CN113248398A

CN113248398A - Hapten, artificial antigen and antibody for detecting phenacetin, and preparation method and application thereof

Info

Publication number: CN113248398A
Application number: CN202110479065.9A
Authority: CN
Inventors: 雷红涛; 赖玮; 林建浩; 王锦
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2021-04-29
Filing date: 2021-04-29
Publication date: 2021-08-13
Anticipated expiration: 2041-04-29
Also published as: CN113248398B

Abstract

The invention provides a hapten, an artificial antigen and an antibody for detecting phenacetin, and a preparation method and application thereof, wherein the hapten 1, the artificial antigen 1 and the artificial antigen 2 are prepared, the artificial antigen 2 is used for preparing a specific antibody for detecting phenacetin, the artificial antigen 1 is used as a coating antigen, the antibody has high sensitivity and high specific recognition capability on phenacetin, the detection limit is 2ng/mL, the cross reaction rate on structural analogues is lower than 3%, an immunoassay method of phenacetin is established, and the aim of quickly and accurately detecting phenacetin in cold tea and other foods is fulfilled.

Description

Hapten, artificial antigen and antibody for detecting phenacetin, and preparation method and application thereof

Technical Field

The invention relates to the technical field of food safety detection, in particular to a hapten, an artificial antigen and an antibody for detecting phenacetin, and a preparation method and application thereof.

Background

In 2006, the Guangdong herbal tea is successfully listed after the first national-grade non-material culture protection, the development of the herbal tea industry is more vigorous, and the wind tide of the herbal tea starts to be raised even in China. However, when the herbal tea industry is rapidly developed, a lot of problems also occur, and some illegal merchants illegally add antipyretic and analgesic medicines, such as phenacetin, into the herbal tea in order to enhance the curative effect, so that the method not only influences the reputation of the whole herbal tea industry, but also may bring serious harm to the life safety of drinkers. For example, the Guangxi province Sterculia city market supervision and management department examines the herb tea stores in the whole city specially in 2019, and 8 western medicine components including phenacetin are detected in part of the herb tea samples. Phenacetin (Phenacetin) is an acetanilide antipyretic analgesic, has similar pharmacological action to acetaminophen but higher toxicity, and is gradually replaced by acetaminophen to be applied to clinical medicine at present. It has been shown that chronic or excessive administration of the drug damages the kidney, causes hemolytic anemia, and induces cancer.

At present, methods for the phenacetin in herbal tea and other foods are as follows: the determination of 59 compounds such as acetaminophen in beverages, tea and related products (BJS 201713) specifies the preparation of samples for the detection of 59 compounds including phenacetin and the method of liquid chromatography-tandem mass spectrometry. In a patent screening and confirming method for 207 veterinary drugs and additives in animal food (publication number CN111060638A), a rapid detection method for simultaneously detecting 18 types of 207 compounds including phenacetin in the animal food within 22min is established by using an ultra-high performance liquid chromatography-quadrupole/electrostatic field orbitrap high-resolution mass spectrometry technology; in the literature, the HPLC-MS/MS method is used for measuring 19 illegally added medicines in the herbal tea.

However, although these methods can accurately quantify phenacetin in a sample, the pretreatment is relatively complex, the detection period is long, the equipment is expensive, the professional requirements on operators are high, and the method can only be operated in a laboratory, and is difficult to meet the food supervision requirement of rapid field detection. Therefore, a method for rapidly and accurately detecting phenacetin in food is urgently needed to be developed.

Disclosure of Invention

The invention aims to solve the technical problem of overcoming the defects and shortcomings of a phenacetin detection method in the prior art, and provides a hapten, an artificial antigen and an antibody for detecting phenacetin, and a preparation method and application thereof.

The invention aims to provide a hapten 1 for detecting phenacetin.

The invention also aims to provide application of hapten 1 and 4- (4-acetamidophenyl) butyric acid as hapten (hapten 2) in preparation of artificial antigen for simultaneously detecting phenacetin.

The invention also aims to provide an artificial antigen 1 and an artificial antigen 2 for detecting phenacetin.

The invention also aims to provide application of the hapten 1 or the artificial antigen 2 in preparing an artificial antibody for detecting phenacetin.

The invention also aims to provide an antibody for detecting phenacetin.

The invention also aims to provide an artificial antigen group for immunodetection of phenacetin.

The invention also aims to provide a kit for detecting phenacetin.

The invention also aims to provide an immunoassay method for detecting phenacetin.

The above purpose of the invention is realized by the following technical scheme:

the invention provides a hapten 1 for detecting phenacetin, the structural formula of the hapten 1 is shown as a formula (I),

hapten 1 was named 4- ((4-acetamidophenyl) methylbenzoic acid using the systematic nomenclature.

