WO2022077824A1 - Sodium picosulfate hapten, aritifical antigen, antibody, preparation method for same, and applications thereof - Google Patents

Abstract

Disclosed are a sodium picosulfate hapten, an artificial antigen, an antibody, a preparation method for same, and applications thereof. Sodium picosulfate haptens PC1, PC2, PC3, and PC4 are prepared first, a hapten conjugated carrier protein is applied to produce the artificial antigen, and an immunized animal acquires a sodium picosuilfate antibody. Preferably, a PC1-lactoferrin conjugate serves as an immunogen, the antibody acquired by immunization provides highly sensitive and highly specific recognition capability with respect to sodium picosulfate, IC50 being 5 ng/mL, and the cross-reaction rates with respect to structural analogs being less than 10%. Also disclosed are a sodium picosulfate enzyme-linked immunosorbent assay reagent kit, a colloidal gold immunochromatographic assay reagent kit, and applications thereof in food products and/or healthcare products.

Description

A kind of sodium picosulfate hapten, artificial antigen, antibody and preparation method and application thereof technical field

The invention relates to the technical field of food and/or health care product safety detection, and more particularly, to a sodium picosulfate hapten, artificial antigen, antibody, and a preparation method and application thereof.

Background technique

Sodium Picosulfate is a stimulant laxative, which can treat constipation by stimulating intestinal peristalsis and secretion, and inhibiting the absorption of water in the intestinal lumen. Due to its significant laxative effect, some unscrupulous merchants add sodium picosulfate to enzyme food or health food claiming to have weight loss function in order to achieve weight loss, bodybuilding and other effects. However, long-term consumption of excessive sodium picosulfate It may cause disturbance of the intestinal system or acute damage to the gastric mucosa.

At present, the most common detection method for sodium picosulfate in food is mainly high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The Chinese Patent Publication No. CN110501438A, "A Method for Detecting Sodium Picosulfate in Slimming Tea", discloses a method for detecting sodium picosulfate in slimming tea using triple quadrupole tandem mass spectrometry. However, although this method can accurately quantify sodium picosulfate in the sample, and the results are stable, it has disadvantages such as relatively complex pretreatment, long detection cycle, expensive equipment, and certain professional requirements for operators, making it difficult to achieve rapid on-site purpose of detection. Therefore, there is an urgent need to develop a rapid and simple method for the rapid detection of sodium picosulfate in weight loss health food.

Compared with existing chromatography-based methods, immunodetection methods based on antigen-antibody specific molecular recognition have greater advantages in on-site detection, showing rapidity, sensitivity, simplicity, etc. Less demanding. The key to the development of immunoassay methods is to design a suitable sodium picosulfate hapten and prepare an antibody with high sensitivity and specificity. related reports.

SUMMARY OF THE INVENTION

The primary purpose of the present invention is to overcome the above-mentioned defects and deficiencies existing in the prior art, and to provide two sodium picosulfate haptens.

The second object of the present invention is to provide two artificial antigens of sodium picosulfate.

The third object of the present invention is to provide a sodium picosulfate antibody.

The fourth object of the present invention is to provide an artificial antigen group for immunodetection of sodium picosulfate.

The fifth object of the present invention is to provide a sodium picosulfate immunoassay kit.

A kind of sodium picosulfate hapten, is characterized in that, the structural formula of described sodium picosulfate hapten is as shown in formula (I) or formula (III):

The sodium picosulfate hapten of the formula (I) is named 4-((4-(carboxymethoxy)phenyl)(pyridin-2-yl)methyl)phenylsodium sulfate using systematic nomenclature;

The sodium picosulfate hapten of the formula (III) is named 4-((4-((5-carboxypentyl)oxy)phenyl)(pyridin-2-yl)methyl)benzene using systematic nomenclature Sodium sulfate.

