Antibody kit for detecting depression and application thereof

Abstract

The invention relates to an antibody kit for detecting depression and application thereof. The kit comprises a specific antibody, preferably a monoclonal antibody, aiming at human CX3CL1, human L-selectin (SELL) and human RAP1B protein. The antibody is labeled by colloidal gold and is fixed in an immunochromatography detection device to form a chromatography test strip comprising a sample pad, a colloidal gold pad, an NC membrane (coating antigen) and a water absorption pad. According to the kit, the rapid in-vitro diagnosis of depression is realized by simultaneously detecting the biomarker combination of CX3CL1, SELL and RAP1B in peripheral blood. The invention provides a depression diagnosis scheme based on multi-target cooperative detection for the first time, has the advantages of high sensitivity (the antibody titer is greater than 500,000), convenience in operation (detection is completed within 15 minutes) and low cost, and is suitable for clinical large-scale screening.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical detection technology, and specifically relates to an in vitro diagnostic kit for depression based on the synergistic effect of multiple target antibodies and its application. Background Art

[0002] The diagnosis of depression has long relied on subjective rating scales (such as HAMD and PHQ-9), lacking objective biological indicators, resulting in misdiagnosis rates exceeding 30% (Trivedi et al., 2006). Recent studies have found significant associations between peripheral blood inflammatory factors (such as IL-6 and TNF-α) and neuroplasticity-related proteins (such as BDNF) and depression (Dowlati et al., 2010). However, the sensitivity and specificity of these single markers are insufficient (AUC < 0.7), making them inadequate for clinical diagnosis. Although ELISA and chemiluminescence techniques can quantitatively detect serum markers, these techniques are complex, time-consuming (> 2 hours), and costly, making them difficult to use for primary care screening.

[0003] While colloidal gold immunochromatographic technology offers the advantages of rapidity (<15 minutes) and portability, existing depression diagnostics only target a single target (such as the SERT protein), resulting in limited diagnostic efficacy (Patel et al., 2019). Studies have shown that depression is a syndrome characterized by disturbances in multiple pathways, necessitating the use of multi-target collaborative testing (such as combined testing of inflammation, adhesion, and signaling pathways) to improve accuracy (Menke, 2019). However, no technology exists for the rapid, simultaneous detection of such a combination of markers. CX3CL1 (chemokine), L-selectin (adhesion molecule), and RAP1B (signaling protein) regulate neuroinflammation, leukocyte migration, and neuronal synaptic function, respectively. Abnormal expression of these three markers is significantly associated with depression pathology (Skaper et al., 2014; Wang et al., 2018). However, there is a lack of high-affinity antibodies: existing commercial antibodies have low potency (<10^5) against low-abundance targets (such as RAP1B), which makes it difficult to meet the sensitivity requirements of colloidal gold detection; there is a lack of multi-detection process: aggregation is prone to occur when colloidal gold labels multiple antibodies, and multiple detection lines in the chromatography device are prone to cross-reaction (Zhou et al., 2021).

[0004] Therefore, there is an urgent need to develop a solution that integrates high-titer antibodies, optimized colloidal gold labeling process and simultaneous detection of multiple targets. Summary of the Invention

[0005] To address the above problems, the present invention provides a depression diagnostic kit based on colloidal gold immunochromatography technology, the core of which is to simultaneously detect three key biomarkers in human peripheral blood: chemokine CX3CL1, adhesion molecule L-selectin (SELL) and signaling protein RAP1B.

[0006] The present invention provides an antibody kit for detecting depression, characterized in that it comprises the following components: anti-human CX3CL1 monoclonal antibody, anti-human L-selectin (SELL) monoclonal antibody and anti-human RAP1B monoclonal antibody;

[0007] In certain embodiments, the above-mentioned antibodies are preferably labeled with colloidal gold, wherein the colloidal gold particle size is 40-70 nm; the immunochromatographic detection device comprises a sample pad, a colloidal gold pad, an antigen-coated NC membrane, and a water-absorbing pad attached in sequence; the NC membrane is immobilized with CX3CL1, SELL, and RAP1B recombinant proteins as detection line antigens.

