CN115902205A - Reagent for detecting nonyl phenol, preparation method thereof and ELISA kit - Google Patents

Abstract

The invention belongs to the technical field of analytical chemistry, and discloses a reagent for detecting nonyl phenol, which comprises a standard product, a primary antibody reagent, a secondary antibody reagent, a sample diluent, an antibody diluent, a concentrated washing solution, a substrate solution and a stop solution, wherein the reagent is detected under the assistance of an enzyme label plate and a plate sticker, and also discloses a method for preparing the reagent and an ELISA kit for placing the reagent.

Description

Reagent for detecting nonyl phenol, preparation method thereof and ELISA kit

Technical Field

The invention relates to the technical field of analytical chemistry, in particular to a reagent for detecting nonyl phenol, a preparation method thereof and an ELISA kit.

Background

Nonylphenol (NP) is mainly used in chemical industry as a representative of Environmental Endocrine Disruptors (EEDs), has accumulative property and lipophilicity, and can interfere normal work of the endocrine system of a human body, damage the nervous system and the reproductive system, destroy the antioxidant system, damage DNA, and influence the survival and multiplication of organisms. Currently, the residue analysis of nonyl phenol is mainly carried out by using chromatography and immunological methods, such as GC (gas chromatography) and HPLC (high performance liquid chromatography), ELISA (enzyme-linked immunosorbent assay).

The application of the physicochemical analysis technologies to analysis of nonyl phenol residues in samples such as environment, biology and the like has high requirements on the degree of instrumentation, complex pretreatment processes such as separation, extraction, purification, derivatization and the like are required, simultaneous detection of large samples cannot be realized, the requirements on the professional performance of operators are high, a large amount of organic solvents are required in the pretreatment process, and new environmental pollution is caused. Chromatography is not suitable for on-site monitoring and large-scale sample screening due to complicated instrumentation and cumbersome procedures. The common immunology method has the disadvantages of long detection time, high cost and being not beneficial to popularization and use in basic units. In the common detection method of the ELISA kit: the direct method is characterized in that the first antibody needs to be marked, the sensitivity is low, and the specificity is general; the cross probability of the indirect method is improved; the double antibody sandwich method requires multiple epitopes. And are not suitable for small molecule detection.

At present, a competition method is mainly applied to detection of small molecule antigens, because small molecules only have one antigen site and cannot be simultaneously combined with two antibodies, a double antibody sandwich method cannot be adopted for detection, small molecule immunochromatography is widely applied to detection of drugs at present, and good results are obtained in the aspects of pesticide residue, antibiotic residue and hormone detection caused by drug abuse in recent years.

Disclosure of Invention

The invention aims to provide a reagent for detecting nonyl phenol, a preparation method thereof and an ELISA kit, which are used for batch and rapid detection of small molecular residues of nonyl phenol.

In order to realize the purpose of the invention, the technical scheme is as follows:

in a first aspect, the reagent for detecting nonyl phenol comprises a standard substance, a primary antibody reagent, a secondary antibody reagent, a sample diluent, an antibody diluent, a concentrated washing solution, a substrate solution and a stop solution, and is detected with the aid of an enzyme label plate and a plate sticker.

The invention is further configured to: the secondary antibody reagent is a goat anti-mouse secondary antibody.

In a second aspect, a method for preparing the reagent is disclosed, which specifically comprises the following steps:

1) Taking out the primary antibody, diluting by 100 times with an antibody diluent, and fully and uniformly mixing;

2) Taking out the standard product and fully mixing to obtain a standard product S1;

3) Taking out 4 centrifugal tubes of 1.5ml, sequentially arranging, adding 500 mul of sample diluent into each centrifugal tube, sucking 250 mul of standard substance S1 into a first centrifugal tube S2, lightly blowing and uniformly mixing, sucking 250 mul from the first centrifugal tube S2 into a second centrifugal tube S3, lightly blowing and uniformly mixing, and so on to dilute the standard substance by 3 times;

4) Extracting a sample;

5) Diluting the sample by using 0.02M PB and the sample according to the proportion of 3;

6) Extracting a body cavity liquid sample, firstly diluting the body cavity liquid sample with methanol according to the proportion of 2 to 3, and then diluting the body cavity liquid sample by 3 times with a sample diluent;

7) Diluting the concentrated washing solution by 25 times with deionized water;

8) Using the antibody diluent to react with a goat anti-mouse secondary antibody reagent according to the ratio of 100: dilution was performed 1 fold.