As a preferred embodiment, the preparation method of the 4-((4-(carboxymethoxy)phenyl)(pyridin-2-yl)methyl)phenylsulfate (PC1) is as follows: Water dichloromethane is used as solvent, 2-(4-((4-hydroxyphenyl)(pyridin-2-yl)methyl)phenoxy)acetate is mixed with triethylamine, chlorosulfonic acid is added, and The reaction was carried out under stirring at room temperature, and 4-((4-(2-ethoxy-2-oxyethoxy)phenyl)(pyridin-2-yl)methyl)phenylsulfuric acid was obtained by separation and purification. Then 4-((4-(2-ethoxy-2-oxyethoxy)phenyl)(pyridin-2-yl)methyl)phenylsulfuric acid was dissolved in methanol, and 1 mol/L of hydrogen peroxide was added. The sodium aqueous solution was stirred at room temperature for 3 to 5 hours, and after the reaction was completed, the pH was adjusted to 6 to 7 with hydrochloric acid with a concentration of 1 mol/L, to obtain 4-((4-(carboxymethoxy)phenyl)(pyridine-2 -yl)methyl)phenylsulfate (PC1).

More preferably, the molar ratio of ethyl 2-(4-((4-hydroxyphenyl)(pyridin-2-yl)methyl)phenoxy)acetate to triethylamine is 1:2-5.

Most preferably, the molar ratio of the ethyl 2-(4-((4-hydroxyphenyl)(pyridin-2-yl)methyl)phenoxy)acetate to triethylamine is 1:4.

More preferably, the molar ratio of ethyl 2-(4-((4-hydroxyphenyl)(pyridin-2-yl)methyl)phenoxy)acetate, triethylamine and chlorosulfonic acid is 1～ 1.5:2～5:1～2.

Most preferably, the mol ratio of described 2-(4-((4-hydroxyphenyl)(pyridin-2-yl)methyl)phenoxy)acetate, triethylamine and chlorosulfonic acid is 1: 4:1.5.

More preferably, the molar ratio of the 4-((4-(2-ethoxy-2-oxyethoxy)phenyl)(pyridin-2-yl)methyl)phenylsulfuric acid to methanol is 1～ 2:1 to 3.

Most preferably, the mol ratio of described 4-((4-(2-ethoxy-2-oxyethoxy) phenyl) (pyridin-2-yl) methyl) phenylsulfuric acid and methanol is 1: 1.

The structural formula of the ethyl 2-(4-((4-hydroxyphenyl)(pyridin-2-yl)methyl)phenoxy)acetate is

The structural formula of the 4-((4-(2-ethoxy-2-oxyethoxy) phenyl) (pyridin-2-yl) methyl) phenyl sulfuric acid is

As a preferred embodiment, the preparation of sodium 4-((4-((5-carboxypentyl)oxy)phenyl)(pyridin-2-yl)methyl)phenyl sulfate (PC3) The method is to use dichloromethane as a solvent, mix ethyl 6-(4-((4-hydroxyphenyl)(pyridin-2-yl)methyl)phenoxy)hexanoate and triethylamine, add chlorosulfonic acid The acid was reacted under stirring at room temperature for 3 to 5 h, and ethyl 6-(4-(pyridin-2-yl(4-(sulfooxy)phenyl)methyl)phenoxy)hexanoate was obtained by separation and purification. Dissolve ethyl 6-(4-(pyridin-2-yl(4-(sulfooxy)phenyl)methyl)phenoxy)hexanoate in methanol, add 1 mol/L aqueous sodium hydroxide solution at room temperature The reaction was stirred for 3 to 5 hours, and after the reaction was completed, the pH was adjusted to 6 to 7 with 1 mol/L hydrochloric acid.

More preferably, the molar ratio of ethyl 6-(4-((4-hydroxyphenyl)(pyridin-2-yl)methyl)phenoxy)hexanoate to triethylamine is 1:2～5 .

Most preferably, the molar ratio of ethyl 6-(4-((4-hydroxyphenyl)(pyridin-2-yl)methyl)phenoxy)hexanoate to triethylamine is 1:4.

More preferably, the molar ratio of ethyl 6-(4-((4-hydroxyphenyl)(pyridin-2-yl)methyl)phenoxy)hexanoate, triethylamine and chlorosulfonic acid is 1 ~1.5:2~5:1~2.

Most preferably, the molar ratio of ethyl 6-(4-((4-hydroxyphenyl)(pyridin-2-yl)methyl)phenoxy)hexanoate, triethylamine and chlorosulfonic acid is 1 :4:1.5.

More preferably, the molar ratio of the 4-(((4-((6-ethoxy-6-oxyhexyl)oxy)phenyl)(pyridin-2-yl)methyl)phenylsulfuric acid to methanol It is 1～2:1～3.