The preparation method of the hapten 1 comprises the following steps:

dissolving N- (4-hydroxyphenyl) acetamide, potassium carbonate and methyl 4-bromomethylbenzoate in a solvent, stirring, reacting for 5-8 h, separating and purifying, dissolving a purified product in the solvent, adding an alkaline aqueous solution, stirring, reacting for 5-10 h, and adjusting acid to obtain hapten 1.

Preferably, the solvent is acetone, acetonitrile or methanol.

Preferably, the stirring is all at room temperature.

Preferably, the purified product is dissolved in methanol.

Further preferably, the volume ratio of the purified product to methanol is 1:1.

Preferably, the concentration of the alkaline aqueous solution is 1 mol/L.

Preferably, the pH value of the acid is adjusted to be 5-7 by using 1mol/L hydrochloric acid.

The application of the hapten 1 in preparing the artificial antigen for detecting the phenacetin is also within the protection scope of the invention.

Application of 4- (4-acetamidophenyl) butyric acid as hapten in preparing artificial antigen for detecting phenacetin. The research of the invention shows that 4- (4-acetamidophenyl) butyric acid can be used as hapten to prepare and detect phenacetin artificial antigen;

an artificial antigen 1 for detecting phenacetin, the structural formula is shown as formula (III),

an artificial antigen 2 for detecting phenacetin, the structural formula is shown as formula (IV),

the preparation method of the artificial antigen 1 or the artificial antigen 2 comprises the steps of taking the hapten 1(PNT1) or 4- (4-acetamidophenyl) butyric acid as the hapten 2(PNT2), and coupling the hapten with a carrier protein by an active ester method.

Preferably, the carrier protein is any one or more of bovine serum albumin, keyhole limpet hemocyanin, lactoferrin or egg white albumin.

As a specific embodiment of the above method, the method for preparing the

artificial antigen

1 or 2 comprises the following steps:

(1) dissolving hapten PNT1 or PNT2, N-hydroxysuccinimide (NHS) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) in N, N-Dimethylformamide (DMF), and stirring at room temperature in a dark place for 2-4 h to obtain hapten PNT1 or PNT2 activation solution;

(2) the carrier protein was added to PBS buffer (0.01moL/L, pH 7.4);

(3) slowly and dropwise adding hapten PNT1 or PNT2 activating solution into the carrier protein solution in the step (2), and reacting for 12 hours at 4 ℃;

(4) and (4) dialyzing the reaction solution obtained in the step (3) by using a PBS buffer solution to obtain the

artificial antigen

1 or 2.

Preferably, the mass ratio of PNT1, NHS and EDC in the step (1) is 1: 1-2.

More preferably, the mass ratio of PNT1, NHS to EDC in step (1) is 1:1.1: 1.4.

Preferably, the mass-to-volume ratio of the carrier protein to the PBS buffer in step (2) is 10mg:1 mL.

Preferably, the carrier protein in step (2) is any one or more of Bovine Serum Albumin (BSA), Keyhole Limpet Hemocyanin (KLH), Lactoferrin (Lactoferrin, LF), or chicken ovalbumin (ovabumin, OVA).

More preferably, the carrier protein is one or two of bovine serum albumin and chicken egg white albumin.

Preferably, the dialysis in step (4) is performed 4 times a day for 2 days.

An artificial antigen group for detecting phenacetin takes artificial antigen 1(PNT1-BSA) or artificial antigen 2(PNT2-BSA) with bovine serum albumin as carrier protein as immunogen and takes artificial antigen 1(PNT1-OVA) or artificial antigen 2(PNT2-OVA) with chicken ovalbumin as carrier protein as coating antigen.

Preferably, the artificial antigen group takes artificial antigen 2(PNT2-BSA) with bovine serum albumin as a carrier protein as an immunogen and takes artificial antigen 1(PNT1-OVA) with chicken egg albumin as a carrier protein as an envelope antigen.

The application of the artificial antigen group in detecting phenacetin is also within the protection scope of the invention.

Preferably, the artificial antigen group is used for detecting the use of phenacetin in herbal tea and other foods.

A specific antibody for detecting phenacetin, which is prepared by immunizing animals with artificial antigen 1 or artificial antigen 2.

Preferably, the specific antibody is one or two of a monoclonal antibody and a polyclonal antibody.

The application of the antibody in detecting phenacetin is also within the protection scope of the invention.

Preferably, the antibody is used for detecting phenacetin in herbal tea and other foods.

A kit for detecting phenacetin comprises an artificial antigen 1(PNT1-BSA) or an artificial antigen 2(PNT2-BSA) which takes a carrier protein as bovine serum albumin as an immunogen, an artificial antigen group which takes the artificial antigen 1(PNT1-OVA) or the artificial antigen 2(PNT2-OVA) which takes the carrier protein as chicken egg albumin as an envelope antigen, and a specific antibody prepared by immunizing an animal with the artificial antigen 1(PNT1-BSA) or the artificial antigen 2(PNT 2-BSA).