In a third aspect, an ELISA kit for placing the reagent according to any one of claims 1-2 is disclosed, comprising an upper case and a lower case, characterized in that: the top of box body is equipped with many telescopic links down, many the telescopic link with go up box body fixed connection, it can along with go up the box body the telescopic link up-and-down motion, the placing chamber has been seted up in the box body down, it is equipped with the reagent bottle mounting to go up in the box body, it places the hole to have seted up a plurality of reagent bottles on the reagent bottle mounting, upward be equipped with visor and fixed notch on the box body, the inside wall of visor is equipped with fixed lug, fixed lug with fixed notch is corresponding.

The invention is further configured to: the placing chamber is used for placing the ELISA plate and the plate sticker.

The invention is further configured to: the reagent bottle placing holes include a standard placing hole, a primary antibody placing hole, a secondary antibody placing hole, a sample diluent placing hole, an antibody diluent placing hole, a concentrated washing solution placing hole, a substrate solution placing hole and a stop solution placing hole.

The invention is further configured to: the inner side wall of the protective cover is provided with a groove.

The invention is further configured to: and a handle is fixedly arranged on one side of the protective cover.

The invention is further configured to: and a plurality of column feet are fixedly arranged at the bottom of the lower box body.

The invention is further configured to: and the bottoms of the column bases are all sleeved with protective sleeves.

In conclusion, compared with the prior art, the reagent for detecting nonyl phenol, the preparation method thereof and the ELISA kit provided by the invention realize batch and rapid detection of small-molecule residues of nonyl phenol through a competitive enzyme-linked immunosorbent assay test kit with high sensitivity, high specificity, high accuracy, high precision and simple operation method.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the prior art descriptions will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a schematic structural diagram 1 of an ELISA kit for detecting nonylphenol provided in this example;

FIG. 2 is a schematic structural diagram of an ELISA kit for detecting nonylphenol provided in this example 2;

FIG. 3 is a schematic structural diagram of an ELISA kit for detecting nonylphenol provided in this example 3;

fig. 4 is a schematic diagram of an improved structure of an ELISA kit for detecting nonylphenol provided in this example.

Reference numerals: 1. an upper box body; 2. a lower box body; 3. a telescopic rod; 4. a placing chamber; 5. a reagent bottle fixing part; 6. a reagent bottle placing hole; 61. a standard placement hole; 62. a placement resisting hole; 63. a secondary antibody placement hole; 64. A sample diluent holding well; 65. placing the antibody diluent in a hole; 66. a concentrated washing liquid placing hole; 67. a substrate solution placing hole; 68. a stop solution placement hole; 7. a protective cover; 8. a fixed notch; 9. fixing the bump; 10. A groove; 11. a handle; 12. a column shoe; 13. a protective sleeve.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the present invention will be further described in detail with reference to the accompanying drawings and embodiments, it being understood that the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention.

In the description of the present invention, it should be noted that the terms "front", "back", "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc. indicate orientations or positional relationships based on orientations or positional relationships shown in the drawings, and are only for convenience of description and simplification of description, but do not indicate or imply that the device or element referred to must have a specific orientation, be constructed in a specific orientation, and be operated, and thus, should not be construed as limiting the present invention. Furthermore, the terms "first," "second," and "third" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance.

In the description of the present invention, it should be noted that, unless otherwise explicitly specified or limited, the terms "mounted," "connected," and "connected" are to be construed broadly, e.g., as meaning either a fixed connection, a removable connection, or an integral connection; can be mechanically or electrically connected; the two elements may be directly connected or indirectly connected through an intermediate medium, or may be connected through the inside of the two elements, or may be connected wirelessly or through a wire. The specific meanings of the above terms in the present invention can be understood in a specific case to those of ordinary skill in the art.

The technical features mentioned in the different embodiments of the invention described above can be combined with each other as long as they do not conflict with each other.

In addition, unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be prepared by existing methods.

In a first aspect of this embodiment, a reagent for detecting nonylphenol is provided, where the reagent includes a standard, a primary antibody reagent, a secondary antibody reagent, a sample diluent, an antibody diluent, a concentrated washing solution, a substrate solution, and a stop solution, and the reagent is detected with the aid of an elisa plate and a plate sticker.

