WO2017107540A1 - Antibody-array test kit for detecting proteins related to periodontal disease - Google Patents

Abstract

Provided is an antibody-array test kit for detecting proteins related to periodontal disease, comprising an antibody array; said antibody array uses as its solid-phase support a glass slide treated with a hydrophilic reagent containing an alkyl glycoside, and a specific antibody mixture of various periodontal disease-related proteins is spotted onto the solid-phase support under conditions of 22-24°C and 30-40% humidity. The test kit is capable of detecting a plurality of proteins related to periodontal disease and commonly used for clinical purposes, and has the advantages that it is sensitive, high-throughput, uses a small amount of specimen, and may be popularized and used on a large scale in conventional laboratories.

Description

Antibody chip kit for detecting periodontal disease related proteins Technical field

The present invention relates to the field of biodetection technology, and in particular to an antibody chip kit for detecting periodontal disease related proteins.

Background technique

Periodontal disease refers to diseases that occur in dental support tissues (periodontal tissue), including gingival disease involving only gingival tissue and periodontitis affecting deep periodontal tissues (periodontal, alveolar, cementum) Big class. Periodontal disease is a common oral disease, one of the main causes of tooth loss in adults, and a major oral disease that harms human teeth and general health. The early symptoms of periodontal disease are not easy to attract attention, resulting in long-term chronic infection of periodontal tissues, repeated attacks of inflammation, not only impairing the function of the oral chewing system, but also seriously affecting health.

Periodontal disease is a disease from simple gum inflammation to soft tissue and tooth bone tissue damage, which can lead to tooth loss in the worst case. Therefore, early diagnosis and prevention of periodontitis is essential. Current clinical tests including periodontal pocket probing, plaque and X-ray imaging diagnosis can be used as indicators of periodontal disease symptoms, but these tests do not indicate the development of the disease. Therefore, it is necessary to develop a new detection method to reflect the condition of the disease and effectively prevent and monitor the occurrence of the disease. Gingival sulcus (GCF) refers to a liquid that penetrates into the sulcus from the gingival connective tissue through the intraepithelial epithelium and the combined epithelium. People with healthy gums have a very small amount of gingival crevicular fluid, and the tissue fluid penetrates into the sulcus by the osmotic gradient in the tissue. Because there are usually inflammatory cells in the gingival crevicular fluid, the other chemical components are not exactly the same as the tissue fluid; the outflow of the gingival crevicular fluid is proportional to the degree of inflammation in the site. The liquid component of the gingival crevicular fluid is mainly derived from serum, and the other components are derived from serum, adjacent periodontal tissues and bacteria. Gingival crevicular fluid includes various electrolytes, proteins, glucose, enzymes, etc. It also contains white blood cells, exfoliated epithelial cells, and bacteria and other microorganisms. The gingival crevicular fluid contains a variety of enzymes, among which aspartate aminotransferase, alkaline phosphatase, collagenase and the like have a certain relationship with the severity and activity period of periodontal disease. Increased gingival crevicular fluid is one of the main manifestations of gingivitis in the early stage, often preceded by changes in clinical characterization. When the inflammation of the gums is obvious, the gingival crevicular fluid is significantly increased. The gingival crevicular fluid can be collected and detected by non-invasive means such as test strips, so the protein in the gingival crevicular fluid can be used as an ideal marker for disease detection. Periodontal disease-associated proteins include inflammatory cytokines (eg, IL-1β, IL-6, IL-8, IL-10, IL-12, IFNG, TNFa, and CRP), cytokines associated with bone metabolism (eg, OPG, OPN) , RANK, RANKL and) and enzymes (such as alkaline phosphatase and aspartate aminotransferase).

Common methods for detecting periodontal disease include measuring the depth of probing of the periodontal pocket, examining the X-rays of the teeth attached to the alveolar bone and referencing the bleeding. Each of the above methods has advantages and disadvantages, and in practical applications, selection is made according to respective experimental purposes and laboratory conditions. These methods rely heavily on the subjective identification of the dentist. The depth of probing is only opposite The measurement of the loss of attachment loss, which is of little help in the current development of periodontitis or its future development. Exploring bleeding can indicate a recovery process rather than a disruption process. Recently, many methods for assessing the activity of periodontal disease have also been developed. However, none of the above methods provides a sufficiently sensitive and specific assay to diagnose periodontitis and its mode of destruction. One result of the non-specific approach is that several patients who are not actually suffering from periodontitis will be treated. Clinical observations such as probing depth are not reliable enough because deep periodontal pockets do not necessarily contain ongoing inflammation, and radiographic evaluation must be combined with detailed clinical observations to give a correct diagnosis, only in the presence of pathogens in the periodontal pocket. Disease activity cannot be accurately reflected. Moreover, the diagnosis based on the enzymatic method for detecting host or bacterial-derived proteins developed to date is not specific enough because immunological detection is relatively simple, rapid, and reproducible, but the false positive rate is high, and only one correlation can be detected at a time. protein.