Preferably, the kit comprises an artificial antigen group which takes artificial antigen 2(PNT2-BSA) with bovine serum albumin as a carrier protein as immunogen, artificial antigen 1(PNT1-OVA) with chicken ovalbumin as a carrier protein as coating antigen and a specific antibody prepared by immunizing animals with artificial antigen 2(PNT 2-BSA).

The phenacetin colloidal gold rapid detection card is characterized by comprising a lining plate, and a sample pad, a gold-labeled conjugate pad, a cellulose membrane and a water absorption pad which are sequentially arranged on the lining plate, wherein a specific antibody prepared by immunizing animals with colloidal gold-labeled artificial antigen 1(PNT1-BSA) or artificial antigen 2(PNT2-BSA) is adsorbed in the gold-labeled conjugate pad, a stealth detection line and a stealth quality control line are printed on the cellulose membrane, the stealth detection line is printed by adopting a coating antigen solution, and the stealth quality control line is printed by adopting a goat anti-rabbit antibody; the envelope antigen is artificial antigen 1(PNT1-OVA) or artificial antigen 2(PNT2-OVA) which takes the carrier protein as the egg white albumin.

Preferably, a specific antibody prepared by immunizing an animal with a colloidal gold-labeled artificial antigen 2(PNT2-BSA) is adsorbed in the gold-labeled conjugate pad; the envelope antigen is artificial antigen 1(PNT1-OVA) taking carrier protein as chicken ovalbumin.

An immunoassay method for detecting phenacetin uses artificial antigen 1(PNT1-OVA) or artificial antigen 2(PNT2-OVA) with carrier protein as chicken ovalbumin as coating antigen, and uses artificial antigen 1(PNT1-BSA) or artificial antigen 2(PNT2-BSA) with carrier protein as bovine serum albumin as immunogen to prepare antibody for detecting antibody.

Preferably, the detection is carried out by taking artificial antigen 1(PNT1-OVA) with the carrier protein being chicken ovalbumin as a coating antigen and taking artificial antigen 2(PNT2-BSA) with the carrier protein being bovine serum albumin as an immunogen to immunize animals to prepare a specific antibody serving as a detection antibody.

Such immunoassay methods include, but are not limited to, enzyme immunoassay, immunochromatography, immunosensing, immunocolloidal gold, and the like.

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

the hapten 1 is prepared, 4- (4-acetamidophenyl) butyric acid is used as the hapten 2, the hapten 1 and the hapten 2 are coupled with carrier protein to obtain

artificial antigens

1 and 2, the artificial antigen 2(PNT2-BSA) is used for preparing a specific antibody for detecting phenacetin, the artificial antigen 1(PNT1-OVA) is used as an envelope antigen, the antibody has high sensitivity and high specificity recognition capability on the phenacetin, the half inhibition concentration is 24ng/mL, the detection limit is 2ng/mL, the cross reaction rate on a structural analogue is lower than 3 percent, the antibody has extremely high specificity on the phenacetin, the interference of the analogue can be effectively eliminated, and a core reagent is provided for establishing an immunodetection method of the phenacetin. The invention realizes the application of the antibody for detecting the phenacetin in the immune rapid detection of the phenacetin in herbal tea and other foods. The colloidal gold rapid detection kit developed by the invention can specifically recognize phenacetin and has high detection sensitivity on phenacetin.

Drawings

FIG. 1 is a UV spectrum of BSA, PNT1 and PNT 1-BSA.

FIG. 2 is a UV spectrum of OVA, PNT1 and PNT 1-OVA.

FIG. 3 is a UV spectrum of BSA, PNT2 and PNT 2-BSA.

FIG. 4 is a UV spectrum of OVA, PNT2 and PNT 2-OVA.

FIG. 5 is a standard curve for phenacetin antibody ELISA.

FIG. 6 is a schematic diagram of the assembly of the rapid test strip.

FIG. 7 is a schematic diagram of the test result of the rapid test strip.

Detailed Description

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

Example 1 Synthesis and characterization of phenacetin hapten

1 artificial hapten is designed on the basis of test data by utilizing the structural characteristics of phenacetin.

1. Synthesis and identification of hapten PNT1

Taking N- (4-hydroxyphenyl) acetamide (1moL) and potassium carbonate (3moL), taking acetone as a solvent, reacting with 4-bromomethyl benzoate (1.2moL) for 5-8 h under stirring at room temperature, and separating and purifying to obtain 4- ((4-acetamidophenoxy) methyl benzoate. Dissolving methyl 4- ((4-acetaminophenoxy) methyl) benzoate in methanol, wherein the volume ratio of the methyl 4- ((4-acetaminophenoxy) methyl) benzoate to the methanol is 1:1, adding 1mol/L sodium hydroxide aqueous solution, stirring at room temperature, reacting for 5-10 h, and adjusting the pH to 5-7 by using 1mol/L hydrochloric acid after the reaction is finished to obtain the hapten PNT 1.

