CN105136915B - Immunoaffinity method - Google Patents

Abstract

The invention discloses an immunoaffinity method. In the present invention, according to the specific target object of the analysis object, the corresponding antibody matrix is filled in the immunoaffinity column. Secondly, the antibody active solution is poured to maintain the antibody activity. The highest liquid level of the active liquid is lower than the highest liquid level scale line of the immunoaffinity column, and the upper and lower plugs are tightened, and placed in a suitable ambient temperature for use. Then, according to the concentration range of the target, select a single immunoaffinity column or a combination of multiple immunoaffinity columns to determine the number of immunoaffinity columns. Finally, the eluent of the target substance is added, and the eluate flowing out from the immunoaffinity column is collected and analyzed. The immunoaffinity column adopts telescopic helical combination immunoaffinity column. The invention increases the volume of the active liquid, enhances the overall sealing performance of the immunoaffinity, and at the same time ensures sufficient antibody active liquid, prevents overflow, liquid leakage and the like from occurring.

Description

an immunoaffinity method

technical field

The present invention relates to an immunoaffinity method.

Background technique

As a sample pretreatment technology, solid phase extraction columns have been widely used in laboratories. With the continuous development of solid phase extraction technology, a new type of extraction column - immunoaffinity column is quietly replacing the traditional solid phase extraction column. Immunoaffinity column (IAC) is based on the antigen-antibody reaction, a separation and extraction column made of biomacromolecules with the characteristics of specific recognition and reversible binding to a class of biomacromolecules. It has unique selection specificity and good adsorption Purification performance. When the treated sample extract slowly passes through the immunoaffinity column, the analyte in the sample extract binds to the antibody in the column, while other impurities that are not bound are removed after washing, and then the column is washed with the eluent. Collect analytes. Compared with traditional solid-phase extraction columns, immunoaffinity columns have stronger selectivity for target objects, better adsorption and purification effects, and consume less reagents.

The following problems exist when using the above-mentioned immunoaffinity column for immunoaffinity:

1. Immunoaffinity Since the antibody matrix is used instead of the traditional solid-phase packing, in order to maintain the activity of the antibody in the column, the antibody needs to be placed in the active solution. However, the current immunoaffinity column is usually small in size and cannot have enough volume to store the active liquid, and the active liquid often overflows and leaks.

2. The number of antibodies in a single immunoaffinity column is fixed, which also determines that a single immunoaffinity column can accommodate a limited target concentration. When the target exceeds the antigen limit that the antibody in the column can bind, the target will be lost, and the concentration of the target cannot be analyzed effectively.

3. In the process of using the immunoaffinity column, the turbidity of the analytical sample solution often occurs, which leads to the clogging of the affinity column, which affects the antibody-antigen binding ability of the affinity column, and even subsequent steps such as rinsing and elution cannot be carried out normally.

The above factors limit the popularization and commercialization of immunoaffinity columns, and a simple and effective device is needed to solve these problems.

Contents of the invention

The invention provides an immunoaffinity method aiming at the deficiencies of the prior art.

The technical scheme that the present invention solves technical problem to take is:

The inventive method is specifically:

First, according to the specific target of the analysis object, fill the corresponding antibody matrix in the immunoaffinity column, specifically: first put the lower sieve plate in the empty column cavity, then fill in the antibody matrix, and then put the upper sieve plate, Press down on the upper sieve plate to make the antibody matrix evenly distributed and play a role in fixing the antibody filling area.

Secondly, the antibody active solution is poured to maintain the antibody activity. The highest liquid level of the active liquid is lower than the highest liquid level scale line of the immunoaffinity column, and the upper and lower plugs are tightened, and placed in a suitable ambient temperature for use.

Then, according to the concentration range of the target, select a single immunoaffinity column or a combination of multiple immunoaffinity columns to determine the number of immunoaffinity columns; specifically: take out the immunoaffinity column to be used, and remove the upper and lower plugs , fix the immunoaffinity column at a suitable position; after the active solution has flowed out completely, add the sample solution to be analyzed according to the experimental needs; after confirming that the analysis sample solution has flowed out completely, add an appropriate amount of eluent, squeeze it dry and discard it.

Finally, the eluent of the target substance is added, and the eluate flowing out from the immunoaffinity column is collected and analyzed.

The immunoaffinity column adopts a telescopic helical combined immunoaffinity column, and the telescopic helical combined immunoaffinity column includes an upper plug, a lower plug and a sample loading tube.

The upper plug has external threads in the circumferential direction, and the upper plug is matched with the inlet thread of the sample loading tube.

The lower plug has an internal thread in the circumferential direction, and the lower plug is matched with the outlet thread of the sample loading tube.

The sample loading tube includes an inlet, a column cavity and an outlet. The inlet has internal threads in the circumferential direction, which is used to combine and fix the outlet of the upper plug or to fix another immunoaffinity column in combination; the outlet has external threads in the circumferential direction for Combine to fix the lower plug or combine to fix the inlet of another immunoaffinity column.