Most preferably, the molar ratio of the 4-(((4-((6-ethoxy-6-oxyhexyl)oxy)phenyl)(pyridin-2-yl)methyl)phenylsulfuric acid to methanol 1:1.

The structural formula of the ethyl 6-(4-((4-hydroxyphenyl)(pyridin-2-yl)methyl)phenoxy)hexanoate is

The structural formula of ethyl 6-(4-(pyridin-2-yl(4-(sulfooxy)phenyl)methyl)phenoxy)hexanoate is

A kind of sodium picosulfate hapten, is characterized in that, the structural formula of described sodium picosulfate hapten is as shown in formula (II) or formula (IV):

The sodium picosulfate hapten of formula (II) is named 2-(4-((4-hydroxyphenyl)(pyridin-2-yl)methyl)phenoxy)acetic acid by systematic nomenclature.

The sodium picosulfate hapten of formula (IV) is named 2-(4-((4-(benzyloxy)phenyl)(pyridin-2-yl)methyl)phenoxy)acetic acid using systematic nomenclature .

As a preferred embodiment, the preparation method of the 2-(4-((4-hydroxyphenyl)(pyridin-2-yl)methyl)phenoxy)acetic acid (PC2) is as follows: 2- (4-((4-hydroxyphenyl)(pyridin-2-yl)methyl)phenoxy) ethyl acetate, dissolved in methanol, and then added 1 mol/L sodium hydroxide in a volume ratio of 1:1 The aqueous solution was stirred for reaction at room temperature, and after the reaction was completed, the pH was adjusted to 6-7 with 1 mol/L hydrochloric acid to obtain 2-(4-((4-hydroxyphenyl)(pyridin-2-yl)methyl)phenoxy ) acetic acid.

Preferably, the molar ratio of ethyl 2-(4-((4-hydroxyphenyl)(pyridin-2-yl)methyl)phenoxy)acetate to methanol is 1-2:1-3.

Preferably, the molar ratio of ethyl 2-(4-((4-hydroxyphenyl)(pyridin-2-yl)methyl)phenoxy)acetate to sodium hydroxide is 1-1.5:1-2.

As a preferred embodiment, the preparation method of the 2-(4-((4-(benzyloxy)phenyl)(pyridin-2-yl)methyl)phenoxy)acetic acid (PC4) is as follows : Fully dissolve 4-((4-(benzyloxy)phenyl)(pyridin-2-yl)methyl)phenol in DMF, add cesium carbonate and ethyl bromoacetate, and react at 50～60℃ for 3～5h After the reaction, the solvent DMF was removed, extracted with water and ethyl acetate, the organic phases were combined, dried with anhydrous sodium sulfate, and the ethyl acetate was removed by rotary evaporation to obtain the intermediate product 2-(4-((4-(benzyloxy) )phenyl)(pyridin-2-yl)methyl)phenoxy)ethyl acetate. Dissolve ethyl 2-(4-((4-(benzyloxy)phenyl)(pyridin-2-yl)methyl)phenoxy)acetate in methanol, 2-(4-((4-(benzyl)acetate The volume ratio of oxy)phenyl)(pyridin-2-yl)methyl)phenoxy)ethyl acetate to methanol was 1:1, and 1 mol/L aqueous sodium hydroxide solution was added and the reaction was stirred at room temperature for 3～5h , after the reaction is completed, the pH is adjusted to 6-7 with 1 mol/L hydrochloric acid.

More preferably, the molar ratio of 4-((4-(benzyloxy)phenyl)(pyridin-2-yl)methyl)phenol, cesium carbonate and ethyl bromoacetate is 1～2:1～1.5 : 1 to 2.

Most preferably, the molar ratio of 4-((4-(benzyloxy)phenyl)(pyridin-2-yl)methyl)phenol, cesium carbonate and ethyl bromoacetate is 1:1.2:1.

More preferably, the molar ratio of ethyl 2-(4-((4-(benzyloxy)phenyl)(pyridin-2-yl)methyl)phenoxy)acetate to sodium hydroxide is 1～ 2:1-2.