Hapten PNT1 nuclear magnetic results:¹H NMR(600MHz,Methanol-d4)δ8.07–7.98(m,11H),7.55(dp,J＝7.6,0.8Hz,11H),7.48–7.41(m,11H),7.00–6.94(m,11H),5.15(s,11H),4.69(s,1H),3.91(s,1H),2.10(s,16H),1.95(s,3H),1.31(d,J＝19.5Hz,2H).

hapten PNT1 mass spectrometry results: MS: c₁₆H₁₅NO₄：285.10，ESI-[M-H]-:：283.90。

The structural formula of the hapten PNT1 is shown as the formula (I):

the hapten PNT1 was designated 4- (4-acetamidophenyl) methylbenzoic acid using the systematic nomenclature.

2. Synthesis and identification of hapten PNT2

Taking N- (4-hydroxyphenyl) acetamide (1moL) and potassium carbonate (3moL), taking acetone as a solvent, reacting with 4-bromoethyl butyrate (1.2moL) at room temperature for 5-8 h under stirring, and separating and purifying to obtain the 4- (4-acetaminophenoxy) ethyl butyrate. Dissolving ethyl 4- (4-acetaminophenoxy) butyrate in methanol, wherein the volume ratio of the ethyl 4- (4-acetaminophenoxy) butyrate to the methanol is 1:1, adding 1mol/L sodium hydroxide aqueous solution, stirring and reacting at room temperature for 5-10 h, and adjusting the pH to 5-7 by using 1mol/L hydrochloric acid after the reaction is finished to obtain the hapten PNT 2.

Hapten PNT2 nuclear magnetic results:¹H NMR(600MHz,Methanol-d4)δ7.44–7.39(m,7H),6.90–6.84(m,7H),4.00(t,J＝6.2Hz,7H),2.49(t,J＝7.3Hz,7H),2.10(s,10H),2.09–2.01(m,8H),1.30(s,3H),1.25(t,J＝7.1Hz,1H),0.94–0.85(m,2H).

hapten PNT2 mass spectrometry results: MS: c₁₂H₁₅NO₄：237.10，ESI-[M-H]-:：236.50。

The structural formula of the hapten PNT2 is shown as the formula (II):

hapten PNT2 was designated 4- (4-acetamidophenyl) butanoic acid using the systematic nomenclature.

Example 2 Synthesis and characterization of artificial antigens to phenacetin

1. Synthesis of artificial phenacetin antigen

The synthetic method of the phenacetin artificial antigen comprises the following steps:

respectively coupling the PNT1 and the PNT2 in the example 1 as haptens with Bovine Serum Albumin (BSA) and egg white albumin (OVA) by an active ester method, respectively weighing 1moL of the phenacetin haptens, dissolving 1.1moL of NHS and 1.4moL of EDC in 50-200 mu L of DMF, and stirring at room temperature in the dark for 2-4 h to obtain active solutions of the phenacetin haptens PNT1 and PNT 2; 10mg of BSA or OVA was added to 1mL of PBS buffer (0.01moL/L, pH 7.4); slowly and dropwise adding the phenacetin hapten PNT1 and PNT2 activating solution into a BSA or OVA solution, and reacting for 12h at 4 ℃; dialyzing with PBS buffer solution for 2 days, changing dialysate for 4 times every day, and after dialysis, obtaining phenacetin artificial antigens PNT1-BSA, PNT1-OVA, PNT2-BSA and PNT2-OVA, subpackaging in centrifuge tubes, and storing at-20 deg.C for use.

The formula of the PBS buffer solution is as follows: na (Na)₂HPO₄·12H₂O 5.80g，NaCl17.00 g，KCl 0.40g，KH₂PO₄0.40g, adding distilled water to reach the constant volume of 2000 mL.

Of these, the two phenacetin artificial antigens in the best combination are PNT2-BSA as immunogen and PNT1-OVA as coatingen (see example 4 for details).

2. Identification of artificial phenacetin antigens

(1) The synthesized PNT1-BSA was subjected to UV scanning, and the results are shown in FIG. 1.

Specifically, ultraviolet (200-350 nm) scanning identification is carried out on BSA, PNT1 and PNT1-BSA respectively, and the highest absorbance values of the substances before and after coupling are compared, so that the absorption curve of the artificial antigen PNT1-BSA is found to be obviously different from that of carrier protein BSA, the PNT1 has obvious absorption at 230-250 nm, the BSA has a characteristic peak at 230-280 nm, after the coupling reaction, the PNT1-BSA has an obvious absorption peak at 230-250 nm, and obvious displacement can be found by comparing the curve of PNT1 with the curve of the BSA. Since unreacted small molecular components such as the drug and the like are completely dialyzed and removed in the dialysis process after the coupling, the characteristic peak of the coupling product is contributed by the drug molecule combined by the protein, so that the reaction product is a compound of the carrier protein and PNT1, and the coupling is successful.