In view of this, it is necessary to develop a novel periodontal disease related protein detection kit to overcome the problems in the prior art, and achieve high throughput, high sensitivity, high specificity and low cost of various target proteins. Detection.

Summary of the invention

The object of the present invention is to overcome the deficiencies of the prior art and provide an antibody chip kit for detecting periodontal disease related proteins, which can simultaneously detect a plurality of periodontal disease related proteins, and overcomes the cumbersome operation of the prior art. It has the advantages of single detection index and low sensitivity. It has the advantages of low cost, convenience, sensitivity, accuracy, high throughput, low sample consumption, and can be promoted and scaled in ordinary laboratories. .

In order to achieve the above object, the present invention is achieved by the following technical solutions:

An antibody chip kit for detecting periodontal disease related proteins, comprising an antibody chip, a periodontal disease related protein standard mixture, a biotin-labeled periodontal disease-related protein detecting antibody mixture; and a fluorescein Cy3-labeled streptavidin The antibody chip is a solid phase carrier treated with a hydrophilic reagent containing an alkyl glycoside, and the specific antibody mixture of various periodontal disease related proteins is at 22 to 24 ° C, 30 to 40%. Spotted onto a solid support under humidity conditions.

Using a standard tissue slide without space limitation as a carrier, the number of cell adhesion factors detected on the same microarray is not limited by space. In addition, the slide has very low autofluorescence and the fluorescence signal is not diffusible. The fluorescent signals between different points do not interfere with each other, so multiple cell adhesion factors can be detected at the same time. However, treating the standard tissue slides in different ways will affect the background and detection sensitivity of the slides. The present invention finds that the slides treated with the hydrophilic reagent alkyl glycosides have a low background by comparing various methods for treating the slides. The detection sensitivity is high, and it is possible to detect not only a dozen cell adhesion factors at a time, but also the sensitivity of the detected cell adhesion factor can reach a single factor ELISA detection sensitivity.

Preferably, the periodontal disease-related protein detecting antibody mixture is C-reactive protein, interferon gamma, interleukin-1 alpha, interleukin-1 beta, interleukin-2, interleukin-4, interleukin- 6, interleukin-8, interleukin-10, interleukin-12, interleukin-17, macrophage inflammatory protein-1α, matrix metalloproteinase 9, A mixture of specific antibodies to matrix metalloproteinase 13, osteoprotegerin, osteopontin, osteoin, tumor necrosis-related protein, tumor necrosis factor beta 1, tumor necrosis factor alpha.

More preferably, the hydrophilic agent is an alkyl glycoside having a mass fraction of 0.01 to 0.2%, 0.01 to 0.1% of glycerin, and 0.01 to 0.05% of an ultrapure aqueous solution of polyethylene glycol 4000; a hydrophilic reagent-treated slide The method is to soak the slide in the hydrophilic reagent for 3 to 5 minutes and dry it.

Most preferably, the hydrophilic agent is an ultra-pure aqueous solution of 0.1% by mass of alkyl glycoside, 0.05% of glycerol, and 0.01% of polyethylene glycol 4000; and the method of treating the slide with a hydrophilic reagent is to slide the slide Soak in a hydrophilic reagent for 3 minutes and dry.

Preferably, the periodontal disease-related protein detecting antibody mixture is spotted onto a solid phase carrier at 22 ° C, 30% humidity.

Compared with the prior art, the present invention has the following beneficial effects:

Treating standard tissue slides in different ways will affect the background and detection sensitivity of the slides. The present invention finds that the slides treated with the hydrophilic reagent containing alkyl glycosides have a low background by comparing various methods for treating slides. The detection sensitivity is high, and it is possible to detect not only a dozen periodontal disease-related proteins at one time, but also the sensitivity of the detected periodontal disease-related proteins can reach the ELISA detection sensitivity of a single protein. The antibody chip kit of the invention overcomes the defects of cumbersome operation, single detection index and low sensitivity in the prior art, and has the advantages of low cost, convenience, sensitivity, accuracy, high throughput, small sample consumption, and can be promoted and scaled in an ordinary laboratory. And other advantages.

