation at 37°C with gentle stirring for 6 minutes, streptavidin-coated photosensitive microspheres (40μL) were added to each well and further incubated at 37°C in the dark for 6 minutes to promote signal enhancement. Finally, the CL intensity of each well was measured using a LICA reader. Experimental results ( Figure 2) showed that with the increase of the IFN-γ coated antibody coupling volume, the CL signal intensity showed a trend of first increasing and then decreasing. When the coupled antibody volume was 10 μL, that is, when the mass ratio reached 2:5, the CL signal intensity reached a peak value, that is, at this time, the coupling efficiency of the luminescent microspheres and the antibody was optimal.

[0055] 2. Optimization of the dilution volume ratio of the luminescent microsphere-IFN-γ coated antibody complex (coupling mass ratio of 2:5) and the diluent

[0056] In order to evaluate the chemiluminescent (CL) signal intensity of the luminescent microsphere-IFN-γ coated antibody complex at different dilution volume ratios (Dope (stock solution), 1:4, 1:9, 1:14, 1:19, 1:29). 300 pg mL -1 IFN-γ-Ag standard (10 μL) was added, followed by adding different dilution ratios of luminescent microspheres-IFN-γ coated antibody complexes (20 μL) and biotin-IFN-γ labeled antibodies (20 μL), incubated at 37°C for 6 minutes, and then added streptavidin-coated photosensitive microspheres (40 μL) to each well. The cells were incubated at 37°C in the dark for another 6 minutes, and the CL intensity was finally measured using a LICA reader. Experimental results ( Figure 3 ) showed that the CL signal intensity first increased and then decreased with the increase of the dilution volume ratio. When the dilution volume ratio reached 1:19, the CL signal intensity was the highest and the background noise was the lowest. Therefore, 1:19 was determined to be the optimal dilution volume ratio, which was equivalent to diluting the luminescent microsphere-IFN-γ coated antibody complex by 20 times, and the final concentration was reduced to 0.05 mg·mL -1 Subsequent experiments were conducted using this dilution condition.

[0057] 3. Optimization of laser power and irradiation time

[0058] The effects of different laser powers (80mW, 90mW, 100mW, 110mW, 120mW) and irradiation times (600ms, 800ms, 1000ms, 1200ms, 1400ms) on the chemiluminescence (CL) signal intensity were investigated. -1 IFN-γ-Ag standard (10 μL) and luminescent microsphere-IFN-γ coated antibody complex (0.05 mg mL -1 , 20 μL) and biotin-IFN-γ labeled antibody (20 μL) were mixed and incubated at 37°C for 6 minutes, then streptavidin-coated photosensitive microspheres (40 μL) were added and the CL intensity was measured after another incubation. Results ( Figure 4 , Figure 5) showed that when the laser power was set to 110 mW and the irradiation time was 800 ms, the CL signal intensity reached a peak, and further increasing the power and time had limited effect on improving the signal. Therefore, 110 mW and 1200 ms were determined to be the optimal laser power and optimal irradiation time, respectively, and subsequent experiments will be carried out based on these parameters.

[0059] 4. Study on the optimal volume ratio of luminescent microspheres-IFN-γ coated antibody complex and IFN-γ-Ag standard

[0060] The chemiluminescence (CL) signal values ​​of the luminescent microsphere-IFN-γ coated antibody complex and the IFN-γ-Ag standard at different volume ratios (2:5, 1:1, 2:1, 4:1, 5:1) were compared. Different volumes (4, 5, 10, 20, and 25 μL) of 300 pg mL -1 IFN-γ-Ag standard, and fixed with 20μL luminescent microspheres-IFN-γ coated antibody complex and 20μL biotin-IFN-γ labeled antibody. After incubation at 37℃ for 6min, 40μL streptavidin-coated photosensitive microspheres were added to each well and incubated in the dark. Comparing the CL intensity, when the volume ratio of luminescent microspheres-IFN-γ coated antibody complex to IFN-γ-Ag standard was 4:1 ( Figure 6 ), the CL signal was the strongest. Therefore, 4:1 was determined to be the optimal volume ratio, that is, the optimal volume of IFN-γ-Ag standard was 5 μL, and subsequent experiments will be carried out according to this volume ratio.

[0061] 5. Study on the optimal volume ratio of luminescent microsphere-IFN-γ coated antibody complex and biotin-IFN-γ labeled antibody

[0062] The maximum chemiluminescence (CL) signal intensity was obtained by optimizing the volume ratio of luminescent microsphere-IFN-γ coated antibody complex to biotin-IFN-γ labeled antibody (None (0 volume), 2:1, 1:1, 2:3, 1:2, 2:5).