(2) The synthesized PNT1-OVA was subjected to UV scanning, and the results are shown in FIG. 2.

Specifically, ultraviolet (200-350 nm) scanning identification is carried out on OVA, PNT1 and PNT1-OVA respectively, and the highest absorbance values of the substances before and after coupling are compared, so that the absorption curve of the original PNT1-OVA artificially coated is obviously different from that of the carrier protein OVA, a characteristic peak exists at 200nm of PNT1, obvious absorption exists at 230 nm-250 nm of the PNT1, characteristic peaks exist at 230nm and 280nm of the OVA respectively, after coupling reaction, obvious absorption exists at 200nm and 230 nm-250 nm of the PNT1-OVA, and obvious displacement can be seen by comparing the curve of PNT1 with the curve of OVA. Since unreacted small molecular components such as the drug and the like are completely dialyzed and removed in the dialysis process after the coupling, the characteristic absorption of the coupling product is contributed by the protein-bound drug molecules, so that the reaction product is a compound of the carrier protein and PNT1, and the coupling is successful.

(3) The synthesized PNT2-BSA was subjected to UV scanning, and the results are shown in FIG. 3.

Specifically, ultraviolet (200-350 nm) scanning identification is carried out on BSA, PNT2 and PNT2-BSA respectively, and the highest absorbance values of the substances before and after coupling are compared, so that the absorption curve of the artificial antigen PNT2-BSA is found to be obviously different from that of carrier protein BSA, the PNT2 has a characteristic peak at 230nm and obviously absorbs at 240 nm-280 nm, the carrier protein BSA has a characteristic peak at 230nm and 280nm respectively, after coupling reaction, the PNT2-BSA has an obvious absorption peak at 230nm, the BSA curve at 240 nm-280 nm and the PNT2 curve can be compared to see that more obvious absorption occurs, and the characteristic peak at about 250nm is obviously shifted. Since the unreacted small molecule components such as the drug and the like are completely dialyzed and removed in the dialysis process after the coupling, the characteristic absorption is contributed by the protein-bound drug molecules, and the conclusion that the reaction product is a compound of the carrier protein and PNT2 is concluded, and the coupling is successful.

(4) The synthesized PNT2-OVA was subjected to UV scanning, and the results are shown in FIG. 4.

Specifically, OVA, PNT2 and PNT2-OVA are respectively subjected to ultraviolet (200-350 nm) scanning identification, and the highest absorbance values of the substances before and after coupling are compared, so that the absorption curve of the artificial antigen PNT2-OVA is obviously different from that of the carrier protein OVA, PNT2 has a characteristic peak at 230nm and an obvious absorption at 240 nm-280 nm, and carrier protein BSA has a characteristic peak at 230nm and 280nm respectively, after coupling reaction, PNT2-OVA also has an obvious absorption at 240 nm-280 nm compared with the OVA curve, and as unreacted small molecular components such as medicines and the like are completely dialyzed and removed in the dialysis process after coupling, the characteristic absorption at 240 nm-280 nm is contributed by protein-bound medicine molecules, the result shows that the reaction product is a compound of the carrier protein and PNT2, and coupling is successful.

EXAMPLE 3 preparation of antibodies to phenacetin

1. The preparation of polyclonal antibody comprises the following steps:

the prepared artificial antigens PNT1-BSA and PNT2-BSA and an immunologic adjuvant (incomplete Freund's adjuvant is used for the first immunization and incomplete Freund's adjuvant is used for the later booster immunization) are evenly emulsified according to the volume ratio of 1:1, and the New Zealand white rabbits are immunized. The weight of the New Zealand white rabbit is 2.5-3.0 kg, subcutaneous multi-point injection is adopted for the neck and the back, the second immunization is carried out after 3 weeks, and the boosting immunization is carried out once every 3 weeks later. Blood was taken from the ear peripheral vein 1 week after the third booster immunization and serum titers were determined using indirect competition ELISA. When the titer no longer increased, the marginal ear vein was used for boosting. Blood was collected from the heart one week later, and the manner in which the collected blood was used to obtain serum was: bathing at 37 ℃ for 0.5-1.0 h, standing overnight at 4 ℃, sucking the precipitated serum by using a suction tube, centrifuging at 5000-8000 rpm/min at 4 ℃ for 10min, and taking the supernatant. The antiserum is purified to obtain polyclonal antibody by ammonium sulfate precipitation method, and is frozen at-20 deg.C for use.