The column cavity includes a telescopic area and an immunoaffinity area; the telescopic area is set below the inlet, the immunoaffinity area is located above the outlet, and an upper sieve plate, an antibody filling area and a lower sieve plate are arranged in the immunoaffinity area from top to bottom ; The highest liquid level scale line is marked on the outer wall of the column chamber, and the highest liquid level scale line is located below the expansion zone.

Furthermore, if the analysis sample is turbid, tighten the upper plug to squeeze the expansion and contraction zone.

The beneficial effects of the present invention are:

1. Due to the large capacity of the column cavity and the upper and lower plugs screwed on the inlet and outlet, the telescopic spiral combined immunoaffinity column increases the volume of the active liquid and enhances the overall sealing of the immunoaffinity. At the same time, the antibody active liquid also achieves anti-overflow, leakage and other situations.

2. By adopting multiple telescopic helical combined immunoaffinity columns, the number of antibodies in the immunoaffinity process can be multiplied, which fundamentally solves the problem of limited concentration of the target substance that can be accommodated in the immunoaffinity method.

3. The telescopic spiral combined immunoaffinity column has a stretching area. During the use of immunoaffinity, the upper plug can be tightened to the inlet, and the pressure in the column cavity can be increased by squeezing the side or compressing the stretching area downward. The purpose of increasing the flow rate of the column cavity solution and fully removing the residual liquid can be achieved, and at the same time, the blockage of the column cavity can also be prevented.

Description of drawings

Fig. 1 is the structural schematic diagram of telescopic helical combined immunoaffinity column in the present invention;

Fig. 2 is a schematic diagram of using a single immunoaffinity column in the present invention;

Fig. 3 is a schematic diagram of multiple immunoaffinity columns used in the present invention.

detailed description

The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

As shown in FIG. 1 , the retractable spiral combined immunoaffinity column mainly includes the following parts: an upper plug 1 , a lower plug 2 , and a sample loading tube 3 . The upper plug 1 is 1.5-1.8 cm high, with outwardly turned threads, with an outer diameter of 0.8-0.9 cm, screwed to the inlet of the sample tube; the lower plug 2 is 1.6-2.0 cm high, with an inner thread, with an inner diameter of 0.4-0.5 cm, screwed on the outlet of the sample tube. The sample loading tube 3 is 9.0-10.0 cm high, and its outer wall is made of polyethylene, including an inlet 4, a column cavity 5 and an outlet 6. The inlet 4 is 0.9-1.0 cm high, has an internal thread, an inner diameter of 0.9-1.0 cm, and an outer diameter of 1.0-1.1 cm, and is used to fix the upper plug 1 or the outlet 6 of another immunoaffinity column. The outlet 6 is 0.9-1.0 cm high, has an outwardly turned thread, an inner diameter of 0.1-0.2 cm, and an outer diameter of 0.3-0.4 cm, and is used to fix the lower plug 2 or the inlet 4 of another immunoaffinity column. The height of the column chamber 5 is 7.0-8.0 cm, the inner diameter is 1.4-1.5 cm, and the outer diameter is 1.5-1.6 cm. It can accommodate an analysis volume of 6-7 mL. The column chamber 5 includes the expansion zone 7, the immunoaffinity zone 8 and the highest liquid level scale Line 9. The telescoping zone 7 is set at a distance of 0.7-0.8 cm from the entrance 4, the unstretched height is 1.1-1.2 cm, and the thickness is thinner, 0.1-0.2 cm. The immunoaffinity area 8 is located above the outlet 6, with a height of 1.3-1.4 cm, and an upper sieve plate 10, an antibody filling area 11 and a lower sieve plate 12 are arranged from top to bottom. The highest liquid level scale line 9 is located 0.9-1.0 cm below the flexible zone 7 on the outer wall of the column cavity.

Using the above immunoaffinity column, the method of immunoaffinity:

According to the specific target of the analysis object, fill the corresponding antibody matrix, specifically: first put the lower sieve plate in the empty column cavity, then fill in the antibody matrix, then put the upper sieve plate, press down the upper sieve plate to make the antibody The matrix is evenly distributed and acts to immobilize the antibody-filled area.

Then pour in the antibody active solution to maintain the antibody activity. The highest liquid level of the active liquid should be lower than the highest liquid level scale line of the immunoaffinity column, tighten the upper and lower plugs, and place it at a suitable ambient temperature for use. According to the concentration range of the target, select a single immunoaffinity column or use multiple combinations to determine the number of immunoaffinity columns. Take out the immunoaffinity column to be used, remove the upper and lower plugs, and fix the immunoaffinity column in a suitable position (if multiple immunoaffinity columns are used in combination, the combination of immunoaffinity columns is preferred). After the active liquid has completely flowed out, add an appropriate volume of sample liquid to be analyzed according to the needs of the experiment (if the sample to be analyzed is turbid, tighten the upper plug and squeeze the expansion zone). After confirming that the analytical sample solution has completely flowed out, add an appropriate amount of eluent, squeeze it dry and discard it. Finally, an appropriate target eluent is added, and the eluate from the immunoaffinity column is collected and analyzed.