[0063] The experimental steps are as follows: First, 10 μL of 300 pg mL -1 The IFN-γ-Ag standard was added to a 96-well plate, followed by the addition of 20 μL of luminescent microspheres-IFN-γ coated antibody complexes and different volumes (0, 10, 20, 30, 40, 50 μL) of biotin-IFN-γ labeled antibodies. After incubation at 37°C on a rocking platform for 6 minutes, 40 μL of streptavidin-coated photosensitive microspheres were added to each reaction well and incubated for another 6 minutes at 37°C in the dark. Finally, the LICA reader was used to measure the CL intensity of each well to evaluate the effect of different volume ratios on the signal intensity. Experimental results ( Figure 7 ) showed that the CL signal intensity first increased and then decreased with the increase of the volume of biotin-IFN-γ labeled antibody. When the volume ratio of luminescent microsphere-IFN-γ coated antibody complex to biotin-IFN-γ labeled antibody was 1:1, the CL signal intensity reached the highest peak. Based on this, we determined that 1:1 was the optimal volume ratio and used it as the condition for subsequent experiments.

[0064] 6. Study on the optimal volume ratio of luminescent microspheres-IFN-γ coated antibody complexes and streptavidin coated photosensitive microspheres

[0065] To evaluate the CL value of the luminescent microsphere-IFN-γ coated antibody complex and the streptavidin-coated light-sensitive microsphere at the volume ratio (None, 2:1, 1:1, 1:2, 1:3, 1:4). -1 The IFN-γ-Ag standard was added to a 96-well plate. Then 20 μL of luminescent microspheres-IFN-γ coated antibody complex and 20 μL of biotin-IFN-γ labeled antibody were added. The plate was incubated at 37°C for 6 minutes and gently stirred on a rocking platform. Finally, different volumes (0, 10, 20, 40, 60, 80 μL) of streptavidin-coated photosensitive microspheres were added to the reaction system and incubated for another 6 minutes at 37°C in the dark. The CL intensity was then measured on a LICA reader to examine the optimal volume ratio of the luminescent microspheres-IFN-γ coated antibody complex and the streptavidin-coated photosensitive microspheres. The results are shown in Figure 2. Figure 8 As shown in the figure, as the volume of the photosensitive microspheres coated with streptavidin increases, the CL value increases first and then decreases. When the volume ratio of the luminescent microsphere-IFN-γ coated antibody complex and the photosensitive microspheres coated with streptavidin is 1:2, the CL value is the largest, and this ratio is considered to be the optimal volume ratio, that is, the volume of the photosensitive microspheres coated with streptavidin is 40 μL. All subsequent studies were conducted under this volume ratio.

[0066] 7. Optimization of reaction temperature

[0067] The effect of different temperatures (15°C, 25°C, 37°C, and 42°C) on the light signal value during the reaction phase was evaluated. -1 The IFN-γ-Ag standard was mixed with 20 μL of luminescent microspheres-IFN-γ coated antibody complex and 20 μL of biotin-IFN-γ labeled antibody, incubated at each specified temperature for 6 minutes, and then 40 μL of streptavidin-coated photosensitive microspheres were added and incubated for another 6 minutes in the dark at the same temperature. The CL intensity was measured by a LICA reader to determine the optimal incubation temperature. Experimental results ( Fig. 9) showed that the CL value increased with the increase of temperature, and the CL value reached the highest at 37°C. Therefore, 37°C was determined to be the optimal incubation temperature for IFN-γ-Ag detection, and subsequent experiments will be carried out at this temperature.

[0068] 8. Optimization of the reaction time of the two stages

[0069] The effects of different incubation times (15s, 1min, 5min, 10min) on the light signal value were compared. -1 After mixing the IFN-γ-Ag standard with 20 μL of luminescent microspheres-IFN-γ coated antibody complex and 20 μL of biotin-IFN-γ labeled antibody, 40 μL of streptavidin-coated photosensitive microspheres were added to each well and incubated for the same time at 37°C in the dark. The CL intensity was measured by a LICA reader to determine the optimal incubation time. Experimental results ( Fig.10 ) showed that as the incubation time increased, the CL value increased, reaching the highest value at 5 min, and the signal value began to decrease after this time. Therefore, 5 min was determined to be the optimal incubation time for IFN-γ-Ag detection, and subsequent experiments will be carried out according to this time.