[0008] In certain embodiments, the heavy chain CDR sequence of the anti-human CX3CL1 monoclonal antibody is SEQ No. 1-3, and the light chain CDR sequence is SEQ No. 4-6; the heavy chain CDR sequence of the anti-human SELL monoclonal antibody is SEQ No. 7-9, and the light chain CDR sequence is SEQ No. 10-12; the heavy chain CDR sequence of the anti-human RAP1B monoclonal antibody is SEQ No. 13-15, and the light chain CDR sequence is SEQ No. 16-18.

[0009] In certain embodiments, the method for preparing the colloidal gold-labeled antibody comprises: dialysis purification of the above-mentioned antibody in a 5 mM NaCl solution at pH 7.0; mixing 1 mg of antibody: 100 mL of colloidal gold solution and stirring for 30 minutes; sequentially adding a final concentration of 0.5% BSA and 0.1% PEG20000 to block; and resuspending the precipitate with DPBS after centrifugation to obtain a gold-labeled antibody solution.

[0010] In certain embodiments, in the immunochromatographic detection device, the colloidal gold pad is fixed with gold-labeled antibodies by a spraying process with spraying parameters of 2–6 μL / cm and a pressure of 10 PSI; the antigen coating volume on the NC membrane is 0.5–1 μL / cm, and the antigen concentration is 5 mg / mL.

[0011] The present invention provides an application of a kit for preparing an in vitro diagnostic product for depression, characterized in that the kit can distinguish samples of depression patients from healthy subjects by detecting the expression levels of CX3CL1, SELL and RAP1B proteins in human peripheral blood.

[0012] Compared with the prior art, the present invention has the following outstanding advantages:

[0013] High accuracy: Triple-target synergistic detection significantly improves diagnostic specificity;

[0014] Fast and convenient: detection time is less than 15 minutes, requiring only 10 μL of peripheral blood;

[0015] Strong stability: the activity of the colloidal gold probe decreases by less than 5% after being stored at 4°C for 12 months;

[0016] Low cost: No professional equipment is required, suitable for primary medical screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 : Anti-human CX3CL1 monoclonal antibody titer detection results.

[0018] Figure 2 : Affinity detection of anti-human CX3CL1 hybridoma cell supernatant antibody.

[0019] Figure 3 : Activity characterization of anti-human CX3CL1 monoclonal antibody.

[0020] Figure 4 : SDS-PAGE identification of anti-human CX3CL1 monoclonal antibody.

[0021] Figure 5 : Anti-human SELL monoclonal antibody titer detection.

[0022] Figure 6 : Comparison of affinity of anti-human SELL hybridoma cell clones.

[0023] Figure 7 : SDS-PAGE identification of anti-human SELL monoclonal antibody.

[0024] Figure 8 : Anti-human RAP1B monoclonal antibody titer detection.

[0025] Figure 9 : Affinity detection of anti-human RAP1B hybridoma cell clones.

[0026] Figure 10 : SDS-PAGE identification of anti-human RAP1B monoclonal antibody.

[0027] Figure 11 : Preparation results of colloidal gold particles.

[0028] Figure 12 : Depression diagnostic kit test results. DETAILED DESCRIPTION

[0029] In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0030] Example 1 Preparation of anti-human CX3CL1 monoclonal antibody

[0031] Animal Immunization

[0032] According to the sequence information of C-X3-C motifchemokine ligand 1 [Homo sapiens], NCBI Reference Sequence: NC_000016.10, the following sequence region was selected as the antigen fragment:

[0033] MAPISLSWLLRLATFCHLTVLLAGQHHGVTKCNITCSKMTSKIPVALLIHYQQNQASCGKRAIILETRQHRLFCADPKEQWVKDAMQHLDRQAAALTRNGGTFEKQIGEVKPRTTPAAGGMDESVVLEPEATGESSSLEPTPSSQEAQRALGTSPELPTGVTGSSGTRLPPTPKAQDGGPVGTELFRVPPVSTAATWQS SAPHQPGPSLWAEAKTSEAPSTQDPSTQASTASSPAPEENAPSEGQRVWGQGQSPRPENSLEREEMGPVPAHTDAFQDWGPGSMAHVSVVPVSSEGTPSREPVASGSWTPKAEEPIHATMDPQRLGVLITPVPDAQAATRRQAVGLLAFLGLLFCLGVAMFTYQSLQGCPRKMAGEMAEGLRYIPRSCGSNSYVLVPV;

[0034] Recombinant human CX3CL1 protein was expressed in E. coli and used to immunize Balb / c mice. 50 μg of CX3CL1 was added dropwise to an equal volume of complete Freund's adjuvant for emulsification to create a fully emulsified solution, which was then injected subcutaneously at multiple sites, with 100 μl per site. Two weeks after the primary immunization, a secondary immunization was performed subcutaneously using an emulsion prepared with 50 μg of CX3CL1 and an equal volume of incomplete Freund's adjuvant. Two weeks after the secondary immunization, a third immunization was performed using the same procedure. Seven days after the third immunization, a small amount of serum was collected to determine the antibody titer using ELISA.

[0035] Dissolve recombinant human CX3CL1 protein in DPBS at a concentration of 1 μg / mL and add 100 μL / well of the ELISA plate. Incubate overnight at 4°C. Discard the antigen solution and prepare 2% BSA in DPBS as blocking buffer. Add 200 μL / well of blocking buffer to each well and incubate at 37°C for 2 hours. Discard the blocking buffer and wash the plate with 200 μL / well of DPBST. Vortex thoroughly and discard. Repeat three times. Add 100 μL of the test serum diluted in blocking buffer and incubate at 37°C for 1 hour. Discard the sample and wash three times with washing buffer. Dilute goat anti-mouse IgG-HRP in blocking buffer 1:5000 and add 100 μL / well of the plate. Incubate at room temperature for 1 hour. Discard the secondary antibody and wash the plate three times with washing buffer. TMB substrate was added to the microplate at 100 μL / well, incubated at room temperature for 20 min, and the absorbance at 650 nm was read on a microplate reader. Figure 1 As shown, the negative control is the pre-collected serum, recorded as N, the post-immune serum is recorded as P, and the 10 dilution of the post-immune serum is recorded as 10P. The horizontal axis is the log of the antibody titer, and the vertical axis is the OD650 reading. Figure 1 It can be seen that the anti-CX3CL1 serum we obtained has good antigen binding ability, with a titer of 1.019*10 6 .

[0036] Cell fusion and hybridoma cloning

[0037] Two weeks after the three immunizations, the mice were killed and the spleen cells were isolated. The mouse myeloma cells SP2 / 0 and the isolated spleen cells were mixed at a ratio of 1:10 and centrifuged. The supernatant was discarded, the tube wall was tapped to loosen the precipitate slightly, and 1 ml of preheated 45% PEG4000 was slowly added while shaking the centrifuge tube. After standing for 90 seconds, centrifugation was carried out, the supernatant was discarded, and the cells were resuspended in HAT selection medium containing 20% ​​fetal bovine serum. The cell suspension was diluted and added to a 96-well plate at 100ul / well. After each spleen was separated, it could be diluted to 4 96-well plates. After culturing in the incubator for 5 days, HT medium was replaced, and RPMI1640 basal medium was replaced after 2 weeks. When the hybridoma cells covered 10% of the bottom of the well, the supernatant was taken for ELISA to detect the affinity of the specific antibody. The experimental method was consistent with the serum titer determination, and the results were as follows. Figure 2 As shown, the negative control is pre-collected serum, recorded as N, the post-immune serum is recorded as P, and the 10 dilution of the post-immune serum is recorded as 10P. The horizontal axis is the log of the antibody titer, and the vertical axis is the OD650 reading.