The antibody chip kit of the invention is especially suitable for C-reactive protein, interferon gamma, interleukin-1 alpha, interleukin-1 beta, interleukin-2, interleukin-4, interleukin-6, leukocyte mediator素-8, interleukin-10, interleukin-12, interleukin-17, macrophage inflammatory protein-1α, matrix metalloproteinase 9, matrix metalloproteinase 13, osteoprotegerin, osteopontin, bone The combination of active antibodies, tumor necrosis-related proteins, tumor necrosis factor β1, and tumor necrosis factor alpha, specific antibodies to these 20 periodontal disease-associated proteins. Because the sensitivity of specific antibodies against different periodontal disease-related proteins is different in the chip detection system of the present invention, if some low-sensitivity periodontal disease-associated protein antibodies are associated with some highly sensitive The combination of periodontal disease-associated protein antibodies results in the detection of low-sensitivity periodontal disease-associated proteins.

DRAWINGS

Figure 1 is a dot plot of the antibody chip.

Figure 2 is a standard curve of periodontal disease related proteins.

detailed description

The present invention will be further elaborated below in conjunction with the drawings and specific embodiments, which are only used in the embodiments. The invention is not intended to limit the scope of the invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents and the like used are, if not specified, commercially available reagents and materials.

Example 1

Screening of antibody chip carrier: Conventional antibody chips mostly use nitrocellulose membrane as carrier. Since nitrocellulose membrane is multi-layer structure, the washing of the chip is so difficult that the background of the chip is high and the result fluctuates greatly. At the same time, the nitrocellulose membrane is fragile and is not easy to operate on a large scale, and the use of large-scale clinical samples is not widespread. The low cost of traditional slides has brought a breakthrough in the widespread use of diagnostics and research. We screened slides that were not treated by an active method on the market, and the effect of the aldehyde-based and aminated slides was unstable. After a large number of screenings, the temperature and the active reagent components on the surface of the slide are the key to determining the spotting effect of the slide.

The following 5 sets of slides were used: Group 1 was an aminated slide, purchased from Corning, under the trade number UltraGAPS 40019; Group 2 was an aldehydeized slide: The first set of slides was immersed in glutaraldehyde for 40 minutes. Aldylated slides; Group 3 is APES slides: ordinary slides are added to acetone diluted APES for 0.5 to 1 minute, then washed with pure acetone to make APES slides; group 4 is polylysine Slide: The ordinary slide was immersed in PBS diluted polylysine for 0.5 to 1 hour, and then washed with pure water to make a polylysine slide; the fifth group was a hydrophilic reagent-treated slide. : The ordinary slide is soaked with a hydrophilic reagent for 3 to 5 minutes, and dried at room temperature; the hydrophilic reagent is an alkyl glycoside having a mass fraction of 0.01 to 0.2%, 0.01 to 0.1% of glycerin, 0.01 to 0.05%. An ultrapure aqueous solution of polyethylene glycol 4000. Preferably, the hydrophilic agent is an ultrafine aqueous solution of 0.1% by mass of alkyl glycoside, 0.05% of glycerol, and 0.01% of polyethylene glycol 4000; the method of treating the slide with hydrophilic reagent is to slide the slide Soak in a hydrophilic reagent for 3 minutes and dry.

The spotting effect of various slides is not the same. The effect of the slides added with the hydrophilic coating is more obvious, and the spotting effect is higher than that of the untreated ones. The slides which have been subjected to amination and hydrophilic treatment have higher effects than other types. At 22～24°C, the film/slide has a clear effect and the background value is low. At 27～30°C, no matter what kind of film/slide, the spotting effect appears, the temperature and humidity are more High, the more obvious the diffusion. The above five groups of slides were combined with antibodies and then added with secondary antibodies and substrates. The sensitivity screening results under different temperature and humidity conditions are shown in Table 1. The sensitivity index in Table 1 is the lowest detection line in ng/ml.

Table 1

Based on the above indicators, a slide treated with the above-mentioned hydrophilic agent containing an alkyl glycoside was finally selected as a solid phase carrier.