Further, the secondary antibody reagent is a goat anti-mouse secondary antibody, preferably a goat anti-mouse secondary antibody IgG-HRP.

In this example, the elisa plate, the standard, the primary antibody reagent, the secondary antibody reagent, the sample diluent, the antibody diluent, the concentrated washing solution, the substrate solution, the stop solution, and the plate sticker were coated with the nonylphenol antigen.

In the preparation process of the enzyme label plate coated with the nonyl phenol antigen, the coating antigen is a coupling compound of small molecule-BSA and small molecule-OVA, the coating solution is sodium carbonate buffer solution, and the blocking solution is the coating solution containing 1% gelatin.

It should be noted that the immunogen used in the preparation of the nonylphenol monoclonal antibody is a conjugated complex of small molecule-BSA and small molecule-OVA, and the carrier protein may be Bovine Serum Albumin (BSA).

In the specific implementation process, 4 Balb/c mice are immunized by the immunogen, the optimal 2-3 mice are selected for fusion, and a mode of combining liquid fusion and semisolid fusion is adopted to ensure at least 1000-2000 effective clones per fusion. Screening was performed with small molecule conjugate coating. And (4) carrying out competitive condition rescreening on the clones with positive primary screening, and carrying out subcloning on the clones with better competitive condition. And (4) carrying out the same verification scheme in the subcloning stage until cell strains capable of stably secreting positive cells are screened, and simultaneously making backup of all cell strains. The cell lines of the established strain were again verified to confirm the stability of the cell lines. And (4) carrying out subtype identification on positive clones of the fixed strains.

In a second aspect, a method for preparing the reagent is provided, which specifically comprises the following steps:

1) Taking out the primary antibody, diluting by 100 times with an antibody diluent, and fully and uniformly mixing;

2) Taking out the standard product and fully mixing to obtain a standard product S1;

3) Taking out 4 centrifugal tubes (S2-S5) with the volume of 1.5ml, sequentially arranging the centrifugal tubes, respectively adding 500 mul of sample diluent, sucking 250 mul of a standard product S1 into a first centrifugal tube S2, lightly blowing and uniformly mixing, sucking 250 mul from the first centrifugal tube S2 into a second centrifugal tube S3, lightly blowing and uniformly mixing, and so on to dilute the standard product by 3 times;

the sample concentrations of S1-S7 are 16.2ng/ml, 5.4ng/ml, 1.8ng/ml, 0.6ng/ml, 0.2ng/ml, 0ng/ml and 0ng/ml in sequence, wherein S6 and S7 are sample diluents.

4) Extracting a sample;

5) Diluting a sample by using 0.02M PB and the sample according to the proportion of 3;

6) Extracting a body cavity liquid sample, firstly diluting the body cavity liquid sample with methanol according to the proportion of 2 to 3, and then diluting the body cavity liquid sample by 3 times with a sample diluent, namely, the dilution ratio is 1;

it should be noted that, since a sediment is present in the body fluid sample, a centrifugal process is required in the dilution process.

7) Diluting the concentrated washing solution by 25 times with deionized water;

in this embodiment, 240ml of deionized water is measured from a measuring cylinder, poured into a beaker or other clean container, 10ml of concentrated cleaning solution is measured, added uniformly, stirred and mixed well, and prepared before use. The concentrated washing solution may be salted out when stored at low temperature, and may be heated in water bath to aid dissolution when diluted.

8) Using the antibody diluent to react with a goat anti-mouse secondary antibody reagent according to the ratio of 100: the dilution was performed 1 fold.

In this example, 10. Mu.l goat anti-mouse secondary antibody IgG-HRP was added to 990. Mu.l secondary antibody dilution, gently mixed and dispensed within 10 minutes just before use.

The third aspect of this embodiment provides an ELISA kit for detecting nonyl phenol, as shown in fig. 1, including upper box body 1 and lower box body 2, the top of lower box body 2 is provided with a plurality of telescopic links 3, a plurality of telescopic links 3 are fixedly connected with upper box body 1, upper box body 1 can move up and down along with telescopic links 3, placing chamber 4 is provided in lower box body 2, upper box body 1 is provided with reagent bottle fixing part 5, reagent bottle fixing part 5 is provided with a plurality of reagent bottle placing holes 6, upper box body 1 is provided with protective cover 7 and fixed notch 8, the inside wall of protective cover 7 is provided with fixed lug 9, fixed lug 9 corresponds to fixed notch 8, in this embodiment, the number of telescopic links 3 is preferably four, the four corners of upper box body 2 top are distributed, the top of telescopic link 3 is welded with upper box body 1, the welding point is located in the column groove (not shown) of upper box body 1, it should be said that, when upper box body 1 moves down to the limit value (namely, the thin pole of telescopic link 3 is received in the column groove (not shown) of lower box body 2, and then the telescopic link 1 is received in the column groove (not shown).