[0038] Select hybridoma cell A with high antibody affinity and obtain monoclones using the limiting dilution method. Gently blow off the hybridoma cells to be cloned from the culture well, count them, and dilute them to 5 cells / ml with basal culture medium. Add the diluted cell suspension at 100ul / well and culture in a 37℃ 5% CO2 incubator. Monoclonal formation can be seen after 8 days. Take the supernatant and test the antibody activity by ELISA. The antigen is recombinant human CX3CL1 protein, the secondary antibody is goat-anti-mouse IgG-HRP, and the negative control is blank culture medium, which is recorded as N. The results are as follows Figure 3 As shown, the horizontal axis is the logarithm of the supernatant dilution multiple. The supernatant of monoclonal clone No. 1 has good antigen binding activity, with a titer of 100.6741*10 6 Monoclonal clone No. 1 was cultured and cryopreserved in a cryopreservation solution of 10% DMSO + 90% fetal bovine serum.

[0039] Purification of monoclonal antibodies

[0040] Clone 1 cells were expanded into two T75 flasks, and the resulting monoclonal antibody was designated anti-CX3CL1. After 7 days, approximately 30 ml of supernatant was collected and centrifuged at 8000 rpm for 10 minutes at 4°C. The supernatant was collected in a new 50 ml centrifuge tube and filtered through a 0.22 μm filter. After filtration, the pH was measured and adjusted to 7.0-7.5 using neutralization buffer. 500 μl of Protein G magnetic beads were added to the supernatant, mixed thoroughly, and vortexed overnight at 4°C. The next day, the supernatant was centrifuged at 1500 rpm at 4°C for 5 minutes, and the supernatant was discarded. The beads were resuspended in 1 ml of cold DPBS and transferred to a chromatography column. The column was washed three times with 10 ml of DPBS. 3 ml of elution buffer was added to the column, the remaining DPBS was discarded, and the lower end of the column was plugged and incubated for 5-10 minutes. After the eluate flowed out, the elution was repeated once. Add 600 μl of neutralization buffer to the collected eluate and mix thoroughly. Transfer the eluate into a 30 kDa ultrafiltration tube and centrifuge at 4000 rpm and 4°C to concentrate the protein solution. After the volume of the protein solution is reduced to 600 μl, add 5 ml of DPBS and centrifuge again. The centrifugation conditions are the same as above. Repeat 3 times until the buffer is completely replaced with DPBS. Use a spectrophotometer to determine the protein concentration. Take 3 μg of protein for SDS-PAGE identification. The results are as follows Figure 4 As shown, the molecular weight of the antibody is around 150 kDa, which is consistent with the molecular weight of mouse IgG.

[0041] Obtain monoclonal antibody sequences

[0042] Candidate hybridoma clones were lysed with Trizol and total RNA was extracted. This was used as a template for first-strand cDNA synthesis. PCR amplification was then performed using the first-strand cDNA as a template using primers specific for the antibody variable region to obtain nucleic acids for the light and heavy chain variable regions of the antibody corresponding to the hybridoma cells. The nucleic acids were then subjected to agarose gel electrophoresis, excised, and recovered, followed by Sanger sequencing to obtain the antibody variable region sequence. The complete antibody sequence is shown in Table 1.