Example 2

An antibody chip kit for simultaneously quantitatively detecting a plurality of periodontal disease-related proteins: in order to detect the presence or absence of a corresponding receptor in a sample, a slide for immobilizing specific antibodies corresponding to 20 periodontal disease-related proteins is prepared, The specific antibodies corresponding to the 20 kinds of periodontal disease related proteins are common commercial products.

Preparation and storage of antibody chips: 100 to 1000 pl of a specific antibody-containing PBS buffer (containing 0.01 to 10 g/100 ml of bovine albumin) was spotted on a slide using a fully automatic spotting apparatus. The specific chip lattice uses biotinylated bovine IgG as a positive control. Twenty antibodies and two different concentrations of positive controls had four replicates in each array. In this embodiment, the chip array adopts the arrangement shown in FIG. 1 , but in fact, in other embodiments, the chip array may be combined in other arrangements, and is not limited to the one shown in FIG. 1 . form. There are 16 identical chip arrays on each slide. The spotted slides were allowed to stand at room temperature overnight, and then air-dried in a desiccator for 2 hours. The dried slides were fitted with a matching 16-hole frame to divide a slide into 16 cells that did not interfere with each other. The U-shaped frame clamps the 16-hole frame from both sides, and the silicone pad is pressed against the standard slide card so that the standard slide closes the bottom of the 16-hole frame, so that each small lattice on the 16-hole frame is formed. A small reaction well, the 16-hole frame edge is marked with a recessed number from 1 to 16 according to the position of the hole for easy identification. After closing the frame with an adhesive film, the entire chip was packaged in a gas-tight pouch and then stored at 2 ° C to 8 ° C for later use.

Preparation of periodontal disease related protein standards: Standards for preparing periodontal disease related proteins. Twenty kinds of periodontal disease-related proteins were diluted with phosphate buffer containing 0.1% calf albumin and mixed together in a certain amount. After packaging, they were dried by freeze-drying and stored at -80 °C. In the present example, the final use concentration of each periodontal disease-related protein used for the standard curve is shown in Table 2.

Table 2 Concentrations of periodontal disease-related protein standards used as standard curves after gradient dilution

(pg/ml)	Cntrl	Std7	Std6	Std5	Std4	Std3	Std2	Std1
CRP	0	55	165	494	1,481	4,444	13,333	40,000
IFNγ	0	14	41	123	370	1,111	3,333	10,000
IL-1α	0	3	8	25	74	222	667	2,000
IL-1β	0	3	8	25	74	222	667	2,000
IL-2	0	5	16	49	148	444	1,333	4,000
IL-4	0	3	8	25	74	222	667	2,000
IL-6	0	3	8	25	74	222	667	2,000
IL-8	0	1	2	5	15	44	133	400
IL-10	0	3	8	25	74	222	667	2,000
IL-12	0	3	8	25	74	222	667	2,000
IL-17	0	14	41	123	370	1,111	3,333	10,000
MIP-1α	0	14	41	123	370	1,111	3,333	10,000
MMP-9	0	14	41	123	370	1,111	3,333	10,000
MMP-13	0	27	82	247	741	2,222	6,667	20,000
OPG	0	27	82	247	741	2,222	6,667	20,000
OPN	0	137	412	1,235	3,704	11,111	33,333	100,000
Osteoactivin	0	14	41	123	370	1,111	3,333	10,000
RANK	0	137	412	1,235	3,704	11,111	33,333	100,000
TGFβ1	0	137	412	1,235	3,704	11,111	33,333	100,000
TNFα	0	3	8	25	74	222	667	2,000

Example 3

The experimental procedures for quantitative detection of various periodontal disease related proteins using the kit of the present invention are as follows:

1. Complete drying of the slide chip: remove the slide chip from the box, balance it at room temperature for 20-30 minutes, open the package, uncover the seal, and then place the chip in a vacuum dryer or dry at room temperature. 2 hours.

2. Gradient dilution of periodontal disease related proteins according to Table 2.

2.1. Add 500 μl of the sample dilution to the small tube of the standard mixture of periodontal disease-associated proteins and redissolve the standard. Before opening the small tube, centrifuge quickly, gently pump up and dissolve the powder, and mark the tube as Std1.

2.2. Mark 6 clean centrifuge tubes as Std2, Std3 to Std7, and add 200 μl of sample dilution to each vial.

2.3. Extract 100 μl of Std1 and add to Std2 and mix gently. Then extract 100 μl from Std2 and add to Std3, and then dilute to Std7.