[0070] Example 4 Preliminary clinical application

[0071] 1. Sample pretreatment:

[0072] Serum: Venous blood samples are collected in standard test tubes or test tubes with separation gel, naturally coagulated at room temperature, and centrifuged at 2000-4000rpm for 20 minutes. About 0.5mL of separated serum is taken for testing.

[0073] Plasma: Venous blood samples were collected in EDTA anticoagulant tubes, centrifuged at 2000-4000 rpm for 20 minutes, and about 0.5 mL of plasma was taken for testing.

[0074] Serum and plasma samples should be tested within 3 hours after blood collection. If it cannot be tested within 3 hours, it can be stored at 4℃ for 24 hours. If it exceeds 24 hours, the separated plasma or serum must be stored at -20℃. Do not freeze-thaw the samples (no more than three times).

[0075] Detection: Add 5 μL of test serum sample to the wells of a 96-well microtiter plate. Then add 20 μL of luminescent microsphere-IFN-γ coated antibody complex and 20 μL of biotin-IFN-γ labeled antibody. Incubate the plate at 37°C for 5 minutes with gentle agitation on a rocking platform. Finally, add 40 μL of streptavidin-coated photosensitive microspheres to the reaction mixture in all wells and incubate for another 5 minutes at 37°C in the dark. Then measure the CL intensity on a LICA reader.

[0076] 2. Specificity Analysis

[0077] 300 pg·mL was prepared -1 IFN-γ-Ag standard and 12000pg·mL -1 A variety of antigen standards (IL-6, cTnT, P24, tPAI·C, SAA, PCT, CRP) were prepared and stored under frozen conditions. In the experiment, 5 μL of each antigen standard was placed in a 96-well plate, 20 μL of luminescent microspheres-IFN-γ coated antibody complex and 20 μL of biotin-IFN-γ labeled antibody were added, and after incubation at 37°C for 5 minutes, 40 μL of streptavidin-coated photosensitive microspheres were added and incubated for another 5 minutes. The CL intensity was measured using a LICA reader, and the results ( Fig.11 ) showed that only the addition of IFN-γ-Ag significantly changed the CL value, while other antigens had no significant effect. This result confirmed that the method has high specificity for IFN-γ-Ag and provides theoretical support for the clinical application of photochemiluminescence analysis.

[0078] 3. Drawing of standard curve and HOOK effect

[0079] Dilute IFN-γ-Ag standard to 0 to 10000 pg mL using PBS buffer. -1 The chemiluminescence (CL) value was measured by LICA reader. Fig.12 ) shows that the concentration of -1 No HOOK effect was observed in the concentration range of 1.0 to 1000 pg mL -1 The concentration of IFN-γ-Ag standard was 1.37×1.76×1.80, and the linear equation y=3.33x+17.51 ​​was established. The correlation coefficient R 2 The value was 0.9943, indicating a good linear relationship, and the detection limit was 0.32 pg·mL -1 ( Fig.13 ). In addition, this method was applied to the detection of IFN-γ antigen in serum samples. The CL value was determined and the concentration was calculated using a linear equation. The results are shown in Table 1. The photochemiluminescence method not only has similar detection results to the immunofluorescence labeling method, but also has potential advantages in terms of ease of operation and detection speed. It is expected to be a reliable alternative method for the detection of IFN-γ antigen in serum samples.

[0080] Table 1 Detection results of IFN-γ antigen in samples

[0081] Sample No. <![CDATA[Immunofluorescence labeling detection (pg·mL -1 )]]> <![CDATA[LICA detection (pg·mL -1 )]]> Absolute error 1 1.51 1.67 0.107 2 2.91 2.63 0.096 3 1.37 1.38 0.01 4 3.79 3.97 0.048 5 0.74 0.721 0.019 6 1.03 1.009 0.021 7 0.81 0.721 0.089

[0082] 4. Anti-interference test

[0083] In order to evaluate the potential impact of other substances in serum samples on IFN-γ antigen detection, 300 pg mL -1 IFN-γ-Ag standard and other antigen standards (IL-6, cTnT, P24, tPAI·C, SAA, PCT, CRP) with a concentration ratio of (1:10, 1:20, 1:30 and 1:40) were mixed in equal volumes to form an antigen mixture. 5 μL of the mixture was added to a 96-well plate, and then the luminescent microsphere-IFN-γ coated antibody complex and biotin-IFN-γ labeled antibody were added according to standard operations and incubated at 37°C for 5 minutes. After that, streptavidin-coated photosensitive microspheres were added, and the CL intensity was measured using a LICA reader after continued incubation. Experimental results ( Fig.14 ) showed that the CL value did not change significantly in the presence of other antigens, indicating that the detection method has high anti-interference performance.

