A method and kit for improving protein chip detection sensitivity

Abstract

The present invention discloses a method and a kit for improving the detection sensitivity of a protein chip, wherein the method uses a hybridization chain reaction to amplify the signal of the protein chip enzymatic color development, thereby greatly improving the detection sensitivity of a common protein chip kit. The present invention only requires a very small amount of sample to achieve simultaneous rapid detection of multiple indicators, further reducing the detection cost.

Description

Technical Field

[0001] The present invention relates to the field of protein chip detection, and in particular to a method and a kit for significantly improving the detection sensitivity of a protein chip. Background Art

[0002] Protein chip technology is to fix various proteins (such as antigens, antibodies), polypeptides, receptors, ligands, etc. in an orderly manner on a carrier (such as filter membrane, gel, glass slide, nano-beads and microplate) to analyze the components in the sample to be tested that can specifically interact with it. It has many advantages in the fields of application such as food inspection, disease diagnosis, drug screening, agriculture, forestry, animal husbandry, and judicial identification. Patent CN201710613324 discloses a protein chip and a kit for the diagnosis of active tuberculosis. The protein chip contains specific antibodies for 29 proteins, which can specifically screen the patient population, and has the characteristics of high sensitivity and specificity, high throughput, small sample dosage, and easy promotion and scale-up in ordinary laboratories. Although protein chips have so many advantages, their sensitivity is still lower than that of chemiluminescence. For some target proteins with low natural content, they still cannot be accurately detected. This disadvantage greatly limits the application of protein chips. Therefore, the technical problem to be solved by the present invention is to provide a protein chip detection kit that can greatly improve the sensitivity of protein chip detection targets. Summary of the invention

[0003] The present invention provides a protein detection kit, comprising an oligonucleotide SEQ NO.1 for modifying a hybridization chain reaction initiator chain sequence of a labeled antibody, an H1 chain with a sequence of SEQ NO.2, and an H2 chain with a sequence of SEQ NO.3.

[0004] The hybridization chain reaction initiator chain is complementary to a portion of the H1 sequence and identical to a portion of the H2 sequence; and another portion of the H1 sequence is complementary to another portion of the H2 sequence.

[0005] The labeled antibody is an antibody or antiantibody that reacts with the antigen to be detected.

[0006] The 5' end of the H1 chain is labeled, and the 3' end of the H2 chain is labeled. The label may be biotin, different fluorescent substances, or the like.

[0007] The present invention also provides a protein detection method, which uses the above-mentioned protein detection kit to perform protein detection.

[0008] The present invention further provides a method for modifying oligonucleotides on labeled antibodies that can trigger hybridization chain reactions, wherein the labeled antibodies are antibodies or anti-antibodies that react with the antigen to be detected, and the steps are as follows:

[0009] Step 1. Add the labeled antibody to DBCO-NHS and react at room temperature for more than 30 minutes;

[0010] Step 2, adding the product of step 1 to the azide-modified priming chain sequence oligonucleotide SEQNO.1, and reacting at room temperature for more than 30 minutes. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 Shown is a real picture of the high-sensitivity hybridization immunochromatographic tube.

[0012] Figure 2 Shown is a schematic diagram of the signal amplification principle.

[0013] Figure 3 Shown are the results of the high-sensitivity protein chip kit for detecting ultra-low concentration antigens.

[0014] Figure 4 Shown are a common protein chip kit and a high-sensitivity protein chip reagent. DETAILED DESCRIPTION

[0015] 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.

[0016] Example 1 A method for improving the detection sensitivity of a protein chip

[0017] The method for improving the detection sensitivity of protein chip comprises the following steps:

[0018] Step 1 Preparation of protein chip

[0019] S1. Dilute the capture antibody that can react with the antigen to be detected to 0.25 mg / ml for protein chip spotting.

[0020] S2. Use a biochip spotter (model BD-1, Shanghai Biochip Technology Co., Ltd.) to spot the diluted capture antibody on an aldehyde substrate, which is the protein chip of the present invention.

[0021] S3. After the sample is spotted, the protein chip is dried at room temperature overnight (12-16 hours) and can be used for subsequent detection reactions.

[0022] Step 2 Preparation of labeled antibodies

[0023] The labeled antibody is an antibody or antiantibody that can react with the antigen to be detected.