[0043] Table 1 Anti-CX3CL1 monoclonal antibody amino acid sequence

[0044]

[0045] Example 2 Preparation of anti-human L-selectin monoclonal antibody

[0046] Similar to Example 1 above, recombinant human L-selectin (SELL) was used to immunize Balb / c mice multiple times to obtain anti-human L-selectin monoclonal antibodies. SELL was purchased from Shanghai Qincheng Biotechnology Co., Ltd. and expressed in E. coli. It has His and GST tags at the N-terminus. The specific sequence is MIFPWKCQSTQRDLWNIFKLWGWTMLCCDFLAHHGTDCWTYHYSEKPMNWQRARRFCRDNYTDLVAIQNKAEIEYLEKTLPFSRSYYWIGIRKIGGIWTWVGTNKSLTEEAENWGDGEPNNKKNKEDCVEIYIKRNKDAGKWNDDACHKLKAALCYTASCQ PWSCSGHGECVEIINNYTCNCDVGYYGPQCQFVIQCEPLEAPELGTMDCTHPLGNFSFSSQCAFSCSEGTNLTGIEETTCGPFGNWSSPEPTCQVIQCEPLSAPDLGIMNCSHPLASFSFTSACTFICSEGTELIGKKKTICESSGIWSNPSPICQKLDKSFSMIKEGDYNPLFIPVAVMVTAFSGLAFIIWLARRLKKGKKSKRSMNDPY. After immunization, the antibody titer of mouse serum was detected by ELISA. The results are as follows: Figure 5 The negative control is the pre-collected serum, recorded as N, the post-immune serum is recorded as P, and the 10 dilution of the post-immune serum is recorded as 10P. The horizontal axis is the logarithm of the antibody titer, and the vertical axis is the OD650 reading. Figure 5 The antibody titer is greater than 1 million. The antibody affinity of hybridoma cell monoclonal antibody was determined by ELISA method. The results are as follows Figure 6 As shown, clones 1 and 2 have good affinity, with clone 1 having a titer of 1.59*10 6, the titer of clone 2 was 2.14*10 6 Clone 1 was expanded and frozen, and the antibody produced by clone 1 was designated as anti-SELL.

[0047] The anti-SELL antibody purified by affinity chromatography was identified and its molecular weight was detected by SDS-PAGE. The results are as follows: Figure 7 As shown, the actual molecular weight is consistent with the theoretical value of mouse IgG molecular weight.

[0048] The method for obtaining the complete sequence of Anti-SELL is the same as in Example 1, as shown in Table 2.

[0049] Table 2 Anti-SELL monoclonal antibody amino acid sequence

[0050]

[0051]

[0052] Example 3 Preparation of anti-human RAP1B monoclonal antibody

[0053] Similar to Example 1 above, recombinant human RAP1B was used to immunize mice multiple times to obtain anti-human RAP1B monoclonal antibodies. The recombinant human RAP1B used was purchased from Abcam, with the product number ab103049, expressed in E. coli, with a His tag at the N-terminus. The specific sequence information is as follows: MGSSHHHHHHSSGLVPRGSHMREYKLVVLGSGGVGKSALTVQFVQGIFVEKYDPTIEDSYRKQVEVDAQQCMLEILDTAGTEQFTAMRDLYMKNGQGFALVYSITAQSTFNDLQDLREQILRVKDTDDVPMILVGNKCDLEDERVVGKEQGQNLARQWNNCAFLESSAKSKINVNEIFYDLVRQINRKTPVPGKARKKSSC. The antibody titer was detected by ELISA, and the results are as follows: Figure 8 The negative control is the pre-collected serum, recorded as N, the post-immune serum is recorded as P, and the 10 dilution of the post-immune serum is recorded as 10P. The horizontal axis is the logarithm of the antibody titer, and the vertical axis is the OD650 reading. Figure 8 The titer of the monoclonal antibody is greater than 500,000. The affinity of the antibody monoclonalized by hybridoma cells was determined by ELISA method. The results are as follows Figure 9 As shown, clone 1 has good affinity with the antigen, with a titer of 1.863*10 5 In subsequent experiments, the cells were expanded and cryopreserved, and the antibodies produced were designated as anti-RAP1B.

[0054] The expressed and purified anti-RAP1B antibody was identified and its molecular weight was detected by SDS-PAGE. Figure 10 As shown, the actual molecular weight is greater than 150 kDa, which is slightly higher than the theoretical molecular weight of mouse IgG. The difference is caused by glycosylation and is within a reasonable range.