The invention also provides a sodium picosulfate artificial antigen PC1-carrier protein or PC3-carrier protein, which is obtained by coupling the carrier protein with the sodium picosulfate hapten PC1 or PC3;

Its structural formula is shown in formula (V) or formula (VI):

As a preferred embodiment, the preparation method of the artificial antigen PC1-carrier protein of sodium picosulfate specifically comprises the following steps:

(1) Dissolve PC1, NHS and EDC in 50-200 μL DMF, and stir at room temperature in the dark for 2-4 h to obtain PC1 activation solution;

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

(3) Slowly add PC1 activation solution dropwise to the carrier protein solution in step (2), and react at 4°C for 12 hours;

(4) Dialyze with PBS buffer for two days, 4 times a day. After the dialysis, the protein solution was adjusted to 2 mL to obtain 5 mg/mL protein conjugate, that is, sodium picosulfate immunogen PC1-carrier protein. Store in a centrifuge tube at -20°C for later use.

Preferably, the mass ratio of PC1, NHS and EDC in step (1) is 1:1.1-2:1-2.1.

More preferably, the mass ratio of PC1, NHS and EDC in step (1) is 1:1.4:1.6.

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

Preferably, the mass ratio of PC1 in step (1) to the carrier protein in step (2) is 1-2:1-4.

More preferably, the mass ratio of PC1 in step (1) to the carrier protein in step (2) is 1:3.

As a preferred embodiment, the preparation method of the sodium picosulfate artificial antigen PC3-carrier protein is the same as the preparation method of PC1-carrier protein, that is, PC1 is replaced by PC3 for the preparation of PC3-carrier protein.

The invention also provides a sodium picosulfate artificial antigen PC2-carrier protein or PC4-carrier protein, which is obtained by coupling the carrier protein with the sodium picosulfate hapten PC2 or PC4;

Its structural formula is shown in formula (VII) or formula (VIII):

As a preferred embodiment, the preparation method of the artificial antigen PC2-carrier protein of sodium picosulfate specifically comprises the following steps:

(1) Dissolve PC2, NHS and EDC in 50-200 μL DMF, and stir at room temperature in the dark for 2-4 h to obtain PC1 activation solution;

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

(3) Slowly add PC2 activation solution dropwise to the carrier protein solution in step (2), and react at 4°C for 12h;

(4) Dialyze with PBS buffer for two days, 4 times a day. After the dialysis, the protein solution was adjusted to 2 mL to obtain 5 mg/mL protein conjugate, that is, sodium picosulfate immunogen PC1-carrier, which was divided into centrifuge tube and store at -20°C for later use.

Preferably, the mass ratio of PC2 in step (1) to the carrier protein in step (2) is 1-2:1-2.

More preferably, the mass ratio of PC2 in step (1) to the carrier protein in step (2) is 1:1.6.

Preferably, the sodium picosulfate artificial antigen is characterized in that the carrier protein is bovine serum albumin (Bovine serum albumin, BSA), keyhole limpet hemocyanin (Keyhole limpet hemocyanin, KLH), lactoferrin (Lactoferrin, LF) or chicken egg albumin (ovalbumin, OVA) any one or more.

As a preferred embodiment, the preparation method of the sodium picosulfate artificial antigen PC4-carrier protein is the same as the preparation method of PC2-carrier protein, that is, PC2 is replaced by PC4 for the preparation of PC4-carrier protein .

The present invention also provides a sodium picosulfate antibody, which is prepared by using the artificial antigen of sodium picosulfate described in PC1-carrier protein, PC2-carrier protein, PC3-carrier protein or PC4-carrier protein;

Preferably, the sodium picosulfate antibody is any one or more of monoclonal antibodies and polyclonal antibodies.

As a preferably embodiment, the preparation method of the sodium picosulfate polyclonal antibody specifically comprises the following steps:

(1) Combine the prepared immunogen PC1-conjugated lactoferrin (PC1-LF) with an equal amount of immune adjuvant (incomplete Freund's adjuvant for the first immunization, and incomplete Freund's adjuvant for subsequent booster immunizations) agent) emulsified uniformly and immunized animals. New Zealand white rabbits weighing 2.5 to 3 kg were immunized by subcutaneous injection on the back, subcutaneous in various parts, leg muscle and ear vein. The second immunization was performed after 4 weeks, and the booster immunization was performed every 3 weeks thereafter. One week after the third booster immunization, blood was collected from the marginal ear vein, and the serum titer was determined by indirect competitive ELISA. When the titer no longer rises, boost the immunization with the ear vein.