[0084] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing a luminescent microsphere-IFN-γ coated antibody complex, characterized in that: Here are the steps: (1) After washing the luminescent microspheres with an activation buffer, the luminescent microspheres were mixed with the activation buffer, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysulfosuccinimide were added in sequence. After oscillation and mixing, the mixture was reacted in the dark. After the reaction was completed, the supernatant was removed by centrifugation; (2) In step (1), activation buffer and deionized water are added in sequence for washing, coupling buffer is added to the washed luminescent microspheres, and ultrasonic mixing is performed; IFN-γ coated antibody is added, the microspheres are sealed after mixing, and the reaction is protected from light; after the reaction is completed, rapid blocking solution is added, the microspheres are mixed after mixing, and the reaction is protected from light; after the reaction is completed, the supernatant is removed by centrifugation; then washing buffer is added for washing, and the supernatant is removed by centrifugation; and storage buffer is added for mixing and standby use.

2. The preparation method according to claim 1, characterized in that: The mass ratio of the IFN-γ coated antibody to the luminescent microspheres is 1-2:1-5, the concentration of the luminescent microspheres is 1-10 mg / mL, the concentration of the IFN-γ coated antibody is 0.5 mg / mL, the activation buffer is 0.1 mol / L MES buffer, the concentrations of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysulfosuccinimide are 10 mg / mL respectively; and the coupling buffer is 0.1 mol / L borate buffer.

3. The preparation method according to claim 1, characterized in that: The rapid blocking solution is bovine serum albumin BSA, the washing buffer is 0.1 mol / L Na2CO3 or NaHCO3, and the storage buffer is 0.1 mol / L PBS buffer containing 0.05% Proclin-300.

4. A luminescent microsphere-IFN-γ coated antibody complex prepared by the preparation method according to any one of claims 1 to 3.

5. A kit for preparing the luminescent microsphere-IFN-γ coated antibody complex prepared by the preparation method according to any one of claims 1 to 3.

6. A kit for preparing a luminescent microsphere-IFN-γ coated antibody complex prepared by the preparation method according to any one of claims 1 to 3.

7. Use of the luminescent microsphere-IFN-γ coated antibody complex prepared by the preparation method according to any one of claims 1 to 3 or the kit prepared by claim 5 or 6 in detecting interferon-γ.

8. A method for detecting interferon-γ using the luminescent microsphere-IFN-γ coated antibody complex prepared by the preparation method according to any one of claims 1 to 3 or the kit prepared by claim 5 or 6, characterized in that: Add luminescent microspheres-IFN-γ coated antibody complex and biotin-IFN-γ labeled antibody to the IFN-γ antigen standard, incubate at 15-42°C for 5s-10min with gentle stirring; then, add streptavidin-coated photosensitive microspheres to the reaction mixture, incubate at 15-42°C in the dark for another 15s-10min, and measure the CE intensity.

9. The detection method according to claim 8, characterized in that: The preparation process of the streptavidin-coated photosensitive microspheres is as follows: after centrifuging the suspended photosensitive microspheres to remove the supernatant, adding MES solution for washing and then adding MES solution, mixing the suspended microspheres, adding 10 mg / mL of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 10 mg / mL of N-hydroxysulfosuccinimide, centrifuging after mixing, resuspending with PBS buffer, adding streptavidin, shaking and mixing overnight, adding BSA for blocking, centrifuging, adding TBS-T, shaking and centrifuging, and storing the suspended microspheres in TBS-T.

10. The detection method according to claim 8, characterized in that: The dilution volume ratio of the luminescent microsphere-IFN-γ coated antibody complex is 1:4-29. When measuring CE intensity, the laser power is 80-120 mW, the irradiation time is 600-1400 ms, the volume ratio of the luminescent microsphere-IFN-γ coated antibody complex to the IFN-γ antigen standard is 1-5:1-5, the volume ratio of the luminescent microsphere-IFN-γ coated antibody complex to the biotin-IFN-γ labeled antibody is 1-2:1-5; the volume ratio of the luminescent microsphere-IFN-γ coated antibody complex to the streptavidin-coated photosensitive microsphere is 1-2:1-4.

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