2.4. Extract 100 μl of the sample dilution into another new centrifuge tube labeled CNTRL as a negative control.

Note: Because the initial concentration of each periodontal disease-associated protein is different, after a gradient dilution of Std1 to Std7, The serial concentration of each periodontal disease-related protein is different. In this example, the concentration of the gradient periodontal disease-related protein dilution is shown in Table 2.

3, chip operation process

3.1. Add 100 μl of sample dilution to each well and incubate for 30 minutes on a shaker at room temperature to block the quantitative antibody chip.

3.2. Remove the buffer from each well, add 100 μl of standard and sample to the wells and incubate overnight at 4 °C on a shaker.

Note: Different samples are not incubated in the same amount: plasma and serum are diluted 1:1 with sample dilution before use; cell supernatant can be used as stock solution; cell or tissue lysate is determined by protein concentration and added in an amount of 5-50 ug.

3.3. Washing: Draw the standard or sample in each well, wash 1× washing solution I 5 times, shake each time for 5 minutes at room temperature, 150μl of 1× lotion I per well, wash clean every time. For the solution, dilute 20x lotion I with deionized water. Drain 1 × lotion I in each well, add 1× lotion II and wash 2 times, shake each time for 5 min at room temperature, 150 μl of 1× lotion II per well, and wash the lotion every time. Dilute 20 x lotion II with deionized water.

3.4 Incubation of the test antibody mixture: Centrifuge the antibody mixture tubules, then add 1.4 ml of the sample dilution, mix well and centrifuge again quickly. 80 μl of the detection antibody was added to each well and incubated for 2 hours on a shaker at room temperature.

3.5 Washing: Remove the detection antibody from each well, wash 1× wash solution I 5 times, shake each time for 5 minutes at room temperature, 150 μl of 1× wash solution I per well, wash the wash solution every time, then Add 1× washing solution II and wash twice, each time 5 min shaking at room temperature, 150 μl of 1× lotion II per well, and wash the washing solution every time.

3.6 Incubation of Cy3-streptavidin: Centrifuge the Cy3-streptavidin tubule, then add 1.4 ml of the sample dilution, mix well and centrifuge again quickly. 80 μl of Cy3-streptavidin was added to each well, and the slides were wrapped in aluminum foil to protect from light and incubated for 1 hour on a shaker at room temperature.

3.7 Cleaning: Remove Cy3-streptavidin from each well, wash 1× wash solution I 5 times, shake each time for 5 min at room temperature, 150 μl of 1× lotion I per well, and wash each time. Clean lotion.

3.8 Fluorescence detection

1) Remove the slide frame, taking care not to touch the side of the slide to print the antibody.

2) Place the slide in the slide cleaning tube, add about 30ml of 1× lotion I, cover the whole slide, shake for 15min on the shaker at room temperature, discard 1× lotion I, add about 30ml of 1 × Wash II, shake on a shaker at room temperature for 5 min.

3) Remove the residual lotion from the slide. The slides were placed in a slide cleaning tube/drying tube, and the lid was not capped and centrifuged at 1000 rpm for 3 min.

4) Scan the signal with a laser scanner such as AxonGenePix using Cy3 or green channel (excitation frequency = 532 nm).

3.9 chip data extraction and analysis software for data analysis

1) Use the GenePix software to read the fluorescence value of the biochip.

2) The value selected after reading is the median reading of the background around the deduction point (F532Median-LocalBackground). A standard curve for each periodontal disease-associated protein using specific quantitative chip software is shown in Figure 2.

Example 4

Test system cross-reaction test.

The accuracy of antibody chip experiments is limited to a certain extent by the source, purity and specificity of the selected antigen or antibody, and the antigenic and immunogenicity of the protein and antibody organisms, heterologous antibodies Interference with rheumatoid factors and autoantibodies, high-volume screening of rare antibodies, and the need to screen for antibodies requires extensive experimental validation.

The cross-reaction test between antibody pairs was carried out according to the following method. Different chips were first incubated with a single antigen with a specific antigen concentration of 100 ng/ml. After washing, the corresponding antibody was reacted with each antigen. Finally, the cells were incubated with Cy3-streptavidin, and the chip was read after scanning. The experimental results of Table 3 can be obtained by taking the capture antibody of each antigen as the horizontal axis and the added antigen and the corresponding detection antibody as the vertical axis.