(2) One week after boosting immunization, blood was collected from the heart, water bathed for 0.5 to 1 h, centrifuged at 4°C and 10000 rpm for 15 min, and the supernatant was taken as antiserum. The antiserum was purified by ammonium sulfate precipitation to the polyclonal antibody and stored at -20°C for future use.

The present invention also provides any one or more of the sodium picosulfate artificial antigens and/or sodium picosulfate antibodies in food and/or health care products containing sodium picosulfate for rapid immune detection, and/or Application in the preparation of sodium picosulfate immunoassay products.

The present invention also provides an artificial antigen group for immunodetection of sodium picosulfate, the artificial antigen group contains PC1-carrier protein or PC3-carrier protein as immunogen and PC2-carrier protein or PC4 as coating original - Carrier protein.

More preferably, the immunogen in the artificial antigen group is the artificial antigen of the sodium picosulfate hapten PC1 coupled lactoferrin (PC1-LF); the coating was originally the sodium picosulfate hapten PC2 coupled chicken egg Artificial antigen for albumin (PC2-OVA).

The invention also provides the application of the artificial antigen group in the rapid immune detection of sodium picosulfate in food and/or health care products, or in the preparation of sodium picosulfate immune detection products.

The present invention also provides a sodium picosulfate immunoassay kit, which is prepared from the above artificial antigen group.

Preferably, the sodium picosulfate immunoassay kit is an enzyme-linked immunosorbent assay kit and/or a colloidal gold rapid detection kit.

More preferably, the sodium picosulfate immunoassay kit is an enzyme-linked immunosorbent assay kit containing:

ELISA plate coated with artificial antigen, sodium picosulfate standard solution, sodium picosulfate antibody, enzyme conjugate concentrate, enzyme conjugate dilution, substrate color developing solution, stop solution, washing solution; The artificial antigen is the above-mentioned coated original PC2-carrier protein; the enzyme conjugate is the sodium picosulfate antibody labeled with horseradish peroxidase.

Further preferably, the sodium picosulfate antibody is a sodium picosulfate polyclonal antibody prepared by using the immunogen PC1-LF.

Further preferably, the coating is originally PC2-OVA.

Preferably, the sodium picosulfate immunoassay kit is a colloidal gold rapid detection kit, and the colloidal gold rapid detection kit contains:

Bottom plate and sample pads, binding pads, cellulose membranes and water-absorbing pads arranged in sequence on the bottom plate, the above-mentioned sodium picosulfate antibody labeled with colloidal gold is adsorbed in the binding pad, and invisible detection lines are printed on the cellulose membrane and invisible quality control line, the invisible detection line is printed with artificial antigen solution, and the invisible quality control line is printed with goat anti-rabbit antibody; the artificial antigen is that the artificial antigen is the above-mentioned coated original PC2-carrier protein .

Further preferably, the sodium picosulfate antibody is a sodium picosulfate polyclonal antibody prepared by using the immunogen PC1-LF.

Further preferably, the coating is originally PC2-OVA.

The present invention also provides the application of the sodium picosulfate immunological detection kit in the rapid immunological detection of sodium picosulfate in food and/or health care products.

Preferably, the enzyme-linked immunosorbent assay kit and/or the colloidal gold rapid detection card in the sodium picosulfate immunoassay kit is provided for the rapid immunoassay of sodium picosulfate in food and/or health products applications in .

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

The invention prepares the sodium picosulfate haptens PC1, PC2, PC3 and PC4, and uses the haptens PC1, PC2, PC3 and PC4 to couple with the carrier protein to obtain artificial antigens, wherein the hapten PC1 or PC3 is coupled with the carrier protein to obtain immunity The original; PC2 or PC4 is coupled to the carrier protein to obtain the coating original, and the immunogen is used to prepare the sodium picosulfate antibody, which has high sensitivity and high specificity for the recognition of sodium picosulfate, and the half inhibitory concentration is 5ng/mL. , the minimum detection limit is 0.10ng/mL, and the cross-reaction rates to structural analogs are all less than 10%, indicating that the sodium picosulfate antibody has extremely high specificity for sodium picosulfate and can effectively exclude its analogs The interference of sodium picosulfate provides a core reagent for the establishment of an enzyme-linked immunosorbent assay for sodium picosulfate.