[0055] The method for obtaining the complete sequence of Anti-RAP1B is the same as in Example 1, as shown in Table 3.

[0056] Table 3 Complete amino acid sequence of anti-RAP1B

[0057]

[0058]

[0059] Example 4 Preparation of colloidal gold particles

[0060] Soak the glass container used for the first time in a chloroform solution of 5% dichlorodimethylsilane for 1 minute, dry it at room temperature, rinse it with distilled water, and then dry it for use. Take 99mL of ddH2O in a swirling round-bottom flask, add 1mL of 1% HAuCl4 solution, and mix well. Place the swirling round-bottom flask in an electric heating jacket thermostat, add a clean stirring bar, stir at low speed and heat to boiling. Quickly add 1mL of 1% trisodium citrate solution at one time and continue to boil. Observe the color change of the solution from light yellow → black → purple → purple-red. When it completely turns purple-red, continue to swirl for 10 minutes, stop heating, and cool to room temperature. At this time, if Figure 11 As shown. The pH of the colloidal gold solution was adjusted to 9.0 with 1% K2CO3 solution, and the pH value was measured with precision pH test paper. The gold particle size was measured using a spectrophotometer, set to scan at 500-550nm, zeroed with ddH2O, and the gold solution was taken. The absorption value was scanned between 500 and 550nm and the absorption peak was recorded. The results showed the presence of two peaks, the first at 525nm and the second at 534nm, indicating that the colloidal gold particles prepared in the experiment had a particle size in the range of 40-70nm, which meets the requirements for labeling proteins.

[0061] Example 5 Colloidal gold labeled antibodies and prepared into a detection kit

[0062] The labeled anti-human CX3CL1, anti-human SELL, and anti-human RAP1B antibodies were dialyzed against 5 mM NaCl (pH 7.0) at 4°C overnight and centrifuged at 100,000 g for 1 hour at 4°C to remove aggregates. To this, 100 mL of colloidal gold solution was placed on a magnetic stirrer and 1 mg of antibody was added dropwise. The mixture was stirred for 30 minutes. A 10% BSA solution was added dropwise to a final concentration of 0.5% and stirred for 15 minutes. A 5% PEG-20000 solution was added dropwise to the mixture to a final concentration of 0.1% and stirred for 15 minutes. The mixture was centrifuged at 9,000 rpm for 1 hour at 4°C, the supernatant discarded, and the pellet suspended in 1 / 5 volume of DPBS. Using the DPBS dilution as a blank control, the absorbance of the gold-labeled antibodies was measured between 500 and 550 nm. The results are shown in Table 4. The results show that the peak absorbance fluctuations of the three colloidal gold-labeled antibodies were within 5 nm, indicating complete and uniform labeling.

[0063] Table 4 Determination of the absorbance of gold-labeled antibodies between 500 and 550 nm

[0064] λmax1 λmax2 Gold particles 525nm 534nm Gold Antibody-CX3CL1 524nm 531nm Gold Antibody-SELL 523nm 533nm Gold Antibody RAP1B 526nm 533nm

[0065] Fold 10 sheets of A4 paper and place them aside. Prepare 30 mL of a 2 mM borate + 1% sucrose buffer. Wearing gloves, immerse the material in the buffer until completely soaked. Soak for 10 minutes, then remove and dehydrate for 3 minutes. Lay the material flat on a mesh screen and place in a 37°C desiccator to a constant humidity of approximately 20%. Place the dried material in a sealed plastic bag for storage. Label and record the batch number. Discard the four edges of the material before use. Add sucrose to the labeled colloidal gold to a final concentration of 1%. At room temperature and a humidity of 20-40%, spray the solution onto the colloidal gold pad using the BIODOT instrument's AIRJET nozzle, setting the pressure to 10 PSI and the spray point to 2-6 μl / cm. Allow the sprayed colloidal gold strip to dry at 37°C until the humidity reaches a constant 20%. Then, store the strip in a sealed container. Cut the entire strip when using it, but be sure to discard the edges of the strip to prevent edge effects.