[0024] The labeled antibody was diluted to 0.1-1 mg / ml, 100 μl was added with 0.1-10 μl DBCO-NHS (10 μM / L), and the reaction was carried out at 4-25°C for 30 min-12 h. The solution was desalted twice with a 10 kd ultrafiltration tube, and the solution was replaced with PBS buffer. 0.1-10 μl of azide-modified oligonucleotide containing the primer chain sequence (10 μM / L) was added, and the reaction was carried out at 4-25°C for 30 min-12 h.

[0025] The oligonucleotide sequence is SEQ NO.1.

[0026] Step 3 Preparation of immunochromogenic strip tubes

[0027] Take the 8-piece strip tube that can be used with the fully automatic hybridization instrument (model BR-526-24, Shanghai Bio-Tech Co., Ltd.), which is a high-sensitivity hybridization immunochromogenic tube. Figure 1 , add reagents according to the table below:

[0028] Table 1 Reagent table for 8-tube strips

[0029]

[0030] The 5' end of the H1 chain carries a marker, and the sequence of the H1 chain is SEQ NO.2, one part of which is complementary to the sequence of the priming chain, and the other part is complementary to the sequence of the H2 chain; the 3' end of the H2 chain carries a marker, and the sequence of the H2 chain is SEQ NO.3, one part of which is complementary to the sequence of the H1 chain, and the other part is identical to the sequence of the priming chain.

[0031] The markers carried by the H1 chain and the H2 chain may be biotin, different fluorescent substances, etc.

[0032] Step 4 Protein chip detection method

[0033] Take 250 μl of the liquid sample to be tested or 250 μl of the sample diluted with the sample diluent and add it to the 5th well of the high-sensitivity hybridization immunochromatographic tube.

[0034] The high-sensitivity hybridization immunochromatographic tube was placed on the reagent tray in the fully automatic hybridization instrument (model BR-526-24, Shanghai Bio-Tech Co., Ltd.), the protein chip prepared above was placed in the chip card slot of the fully automatic hybridization instrument and the reaction chamber cover was closed.

[0035] The reaction program was set up in the automatic hybridization instrument as follows:

[0036] Step 1: 200 μl well 8, 25 degrees, 60 minutes;

[0037] Step 2: 200 μl well 7, 25 degrees, 2 minutes;

[0038] Step 3: 200 μl well 5, 25 degrees, 30 minutes;

[0039] Step 4: 200 μl well 7, 25 degrees, 2 minutes;

[0040] Step 5: 200 μl well 2, 25 degrees, 30 minutes;

[0041] Step 6: 200 μl well 7, 25 degrees, 2 minutes;

[0042] Step 7: 200 μl well 6, 25 degrees, 60 minutes;

[0043] Step 8: 200 μl well 7, 25 degrees, 2 minutes;

[0044] Step 9: 200 μl well 3, 25 degrees, 30 minutes;

[0045] Step 10: 200 μl well 7, 25 degrees, 2 minutes;

[0046] Step 11: 200 μl well 4, 25 degrees, 30 minutes;

[0047] Step 12: 200 μl well 7, 25 degrees, 2 minutes.

[0048] After the reaction, the protein chip was taken out and placed in a biochip reader (model BE-3.0, Shanghai Bio-Tech Co., Ltd.) for result reading and scanning. If the protein chip identification column signal value is greater than 100 and the target signal value is greater than 20, it can be judged as positive, and less than 20 as negative. If the identification column signal value is less than 100, retest.

[0049] Example 2 A protein chip kit for detecting IL-6 and PCT

[0050] The kit contains: protein chip for detecting IL-6 and PCT, 8-link reagent strips and instructions for use.

[0051] Preparation of protein chip: 0.25 mg / ml PCT capture antibody (Chongqing Tansheng, clone number: 10D6) and IL-6 capture antibody (Chongqing Tansheng, clone number: 2F7) were added according to step 1 of Example 1 to complete the spotting of protein chip. Each spot was repeated 3 times, for a total of 2 rows and 9 columns (see Figure 2 ).

[0052] Preparation of 8-strip reagent tubes: Take the 8-strip consumable tubes (Shanghai Bio-Tech Co., Ltd.) of the fully automatic hybridization instrument (model BR-526-24, Shanghai Bio-Tech Co., Ltd.), and pre-fill each well with the following table:

[0053] Table 2 Reagent table for 8-tube strips

[0054]

[0055] Preparation method of IL-6 and PCT labeled antibodies:

[0056] PCT labeled antibody (Chongqing Tansheng, clone 4A1) and IL-6 labeled antibody (Chongqing Tansheng, clone TE5) were diluted with NaHCO3 solution (0.1Mol / L pH=8.3) to a final concentration of 0.5mg / ml, 100μl of each was added with 1μl DBCO-NHS (10uM / L), reacted at 25℃ for 90min, desalted twice with a 10kd ultrafiltration tube, replaced with PBS buffer, added with 1μl azide-modified oligonucleotide containing the initiator chain sequence (SEQ NO.1) (10uM / L), reacted at 25℃ for 30min, replaced with PBS by ultrafiltration, and finally 100μl of product was obtained.