In addition, the present invention utilizes the sodium picosulfate antibody to develop the application of the sodium picosulfate immunodetection kit in the rapid immunological detection of sodium picosulfate in food and/or health care products. The enzyme-linked immune kit and the colloidal gold rapid detection kit developed by the invention can specifically identify the sodium picosulfate, and have high detection sensitivity for the sodium picosulfate.

Description of drawings

Fig. 1 is the synthetic route of sodium picosulfate immunogen PC1-LF.

Figure 2 shows the UV spectra of LF, PC1 and PC1-LF.

Figure 3 shows the UV spectra of LF, PC3 and PC3-LF.

Fig. 4 is the synthetic route of the original PC2-OVA coated with sodium picosulfate.

Figure 5 shows the UV spectra of OVA, PC2 and PC2-OVA.

Figure 6 shows the UV spectra of OVA, PC4 and PC4-OVA

Figure 7 is a standard inhibition curve of sodium picosulfate antibody to sodium picosulfate.

Figure 8 is a schematic side view of a sodium picosulfate colloidal gold immunochromatographic test strip, wherein 1: PVC bottom plate; 2: Sample pad; 3: Binding pad; 4: NC membrane; 5: Detection line (T point); 6 : quality control line (point C); 7: absorbent pad.

FIG. 9 is a diagram showing the determination of the detection result of the sodium picosulfate colloidal gold immunochromatographic test strip.

Detailed ways

The present invention is further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.

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

Example 1 Synthesis and identification of sodium picosulfate hapten

Using the structural characteristics of sodium picosulfate, two artificial haptens were designed on the basis of experimental data.

1. Synthesis and identification of hapten 4-((4-(carboxymethoxy)phenyl)(pyridin-2-yl)methyl)phenylsodium sulfate (PC1)

The specific synthesis steps include:

Take 2-(4-((4-hydroxyphenyl)(pyridin-2-yl)methyl)phenoxy)ethyl acetate (1moL), triethylamine (4moL), use anhydrous dichloromethane as solvent , reacted with chlorosulfonic acid (1.5moL) under stirring at room temperature for 3~5h, and separated and purified to obtain 4-((4-(2-ethoxy-2-oxyethoxy)phenyl)(pyridin-2-yl) ) methyl) phenyl sulfuric acid. 4-((4-(2-Ethoxy-2-oxyethoxy)phenyl)(pyridin-2-yl)methyl)phenylsulfuric acid was dissolved in methanol, 4-((4-(2- The volume ratio of ethoxy-2-oxyethoxy)phenyl)(pyridin-2-yl)methyl)phenylsulfuric acid to methanol was 1:1, and 1mol/L aqueous sodium hydroxide solution was added and stirred at room temperature The reaction was carried out for 3 to 5 hours, and after the reaction was completed, the pH was adjusted to 6 to 7 with 1 mol/L hydrochloric acid, and the result was obtained.

4-((4-(Carboxymethoxy)phenyl)(pyridin-2-yl)methyl)phenylsulfate sodium NMR results: ¹ H NMR (600MHz, Methanol-d ₄ )δ4.07(q,J =7.1Hz,1H),9.74～-1.67(m,9H),3.24(q,J=7.3Hz,11H), 1.37～1.28(m,18H).

Sodium 4-((4-(carboxymethoxy)phenyl)(pyridin-2-yl)methyl)phenyl sulfate MS: MS ^: _C20H17NO7S ^: _415.07 , ESI-[MH] _- ::414.0.

2. Synthesis and identification of hapten 2-(4-((4-hydroxyphenyl)(pyridin-2-yl)methyl)phenoxy)acetic acid (PC2)

The specific synthesis steps include:

Ethyl 2-(4-((4-hydroxyphenyl)(pyridin-2-yl)methyl)phenoxy)acetate, dissolved in methanol, 2-(4-((4-hydroxyphenyl)( The volume ratio of pyridin-2-yl)methyl)phenoxy)ethyl acetate to methanol was 1:1, and 5-10 mL of 1 mol/L sodium hydroxide aqueous solution was added, and the reaction was stirred at room temperature for 3-5 h. After the reaction was completed Adjust the pH to 6-7 with 1 mol/L hydrochloric acid to obtain 2-(4-((4-hydroxyphenyl)(pyridin-2-yl)methyl)phenoxy)acetic acid.