Table 3. Cross-response test results between antibody pairs

CAB\DAB	CRP	IFNγ	IL-10	IL-12p70	IL-17	IL-1α	IL-1β	IL-2	IL-4	IL-6	IL-8	MIP-1α	MMP-13	MMP-8	MMP-9	OPG	OPN	Osteoactivin	RANK	TGFβ1	TIMP-2	TNFα
CRP	1239	241	252	243	376	308	298	272	287	292	269	397	228	265	303	276	276	421	268	257	264	285
IFNγ	655	52743	614	643	937	648	688	732	676	744	707	811	640	653	666	657	610	704	657	758	655	700
IL-10	534	465	42389	471	666	468	518	499	536	556	517	562	390	475	484	467	436	473	500	523	443	453
IL-12p70	235	224	235	14078	242	223	226	224	220	226	218	227	221	244	260	225	249	227	231	251	186	226
IL-17	245	229	227	241	19239	237	242	263	242	260	265	293	226	251	249	244	226	248	253	276	231	242
IL-1α	239	248	240	244	283	15722	253	272	250	284	255	301	250	235	259	247	218	265	250	316	249	344
IL-1β	140	107	134	123	150	134	19474	138	133	127	135	119	117	129	159	125	132	144	132	116	113	131
IL-2	515	557	498	502	812	507	575	14332	540	648	574	702	516	542	508	557	445	548	548	619	550	543
IL-4	205	188	212	234	227	200	199	195	22889	203	193	202	191	226	236	196	200	189	206	238	160	183
IL-6	639	631	641	683	922	624	697	674	693	5661	626	738	582	721	689	699	636	729	688	764	622	662
IL-8	713	683	697	700	995	707	738	741	751	723	3162	791	654	706	723	747	679	751	758	787	651	681
MIP-1α	87	77	85	103	100	91	85	86	83	76	89	6898	85	81	100	89	83	89	77	75	73	79
MMP-13	twenty three	twenty two	29	twenty two	31	32	twenty four	30	20	20	twenty two	28	52426	twenty two	32	78	28	59	114	twenty three	28	141
MMP-8	83	80	89	84	110	96	94	94	98	91	95	121	98	2321	113	110	92	121	101	108	103	109
MMP-9	twenty two	31	20	16	twenty three	19	17	19	17	16	17	twenty one	28	25	37991	twenty two	26	25	14	38	38	twenty three
OPG	18	17	15	13	28	twenty one	twenty three	19	twenty three	twenty one	19	28	27	29	43	3995	28	36	twenty two	twenty three	33	18
OPN	27	twenty one	twenty three	twenty four	30	27	17	twenty one	twenty two	twenty one	20	twenty four	30	twenty two	48	38	10051	45	34	75	30	twenty four
Osteoactivin	16	14	19	16	twenty one	14	16	9	14	12	10	twenty one	61	15	35	14	15	9896	15	twenty three	16	12
RANK	44	46	46	48	52	49	48	53	45	47	43	55	44	55	48	76	52	62	3135	60	45	57
TGFβ1	427	367	433	460	483	432	462	492	488	488	470	561	368	433	493	446	414	477	469	13449	432	484
TIMP-2	28	29	37	26	38	27	28	twenty four	twenty two	28	twenty four	28	39	174	957	33	28	twenty four	39	36	42709	twenty four
TNFα	460	535	471	449	555	480	504	507	518	536	503	599	447	517	496	498	464	514	495	513	498	20924

The experimental results indicate that each antibody pair can specifically recognize its own detection antigen without cross-reacting with other antigens.

Example 5

Media recovery rate of the experimental system.

The suitability of the quantitative antibody chip in different sample media is shown by measuring the media recovery. Different concentrations of each periodontal disease-associated protein were added to 2-fold diluted normal human serum and 2-fold diluted cell supernatant (CM), and the quantitative antibody chip was used to detect the medium recovery rate of the sample = (intervention Periodontal disease-associated protein concentration of the sample - periodontal disease-associated protein concentration of the control sample) / theoretically involved periodontal disease-associated protein concentration. Experiments have shown that the kit of the invention has a recovery rate of 31 to 126% in human serum and cell supernatant.