Further, the placing chamber 4 is used for placing the elisa plate and the plate sticker, and in the specific implementation process, 12 × 8 wells of the elisa plate and 3 plate stickers are placed in the placing chamber 4.

Further, as shown in fig. 2, the reagent bottle placing hole 6 includes a standard placing hole 61, a primary antibody placing hole 62, a secondary antibody placing hole 63, a sample diluent placing hole 64, an antibody diluent placing hole 65, a concentrated washing solution placing hole 66, a substrate solution placing hole 67 and a stop solution placing hole 68, and in a concrete implementation, a standard reagent bottle, a primary antibody reagent bottle, a secondary antibody placing hole 63, a sample diluent placing hole 64, an antibody diluent placing hole 65, a concentrated washing solution placing hole 66, a substrate solution placing hole 67 and a stop solution placing hole 68 are respectively placed therein.

Furthermore, the inner side wall of the protective cover 7 is provided with a groove 10, and in the specific implementation process, the groove 10 plays a role in increasing the space.

Further, a handle 11 is fixedly arranged on one side of the protective cover 7, and in the specific implementation process, the handle 11 plays a role in assisting the opening and closing of the protective cover 7.

Further, as shown in fig. 3, the fixed many column bases 12 that are equipped with in bottom of box body 2 down, the bottom of many column bases 12 all overlaps and is equipped with lag 13, and it can play the effect of protection column base 12, and in this embodiment, the quantity of column base 12 is preferably four, distributes in the four corners of box body 2 bottom down, and at the concrete implementation in-process, box body 2 direct contact ground under can avoiding in the setting of column base 12 to play the effect of box body 2 under the protection.

It should be noted that, in this embodiment, through the arrangement of the upper box body 1, the lower box body 2, the placing chamber 4, the reagent bottle fixing member 5 and the plurality of reagent bottle placing holes 6, the classified placing of each bottle body or plate body in the reagent box is realized, so that the orderliness in the reagent box and the work efficiency of the operator are improved.

In addition, in the specific implementation process, the lower box body 2 in this embodiment may be designed to be a drawing structure, as shown in fig. 4, it should be noted that this improvement can achieve the technical purpose of the present invention, and can also provide the aesthetic benefits of low production cost and simple style.

The fourth aspect of this embodiment provides an operation method of an elisa test for nonylphenol residue analysis, which specifically includes the following steps:

1) Transferring the reagents to room temperature (18-25 ℃) for balancing for at least 30 minutes, and preparing the reagents for standby according to the method;

2) Sample adding: respectively arranging a standard hole and a sample hole to be tested, adding 50 mul of the standard and the sample to be tested into the standard hole and the sample hole to be tested, adding 50ul of primary antibody, slightly shaking and mixing for 2 minutes, covering a plate, sticking and culturing for 30 minutes at room temperature;

3) Discarding the liquid, spin-drying, washing the plate for 5 times, soaking (300 ul/hole) for 20 seconds each time, and spin-drying;

4) Adding 100 mul of HRP-labeled secondary antibody working solution into each hole, covering a new plate, and incubating for 30 minutes at room temperature;

5) Discarding liquid in the holes, spin-drying, washing the plate for 5 times, soaking (300 ul/hole) for 20 seconds each time, and spin-drying;

8) Adding 100 mul of substrate solution into each hole in sequence, and developing for 15 minutes at room temperature in a dark place;

9) Adding 50 mul of stop solution into each hole in sequence to stop the reaction;

10 The optical density (OD value) of each well was measured sequentially at a wavelength of 450nm with a microplate reader within 5 minutes after the reaction was terminated, and the sample concentration was calculated.

The reagent provided in this example should be stored in an environment of 2-8 ℃ in the dark before unsealing, and the effective period thereof is 12 months. After unsealing, the following preservation conditions were followed:

enzyme label plate: after being opened, the product should be put into an aluminum foil bag with a desiccant and stored in a sealed and moisture-proof way at the temperature of 2-8 ℃, and the product can be stored for at most one month at the temperature of 2-8 ℃ within the effective period.