NMR results of 2-(4-((4-hydroxyphenyl)(pyridin-2-yl)methyl)phenoxy)acetic acid: ¹ H NMR (600MHz, Acetone-d ₆ )δ8.53(ddd, J= 4.8,1.9,0.9Hz,3H),7.70(ddt,J=9.5,7.7,1.7Hz,3H),7.24～7.19(m,4H),7.19～7.13(m,7H),7.09～7.03(m, 6H), 6.90～6.85(m, 6H), 6.80～6.75(m, 6H), 5.56(t, J=2.6Hz, 3H), 4.69(s, 6H), 4.20(q, J=7.1Hz, 6H) ),3.64～3.56(m,1H),1.98(dd,J=3.6,0.8Hz,1H),1.45～1.39(m,1H),1.39～1.29(m,9H),1.25(td,J=7.1 ,0.7Hz,11H),1.18～1.11(m,2H),0.93～0.87(m,4H).

Mass spectrum results of 2-(4-((4-hydroxyphenyl)(pyridin-2-yl)methyl)phenoxy)acetic acid:

MS: C20H17NO4: 337.15, ESI-[MH] ^{- :} 336.4.

3. Synthesis and identification of hapten 4-((4-((5-carboxypentyl)oxy)phenyl)(pyridin-2-yl)methyl)phenylsodium sulfate

The specific synthesis steps include:

Take ethyl 6-(4-((4-hydroxyphenyl)(pyridin-2-yl)methyl)phenoxy)hexanoate (1moL), triethylamine (4moL), and anhydrous dichloromethane as solvent, react with chlorosulfonic acid (1.5moL) under stirring at room temperature for 3 to 5 h, and separate and purify to obtain 6-(4-(pyridin-2-yl(4-(sulfooxy)phenyl)methyl)phenoxy ) ethyl hexanoate. Dissolve ethyl 6-(4-(pyridin-2-yl(4-(sulfooxy)phenyl)methyl)phenoxy)hexanoate in methanol, 6-(4-(pyridin-2-yl( The volume ratio of ethyl 4-(sulfooxy)phenyl)methyl)phenoxy)hexanoate to methanol was 1:1, and 1 mol/L aqueous sodium hydroxide solution was added to stir the reaction at room temperature for 3 to 5 hours. After the end, adjust the pH to 6-7 with 1 mol/L hydrochloric acid.

4-((4-((5-carboxypentyl)oxy)phenyl)(pyridin-2-yl)methyl)phenylsulfate sodium NMR results: 1H NMR (600MHz, Acetone-d6)δ4.06( q,J＝7.1Hz,2H),3.32(s,1H),2.86(s,73H),2.20～2.13(m,1H),2.10(s,9H),2.06～2.02(m,11H),1.97 (s,3H),1.88(s,1H),1.70(s,1H),1.60(s,1H),1.60～1.57(m,2H),1.39(s,4H),1.30(d,J=4.0 Hz, 23H), 1.21 (t, J=7.1Hz, 6H), 0.98 (dd, J=12.6, 7.1Hz, 4H), 0.92-0.83 (m, 18H), 0.14 (s, 13H).

Sodium 4-((4-((5-carboxypentyl)oxy)phenyl)(pyridin- ₂ -yl)methyl)phenyl sulfate MS: MS: _C24H25NO7S : _471.13 , ESI ^- [MH] ^- :469.9.