Table 4. Recovery of Quantitative Antibody Chips in Different Media

(pg/ml)	Spiking	CM	CM+Ag	CM%	Serum	Serum+Ag	Serum%
CRP	20,000	0	14,411	72%	7,444	20,945	68%
IFNγ	5,000	391	4,425	81%	27	3,607	72%
IL-1α	1,000	twenty one	746	72%	10	654	64%
IL-1β	1,000	0	631	63%	0	513	51%
IL-2	2,000	432	2,536	105%	49	2,152	105%
1L-4	1,000	14	1,244	123%	10	1,361	135%
IL-6	1,000	1,726	2,436	71%	27	1,023	100%
IL-8	1,000	159	1,156	100%	6	956	95%
IL-10	1,000	9	726	72%	1	590	59%
IL-12	1,000	2	1,198	120%	2	857	85%
IL-17	5,000	0	3,461	69%	2	3,255	65%
MIP-1α	5,000	110	7,134	140%	0	3,934	79%
MMP-9	5,000	15	2,587	51%	3,368	4,067	14%
MMP-13	10,000	129	9,919	98%	6	6,128	61%
OPG	10,000	34,602	39,009	44%	twenty three	12,799	128%
OPN	50,000	958	47,043	92%	203	28,861	57%
Osteoactivin	5,000	89	4,993	98%	2,493	4,538	41%
RANK	25,000	68	19,119	76%	twenty one	17,503	70%
TGFβ1	50,000	313	54,434	108%	0	60,182	120%
TNFα	1,000	42	765	72%	16	1,301	128%

In summary, the present invention discloses an antibody chip kit for simultaneously quantitatively detecting a plurality of periodontal disease related proteins. On the one hand, the kit overcomes the shortcomings of the conventional ELISA in the detection of receptors, such as single, time-consuming, labor-intensive, consumables, etc., and overcomes the weaknesses of low-throughput and poor repeatability in the existing multi-protein detection technology. The system uses standard tissue slides as a surface carrier to perform multiple sandwich ELISA reactions on the surface of the slide. At the same time, the specifications of the antibody chip conform to the size of the standard 96-well microtiter plate, making high-throughput sample manipulation possible. In addition, we have also confirmed that the concentration of the receptor detected by the invention can achieve the detection sensitivity and accuracy of a single ELISA. Finally, by measuring the medium recovery rate of the chip in different samples, it is confirmed that the chip kit of the invention can be applied to different biological samples such as serum and cell supernatant.

Claims (5)

1. An antibody chip kit for detecting various periodontal disease related proteins, comprising an antibody chip, a periodontal disease related protein standard mixture, a biotin-labeled periodontal disease-related protein detecting antibody mixture; and a fluorescein Cy3-labeled streptavidin Avidin; characterized in that the antibody chip is a solid phase carrier treated with a hydrophilic reagent containing an alkyl glycoside, and a specific antibody mixture of various periodontal disease-related proteins is at 22 to 24 ° C, 30 Spot to the solid support under ~40% humidity conditions.
2. The kit according to claim 1, wherein the periodontal disease-related protein detecting antibody mixture is C-reactive protein, interferon gamma, interleukin-1 alpha, interleukin-1 beta, and interleukin-2. , interleukin-4, interleukin-6, interleukin-8, interleukin-10, interleukin-12, interleukin-17, macrophage inflammatory protein-1 alpha, matrix metalloproteinase 9 , a mixture of specific antibodies such as matrix metalloproteinase 13, osteoprotegerin, osteopontin, osteoin, tumor necrosis-related protein, tumor necrosis factor β1, and tumor necrosis factor alpha.
3. The kit according to claim 1, wherein the hydrophilic agent is an alkyl glycoside having a mass fraction of 0.01 to 0.2%, 0.01 to 0.1% of glycerin, and 0.01 to 0.05% of polyethylene glycol 4000. Ultra-pure aqueous solution; hydrophilic reagent treatment of the slide is to soak the slide in the hydrophilic reagent for 3 to 5 minutes, and dry it.
4. The kit according to claim 1, wherein the periodontal disease-related protein detecting antibody mixture is spotted onto a solid phase carrier at 22 ° C under a humidity of 30%.
5. The kit according to claim 3, wherein the hydrophilic agent is an alkyl glycoside having a mass fraction of 0.1%, 0.05% glycerol, and 0.01% of an ultrapure aqueous solution of polyethylene glycol 4000; The reagent is used to treat the slide by soaking the slide in a hydrophilic reagent for 3 minutes and drying it.

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