Goat anti-mouse secondary IgG-HRP, standard and primary antibody: the effective period can be preserved at 2-8 deg.C for at most one month, and if not used recently, it is preferably preserved at-20 deg.C.

Sample diluent, antibody diluent, concentrated wash, substrate solution and stop solution: the shelf life can be up to one month at 2-8 deg.C.

In conclusion, the invention has the following beneficial effects:

1) Bisphenol A is used as a coating antigen, and the detection range is as follows: IC50 is approximately equal to 125ppb, nonyl phenol is used as coating antigen, and the detection range is as follows: IC50 ≈ 427ppb;

2) The precision of the invention is as follows: the intra-batch difference CV% is less than 10%, and the inter-batch difference CV% is less than 10%, which is expected;

3) The stability of the invention is as follows: through measurement, the reagent is stored at the recommended temperature within the validity period, the thermal stability is reduced by less than 30 percent within 7 days, and the thermal stability is qualified.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.

Claims (10)

1. A reagent for detecting nonyl phenol is characterized in that: the reagent comprises a standard product, a primary antibody reagent, a secondary antibody reagent, a sample diluent, an antibody diluent, a concentrated washing solution, a substrate solution and a stop solution, and the reagent is detected with the assistance of an ELISA plate and a plate sticker.

2. The reagent of claim 1, wherein the secondary antibody reagent is a goat anti-mouse secondary antibody.

3. A method for preparing a reagent according to any one of claims 1 to 2, comprising in particular the steps of:

1) Taking out the primary antibody, diluting by 100 times with an antibody diluent, and fully and uniformly mixing;

2) Taking out the standard product and fully mixing to obtain a standard product S1;

3) Taking out 4 centrifugal tubes of 1.5ml, sequentially arranging, adding 500 mul of sample diluent into each centrifugal tube, sucking 250 mul of standard substance S1 into a first centrifugal tube S2, lightly blowing and uniformly mixing, sucking 250 mul from the first centrifugal tube S2 into a second centrifugal tube S3, lightly blowing and uniformly mixing, and so on to dilute the standard substance by 3 times;

4) Extracting a sample;

5) Diluting a sample by using 0.02M PB and the sample according to the ratio of 3;

6) Extracting a body cavity fluid sample, diluting the body cavity fluid sample by using methanol according to a ratio of 2 to 3, and then diluting the body cavity fluid sample by using a sample diluent by 3 times;

7) Diluting the concentrated washing solution by 25 times with deionized water;

8) Using the antibody diluent to react with a goat anti-mouse secondary antibody reagent according to the ratio of 100: dilution was performed 1 fold.

4. An ELISA kit for placing reagents according to any of claims 1-2, comprising an upper case and a lower case, characterized in that: the top of box body is equipped with many telescopic links down, many the telescopic link with go up box body fixed connection, it can along with go up the box body the telescopic link up-and-down motion, the placing chamber has been seted up in the box body down, it is equipped with the reagent bottle mounting to go up in the box body, it places the hole to have seted up a plurality of reagent bottles on the reagent bottle mounting, upward be equipped with visor and fixed notch on the box body, the inside wall of visor is equipped with fixed lug, fixed lug with fixed notch is corresponding.

5. The ELISA kit for detecting nonyl phenol of claim 4 wherein the holding chamber is used to hold an ELISA plate and a plate sticker.

6. The ELISA kit for detecting nonylphenol of claim 5 wherein the reagent bottle placing holes comprise a standard placing hole, a primary antibody placing hole, a secondary antibody placing hole, a sample diluent placing hole, an antibody diluent placing hole, a concentrated washing solution placing hole, a substrate solution placing hole and a stop solution placing hole.

7. The ELISA kit for detecting nonyl phenol of claim 4 wherein the interior sidewall of the cover is notched.

8. The ELISA kit for detecting nonyl phenol of claim 7 wherein a handle is affixed to one side of the protective cover.

9. The ELISA kit for detecting nonyl phenol of claim 4 wherein a plurality of legs are affixed to the bottom of the lower case.

10. The ELISA kit for detecting nonyl phenol of claim 9, wherein a plurality of said legs are provided with a protective sleeve at the bottom.

Images