2. The monoclonal antibody is prepared by the following specific steps:

female Bal b/c mice were immunized with the artificial antigen PNT1-BSA prepared in example 2. Artificial antigens PNT1-BSA and PNT2-BSA are taken and emulsified uniformly with an equal volume of immunologic adjuvant (complete Freund adjuvant is used for first immunization, and Freund incomplete adjuvant is used for boosting immunization), then the mice are immunized by an abdominal subcutaneous multipoint injection method, and blood is taken from the tail part after 1 week of boosting immunization each time to determine the antiserum titer. When the titer is stable and unchanged, selecting the mouse with the best immune effect to strengthen the immunity for one time, and taking splenocytes for fusion after 3 days to prepare the monoclonal antibody.

Example 4 optimization of phenacetin immunogen and coatingen combinations

Respectively mixing the artificial antigens of phenacetin: PNT1-BSA and PNT2-BSA are used for immunizing New Zealand white rabbits, the prepared antibody is used for screening all structural coating antigens, and the antiserum titer and inhibition rate obtained by immunizing the new Zealand white rabbits with immunogen are detected by ELISA. The titers and inhibition rates of the 4 groups of immunogen and coatingen combinations are shown in table 1.

The specific operation steps are as follows:

(1) respectively diluting phenacetin artificial antigens PNT1-OVA and PNT2-OVA to the concentration of 250ng/mL by using coating solution (0.05M carbonate buffer solution, pH 9.6), coating a 96-well enzyme label plate, adding 100 mu L of each well, incubating overnight in a constant-temperature water bath box at 37 ℃, abandoning the coating solution, and washing for 2 times by using PBST (0.01M PBS, 0.06% Tween-20 (v/v));

(2) adding 120 μ L of sealing solution (1% fish glue protein) into each well, sealing at 37 deg.C for 3 hr, discarding sealing solution, clapping, and oven drying at 37 deg.C in drying oven for use;

(3) the phenacetin antibody was diluted with PBST to 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000 while blank control wells (replaced with PBST) were set;

1mg/mL phenacetin was diluted 1000-fold with PBST to 1. mu.g/mL;

the potency is listed as: firstly adding 50 mu L of PBST into each hole, then sequentially adding the antibody diluted by times into the holes according to 50 mu L of each hole, and replacing the last hole with 50 mu L of PBST without adding the antibody;

inhibition column: adding 50 μ L of the drug into each well, sequentially adding the antibody diluted by multiple times into each well according to 50 μ L of the drug, wherein the last well is not added with the antibody and is replaced by 50 μ L of PBST;

incubating at 37 deg.C for 45min, washing for 5 times, and clapping;

(4) adding goat anti-rabbit secondary antibody IgG-HRP (5000-fold dilution), incubating for 30min at 37 ℃, washing for 5 times, and clapping;

(5) adding color developing solution, and performing warm bath at 37 deg.C for 10 min;

(6) adding 10% of H₂SO₄The reaction was stopped and the OD read at 450 nm;

the potency is OD₄₅₀The dilution factor of the antiserum is about 1.0.

Inhibition rate (titer OD value-inhibited OD value)/inhibited OD value 100%

Table 14 titers and inhibition ratios of combinations of immunogens and coatinggens

As can be seen from Table 1, antiserum raised from New Zealand white rabbits immunized with different artificial antigens of phenacetin as immunogens has a certain titer, and the antiserum obtained has inhibitory effect on target analyte phenacetin to different degrees. The antiserum titer 1:128000 and the inhibition rate 87.0% shown by the immunogen and coating antigen structure combination of the number 3 are the optimal combination, and under the combination, the phenacetin antibody not only can specifically recognize target analyte phenacetin, but also has better antibody sensitivity. The antiserum titer and the inhibition rate are higher than those of the combination of the immunogen and the coating antigen structure of the

numbers

1, 2 and 4, so that the combination of the immunogen and the coating antigen structure of the number 3 is the optimal combination. PNT2-BSA is taken as immunogen, PNT1-OVA is taken as coating antigen.

Example 5 establishment of a phenacetin Indirect competitive enzyme-linked immunoassay (ELISA)

1. Establishment of a Standard Curve

The sensitivity of the phenacetin-specific antibody is determined by establishing an ELISA standard curve based on the phenacetin-specific antibody and calculating the concentration IC of the half inhibition amount₅₀To indicate.