Example: Application of immunoaffinity purification of tetrodotoxin in aquatic products.

Fill the tetrodotoxin antibody into the column cavity with a height of 1.2-1.3 cm, cover the sieve plate, inject the active solution, tighten the upper and lower plugs, and store at 0-5°C until use.

Accurately weigh 2.00 g of the fully homogenized horse-faced fish sample into a 50 mL stoppered centrifuge tube, add 10 mL of methanol solution containing 1% acetic acid, vortex for 2 min, ultrasonically extract in a 60°C water bath for 15 min, and cool to room temperature Afterwards, centrifuge at 6000 r/min for 5 min. Pipette 5 mL of the supernatant into another 50 mL centrifuge tube, add 20 mL of PBS solution for dilution, adjust the pH to 7~8 with 1 mol/L sodium hydroxide solution, and wait for purification.

Take out the immunoaffinity column, remove the upper and lower plugs, release the active solution stored in the column, and load the sample solution. After sample loading, rinse the affinity column with 8 mL of water, squeeze out the residual liquid in the column, discard all the above effluent, and finally elute with 4 mL of methanol solution containing 1% acetic acid. Blow dry at 60°C with nitrogen, dissolve with 1 mL of mobile phase at constant volume, pass through a 0.22 μm filter membrane for liquid chromatography-mass spectrometry analysis. According to the above steps, parallel determination was performed 6 times, and the results are shown in Table 1. At the spiked level of 1.50 µg/kg, the average recovery of six determinations was 96.7% with a relative standard deviation of 3.24%. The entire immunoaffinity purification process took 30 min.

Table 1 Results of determination of tetrodotoxin residues in aquatic products by immunoaffinity purification ( n =6)

Experimental results show that the method of the present invention can effectively adsorb the target substance, thereby achieving the effect of purification. At the same time, the whole purification process is short in time, simple in operation, and has high practical application value.

The above disclosures are only preferred embodiments of the present invention, and of course cannot be used to limit the scope of rights of the present invention. Therefore, equivalent changes made according to the requirements of the present invention still fall within the scope of the present invention.

Claims (2)

1. a kind of affine in immunity method, it is characterised in that the method is specifically：

First according to the objectives thing of analysis object, corresponding antibody matrix is filled in immune affinity column, specifically：It is first First lower sieve plate is put in void column chamber, then inserts antibody matrix, place into upper sieve plate, pushing upper sieve plate is distributed antibody matrix Uniformly, and sessile antibody fill area is played a part of；

Next pours into antibody activity liquid and keeps the highest liquid level of antibody activity, active liquid to carve less than the highest liquid level of immune affinity column Degree line, tightens lower end cap, is placed in suitable environment temperature to be used；

Then according to the concentration range of object, single immune affinity column or many immune affinity columns are selected to be applied in combination, really Determine the radical of immune affinity column；The immune affinity column that will be used takes out, and removes upper lower end cap, and fixed immune affinity column is in suitable Position；After active liquid flows out entirely, needed to add sample to be analysed liquid according to experiment；After confirmation analysis sample liquid flows out entirely, plus Enter appropriate leacheate, and extracted and discard；

Object eluent is eventually adding, the eluent that immune affinity column flows out is collected and surveyed；

Described immune affinity column adopts concertina type spiral combined type immune affinity column, the concertina type spiral combined type immunity parent Include upper plug head, lower end cap and loading pipe with post；

The upper plug head circumferentially carries external screw thread, and upper plug head coordinates with loading pipe import screw thread；

The lower end cap circumferentially carries internal thread, and lower end cap coordinates with loading pipe outlet screw thread；

The loading pipe includes import, post chamber and outlet, and import circumferentially carries internal thread, for combining fixed upper plug head or group Close the outlet of another immune affinity column of fixation；Outlet is circumferential to carry external screw thread, fixes for combining fixed lower end cap or combination The import of another immune affinity column；

The post chamber includes breathing zone and affine in immunity area；Breathing zone is arranged at import lower section, and affine in immunity area is located in outlet Side, is provided with from top to bottom upper sieve plate, antibody fill area and lower sieve plate in affine in immunity area；Highest liquid is identified with post cavity outer wall Face graduation mark, the highest liquid level graduation mark is located at breathing zone lower section.

2. a kind of affine in immunity method according to claim 1, it is characterised in that：If analysis sample is more muddy, tighten Upper plug head, extrudes breathing zone.