[0057] Highly sensitive detection of IL-6 and PCT protein chips:

[0058] Take 250 μl of 0.0000125 mg / ml PCT antigen, IL6 antigen and 1×PBS, respectively, and add them into the fifth well of three 8-link reagent strip tubes prepared in this example.

[0059] For the following steps, please refer to step 4 of embodiment 1.

[0060] After the reaction is completed, the protein chip is taken out. The protein chip after the hybridization reaction is placed in a biochip reader (model BE-3.0, Shanghai Bio-Tech Co., Ltd.) for result reading and scanning. From the results, it can be seen that the signal values ​​of the identification column and the target site meet the requirements, and the judgment results are accurate. The signal value of the PBS negative control sample is zero. It shows that the PCT+IL6 high-sensitivity protein chip kit prepared according to the scheme of the present invention can detect PCT and IL-6 antigens as low as 0.0000125 mg / ml in the sample, please see Figure 3 .

[0061] Example 3 Sensitivity comparison experiment:

[0062] S01. Preparation of control 8-link reagent strip tube: This experiment refers to Example 2, the main difference is that the components of wells 2 and 6 of the 8-link reagent strip tube are different. Well 2 of this example contains 250 μl of biotin-modified PCT labeled antibody and biotin-modified IL-6 labeled antibody with a final concentration of 0.0025 mg / ml, instead of PCT and IL-6 labeled antibodies modified with primer sequence oligonucleotides in Example 2. Well 6 of this example contains 250 μl of 1×PBS buffer.

[0063] S02. Comparative experiment: Take 250μl of 0.0000125mg / ml PCT antigen, IL6 antigen and 1×PBS, and add them to the 5th hole of the three control 8-way reagent strips prepared in S01 and the three 8-way reagent strips prepared in Example 2. Place the 8-way reagent strips of this example and Example 2 on the reagent tray of the fully automatic molecular hybridization instrument (model BR-526-24, Shanghai Bioao Technology Co., Ltd.), put the protein chip prepared in Example 2 into the chip card slot and close the reaction chamber pressure cover. Start the reaction program of Example 2. Place the protein chip after the hybridization reaction in a biochip reader (model BE-3.0, Shanghai Bioao Technology Co., Ltd.) for result reading and scanning. For experimental results, please see Figure 4 The protein chip prepared according to the scheme of the present invention (Example 2) can detect PCT and IL-6 antigens as low as 0.0000125 mg / ml in the sample, while the ordinary protein chip (Example 3, without trigger chain, H1) cannot detect the same concentration of PCT and IL-6 antigens in the sample.

[0064] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A protein detection kit, It is characterized in that Include Oligonucleotide SEQ NO.1 for modifying the hybridization chain reaction initiator chain sequence of the labeled antibody; The H1 chain has a sequence of SEQ NO.2, and The sequence is the H2 chain of SEQ NO.

3.

2. The kit according to claim 1, It is characterized in that The SEQ NO.1 is complementary to a part of SEQ NO.2 and is identical to a part of SEQ NO.3; another part of SEQ NO.2 is complementary to another part of SEQ NO.

3.

3. The kit according to claim 1, It is characterized in that The labeled antibody is an antibody or anti-antibody that reacts with the antigen to be detected.

4. The kit according to claim 1, It is characterized in that The 5' end of SEQ NO.2 and the 3' end of SEQ NO.3 are respectively marked with markers.

5. The kit according to claim 4, It is characterized in that The label is selected from biotin and different fluorescent substances.

6. A protein detection method, It is characterized in that Use the protein detection kit according to claim 1.

7. A method for modifying a hybridization chain reaction oligonucleotide on a labeled antibody, wherein the labeled antibody is an antibody or anti-antibody that reacts with an antigen to be detected, It is characterized in that Step 1. Add the labeled antibody to DBCO-NHS and react at room temperature for more than 30 minutes; Step 2, add the product of step 1 to SEQ NO.1 modified with azide, and react at room temperature for more than 30 minutes.

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