The standard curve establishing method specifically comprises the following steps:

(1) the phenacetin polyclonal antibody prepared in example 3 was diluted to 1:8000 with PBST while a blank control well (replaced with PBST) was set;

(2) diluting the phenacetin artificial antigen PNT1-OVA to the concentration of 62.5ng/mL by using a coating solution, coating a 96-hole enzyme label plate, adding 100 mu L of the coating solution into each hole, incubating for 12h in a constant-temperature water bath tank at 37 ℃, discarding the coating solution, washing for 2 times, and patting to dry;

(3) adding 120 μ L of sealing solution (1% fish glue protein solution) into each well, sealing at 37 deg.C for 3 hr, discarding sealing solution, clapping, and oven drying at 37 deg.C in drying oven for use;

(4) diluting phenacetin with PBST to 100000.00, 10000.00, 1000.00, 100.00, 10.00, 0.10, 0.01, 0 ng/mL;

(5) adding 50 mu L of phenacetin diluent into each row, wherein the concentration of the phenacetin diluent is 100000.00, 10000.00, 1000.00, 100.00, 10.00, 0.10 and 0.01ng/mL (three groups are parallel), adding 50 mu L of PBST diluent into a hole with the concentration of 0ng/mL, adding the phenacetin antibody diluent in the step (1), and adding 50 mu L of the phenacetin antibody diluent into each hole. Incubating at 37 deg.C for 40min, discarding the liquid in the well, washing for 5 times, and drying;

(6) adding goat anti-rabbit secondary antibody IgG-HRP (diluted by 5000 times), incubating at 37 deg.C for 30min, discarding the liquid in the well, washing for 5 times, and patting dry;

(7) adding 100 μ L of color developing solution into each well, and incubating at 37 deg.C for 10min for color development;

(8) add 50. mu.L of stop solution (10% H) to each well₂SO₄) Stopping the reaction, and reading the OD value at 450nm by using an enzyme-labeling instrument;

wherein the formula of the PBST is as follows: na (Na)₂HPO₄·12H₂O 14.50g，NaCl 42.50g，KCl 1.00g，KH₂PO₄1.00g, Tween-203.0 mL, adding distilled water to reach 5000 mL.

Preparing 1% fish glue protein solution: for example, 0.01g of fish gelatin protein powder is dissolved in 1mL of PBST, specifically calculated according to the actual dosage.

And establishing an ELISA standard curve by using phenacetin as a standard substance, wherein the detection limit is 2ng/mL, and the half inhibition concentration is 24 ng/mL. As can be seen from fig. 5, the standard curve established using phenacetin as the standard substance has a typical S-shaped curve, and the detection sensitivity is good.

2. Assay for specificity of phenacetin antibodies

The specificity of the phenacetin antibody is determined by carrying out a cross reaction experiment on phenacetin and analogues thereof, the specificity of the antibody is expressed by cross reaction rate (CR), and the smaller the cross reaction, the better the specificity.

Taking phenacetin and analogues thereof as competitive antigens, respectively carrying out serial dilution, adopting an indirect competitive ELISA method for determination, and adopting an experimental method of step reference sensitivity verification to obtain IC of various analogues₅₀The value is obtained. The cross-reactivity (CR) of phenacetin and various analogs was calculated using the following formula:

the results of the cross-reaction experiments with phenacetin and its analogs are shown in table 2.

TABLE 2 Cross-reactivity test results of phenacetin and its analogs

Note: NR indicates no reaction.

From the results of table 2, it can be found that: the cross-reactivity of the phenacetin antibody to phenacetin was 100%, IC₅₀The value is 24ng/mL, and the analogs have no response or the cross reaction rate is less than 3 percent; the phenacetin antibody has extremely high specificity to phenacetin, can effectively eliminate the interference of analogues thereof, and can be specially used for the detection of phenacetin.

The above results illustrate that: the phenacetin antibody prepared by the invention has strong detection specificity to phenacetin.

Example 6 Rapid detection method of phenacetin colloidal gold

1. Preparation of gold-labeled antibody and gold-labeled conjugate pad

Colloidal gold suspension with average diameter of 40nm was prepared by reducing chloroauric acid with trisodium citrate. Under reflux, 100mL of 0.01% chloroauric acid solution was heated to boiling, and 1.1mL of 1% trisodium citrate was added rapidly with constant stirring. Heating and stirring were continued for 5min when the reaction solution became reddish-red in color. After cooling to room temperature, 0.05% sodium azide was added and stored at 4 ℃.

Colloidal gold was labeled with 0.2mol of K before being labeled with the antibody prepared in example 3₂CO₃The solution was adjusted to pH 8.2 and 30. mu.g of antibody-labeled 1mL of colloidal gold solution was determined by classical NaCl titration. Then, the mixture was labeled at the optimum amount, and after 1 hour of labeling, 10% BSA was added under stirring (so that B was finally addedSA concentration of 1%), incubated for 1h, centrifuged at 10000rpm for 25min at 4 ℃ and the supernatant removed. Adding 5% BSA solution with the same volume of colloidal gold solution for resuspension, centrifuging at 4 deg.C and 10000rpm for 25min, and repeating twice. Finally, it was resuspended in 1/5 volumes of colloidal gold solution in TB solution (containing 3% BSA, 3% sucrose, 0.01mol/L sodium borate and 0.05% sodium azide) and stored at 4 ℃. Spraying 4% BSA solution at 8 μ L/cm onto glass wool with XYZ-3000 three-dimensional film spraying instrument, drying at 42 deg.C for 50min in a drying oven, spraying colloidal gold-labeled antibody at 6 μ L/cm onto glass wool, drying at 42 deg.C for 50min in the drying oven, and vacuum drying for storage.