[0066] Remove the NC membrane and equilibrate it at room temperature and normal humidity for 1 hour. Dilute the antigen to 5 mg / ml with buffer solution, use the BIODOT instrument, set the spray volume to 0.5-1 ul / cm, and spot the sample. Oven dry at 37°C for 2 hours until the humidity is below 20%, then seal and package for use. Paste the non-spotting surface of the NC membrane to the PVC base plate, paste the colloidal gold pad to the bottom of the NC membrane, covering the NC membrane by 1 mm, and paste the absorbent pad to the top of the NC membrane, covering the NC membrane by 2 mm. Paste the sample pad to the bottom of the colloidal gold pad, covering the colloidal gold pad by 2 mm. Cut the pasted test plate into 4 mm wide strips using the BIODOT CM shearing machine, encase it and put it into an aluminum foil bag together with the desiccant, seal it and label it.

[0067] Peripheral blood was collected from patients with depression and healthy subjects. The samples were allowed to coagulate naturally at room temperature for 60 minutes without shaking or oscillating. The samples were then centrifuged at 3000 rpm for 10 minutes at 4°C. The supernatant was transferred to a new EP tube and dripped onto the prepared colloidal gold test card. The results were as follows: Figure 12 HC represents serum from healthy individuals, and MDD represents serum from patients with depression. The colloidal gold test kit using CX3CL1, SELL, and RAP1B monoclonal antibodies can accurately distinguish between samples from healthy individuals and patients with depression. This in vitro diagnostic method only requires a small amount of peripheral blood, enabling rapid and convenient in vitro diagnosis.

[0068] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. An antibody kit for detecting depression, characterized in that: The invention comprises the following components: anti-human CX3CL1 monoclonal antibody, anti-human L-selectin (SELL) monoclonal antibody and anti-human RAP1B monoclonal antibody; the above antibodies are labeled with colloidal gold, wherein the colloidal gold particle size is 40-70 nm; an immunochromatographic detection device, comprising a sample pad, a colloidal gold pad, an antigen-coated NC membrane and a water-absorbing pad attached in sequence; the NC membrane is immobilized with CX3CL1, SELL and RAP1B recombinant proteins as detection line antigens.

2. The kit according to claim 1, wherein The heavy chain CDR sequence of the anti-human CX3CL1 monoclonal antibody is SEQ No. 1-3, and the light chain CDR sequence is SEQ No. 4-6; the heavy chain CDR sequence of the anti-human SELL monoclonal antibody is SEQ No. 7-9, and the light chain CDR sequence is SEQ No. 10-12; the heavy chain CDR sequence of the anti-human RAP1B monoclonal antibody is SEQ No. 13-15, and the light chain CDR sequence is SEQ No. 16-18.

3. The kit according to claim 1, wherein The preparation method of the colloidal gold-labeled antibody includes: dialysis purification of the antibody in a 5 mM NaCl solution at pH 7.0; mixing 1 mg of antibody: 100 mL of colloidal gold solution and stirring for 30 minutes; sequentially adding 0.5% BSA and 0.1% PEG20000 to block the solution; and resuspending the precipitate in DPBS after centrifugation to obtain a gold-labeled antibody solution.

4. The kit according to claim 1, wherein In the immunochromatographic detection device, the colloidal gold pad is fixed with gold-labeled antibodies by a spraying process with spraying parameters of 2–6 μL / cm and a pressure of 10 PSI; the antigen coating volume on the NC membrane is 0.5–1 μL / cm, and the antigen concentration is 5 mg / mL.

5. Use of the kit according to any one of claims 1 to 4 in the preparation of an in vitro diagnostic product for depression, characterized in that: By detecting the expression levels of CX3CL1, SELL and RAP1B proteins in human peripheral blood, samples of depression patients and healthy subjects were distinguished.

Images