2. Coupled antigen goat anti-rabbit coated cellulose membrane

An XYZ-3000 three-dimensional film spraying instrument is used for spraying the coating antigen with the concentration of 1mg/mL on the lower side of a cellulose film in an amount of 0.8 mu L/cm to serve as a detection line. Goat anti-rabbit IgG was sprayed onto the upper side of the cellulose membrane at a concentration of 120. mu.g/L in an amount of 0.8. mu.L/cm using an XYZ-3000 three-dimensional spray coater, as a control line, with a space of 8mm between the two lines.

3. Assembly of quick test paper strip

As shown in FIG. 6, the cellulose film 4 was stuck to the middle portion of the backing sheet 1, and the absorbent pad 7 was stuck to the upper side of the cellulose film 4 so as to overlap the cellulose film 4 by 1 mm. The gold-labeled conjugate pad 3 was stuck under the cellulose film 4 with an overlap of 1 mm. The sample pad 2 is stuck under the gold-labeled conjugate pad 3 with an overlap of 2 mm. The assembled test paper board was cut into test paper strips 3.05mm wide with a cutter.

4. Preparation of test sample solution

Weighing 1g (accurate to 0.001g) of sample in a 50mL centrifuge tube, accurately adding 40mL of water, carrying out vortex for 2min, carrying out ultrasonic treatment for 20min, centrifuging for 5min at 8000r/min, and taking supernatant to be tested.

5. Rapid test strip detection and judgment

When the sample solution to be tested is added into the test end of the test strip or the test paper card, the solution to be tested drives the object to be tested and the gold-labeled antibody in the gold-labeled conjugate pad 3 to diffuse together to the cellulose membrane 4 through the siphon action, and finally permeates into the end 7 of the water absorption pad. In the diffusion process, if the sample contains the substance to be detected, the substance to be detected is combined with the gold-labeled antibody, so that the antigen binding site on the gold-labeled antibody is occupied, the combination of the gold-labeled antibody and the invisible detection line 5 (the combination of the hapten and the carrier protein) on the cellulose membrane 4 is prevented, and the invisible detection line 5 is not colored or is weakly colored, namely, the detection sample is positive or weakly positive; if the sample to be detected does not exist in the sample, a clear red line is displayed when the gold-labeled antibody meets the invisible detection line 5 in the upward moving process, and the detection sample is negative. Similarly, the gold-labeled antibody also binds to the invisible quality control line 6 (goat anti-rabbit IgG) on the cellulose membrane 4, so that the invisible control line 6 is red. The presence or absence of the color of the invisible control line 6 indicates the validity or invalidity of the test strip, respectively, and the determination result is shown in fig. 7.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

1. A hapten 1 for detecting phenacetin is characterized in that the structural formula of the hapten 1 is shown as a formula (I),

2. use of hapten 1 as defined in claim 1 for the preparation of an artificial antigen for the detection of phenacetin.

3. An artificial antigen 1 for detecting phenacetin is characterized in that the structural formula is shown as a formula (III),

4.4- (4-acetamidophenyl) butyric acid is used as hapten in the preparation of artificial antigen for detecting phenacetin.

5. An artificial antigen 2 for detecting phenacetin is characterized in that the structural formula is shown as a formula (IV),

6. the method for producing the artificial antigen 1 according to claim 3 or the artificial antigen 2 according to claim 5, wherein the hapten 1 or 4- (4-acetamidophenyl) butyric acid according to claim 1 is used as a hapten, and a carrier protein is coupled by an active ester method.

7. An artificial antigen group for detecting phenacetin is characterized in that carrier protein is used as an artificial antigen 1 or an artificial antigen 2 of bovine serum albumin as immunogen, and carrier protein is used as an artificial antigen 1 or an artificial antigen 2 of chicken ovalbumin as coating antigen.

8. A specific antibody for detecting phenacetin is characterized in that the specific antibody is prepared by immunizing animals with artificial antigen 1 or artificial antigen 2.

9. A kit for detecting phenacetin, comprising the artificial antigen group of claim 7 and the specific antibody of claim 8.

10. An immunoassay method for detecting phenacetin is characterized in that an artificial antigen 1 or an artificial antigen 2 taking a carrier protein as chicken ovalbumin is taken as an envelope antigen, and an antibody prepared by immunizing an animal by taking the artificial antigen 1 or the artificial antigen 2 taking the carrier protein as bovine serum albumin as an immunogen is taken as a detection antibody for detection.