4. Synthesis and identification of hapten 2-(4-((4-(benzyloxy)phenyl)(pyridin-2-yl)methyl)phenoxy)acetic acid

The specific synthesis steps include:

Fully dissolve 4-((4-(benzyloxy)phenyl)(pyridin-2-yl)methyl)phenol in DMF, add cesium carbonate (1.2moL) and ethyl bromoacetate (1.3moL), 50～ The reaction was carried out at 60 °C for 3 to 5 h. After the reaction was completed, the solvent DMF was removed, extracted with water and ethyl acetate, the organic phases were combined, dried with anhydrous sodium sulfate, and the ethyl acetate was removed by rotary evaporation to obtain the intermediate product 2-(4-( (4-(benzyloxy)phenyl)(pyridin-2-yl)methyl)phenoxy)ethyl acetate. Dissolve ethyl 2-(4-((4-(benzyloxy)phenyl)(pyridin-2-yl)methyl)phenoxy)acetate in methanol, 2-(4-((4-(benzyl)acetate The volume ratio of oxy)phenyl)(pyridin-2-yl)methyl)phenoxy)ethyl acetate to methanol was 1:1, and 1 mol/L aqueous sodium hydroxide solution was added and the reaction was stirred at room temperature for 3～5h , after the reaction, adjust the pH to 6-7 with 1 mol/L hydrochloric acid to obtain 2-(4-((4-(benzyloxy)phenyl)(pyridin-2-yl)methyl)phenoxy)acetic acid .

NMR results of 2-(4-((4-(benzyloxy)phenyl)(pyridin-2-yl)methyl)phenoxy)acetic acid: 1H NMR(600MHz, Methanol-d4)δ8.56(s, 1H), 8.46(ddd, J＝5.0, 1.9, 0.9Hz, 3H), 7.77(td, J＝7.7, 1.9Hz, 4H), 7.46～7.41(m, 5H), 7.37(dd, J＝8.4, 6.8Hz,6H),7.34～7.26(m,6H),7.16(dt,J=8.0,1.1Hz,4H),7.05～7.00(m,9H),6.98～6.92(m,6H),6.92～6.86 (m, 6H), 6.70(d, J=8.6Hz, 2H), 5.59(s, 3H), 5.50(s, 1H), 5.23(s, 1H), 5.08(s, 5H), 4.37(s, 5H), 3.89(d, J＝9.0Hz, 2H), 3.66～3.59(m, 2H), 3.37(s, 2H), 2.91(d, J＝7.5Hz, 1H), 2.80(s, 1H), 2.38(s, 1H), 2.21(t, J=7.7Hz, 2H), 2.19～2.14(m, 2H), 2.04(s, 1H), 1.91(s, 3H), 1.61(s, 1H), 1.38 ～1.29(m, 8H), 1.24～1.12(m, 4H), 1.10(s, 1H), 0.92(t, J=6.8Hz, 2H).

2-( ₄ -((4-(benzyloxy)phenyl)(pyridin-2-yl)methyl)phenoxy)acetic acid mass spectrum result: MS: _C27H23NO4 ^: _425.16 , ESI-[MH ] ^- : 424.6.

Example 2 Synthesis and identification of sodium picosulfate artificial antigen

1. Synthesis of Sodium Picosulfate Artificial Antigen

The synthetic method of sodium picosulfate artificial antigen, comprises the following steps:

Taking PC1, PC2, PC3, PC4 as the hapten in Example 1, respectively coupled lactoferrin (LF) and chicken ovalbumin (OVA) by the active ester method, respectively take by weighing the above-mentioned 1moL sodium picosulfate hapten, 1.4moL of NHS and 1.6moL of EDC were dissolved in 50-200μL DMF, and stirred at room temperature in the dark for 2-4h to obtain sodium picosulfate hapten activation solution; 10mg LF or OVA was added to 1mL of PBS buffer (0.01moL/ L, pH=7.4); slowly add sodium picosulfate hapten activation solution dropwise to LF solution, react at 4°C for 12 h; dialyze with PBS buffer for 3 days, 3 times a day, after dialysis, get picosulfate Sodium artificial antigen, aliquoted into centrifuge tubes, and stored at -20°C for use.

Among them, the formula of PBS buffer: Na ₂ HPO ₄ ·12H ₂ O 2.90g, NaCl 8.50g, KCl 0.20g, KH ₂ PO ₄ 0.20g, add distilled water to make up to 1000mL.

Among them, the best combination of two artificial antigens of sodium picosulfate is artificial antigen 4-((4-(carboxymethoxy)phenyl)(pyridin-2-yl)methyl)phenylsodium sulfate-LF (PC1 -LF) (see Fig. 1 for the synthetic route) and 2-(4-((4-hydroxyphenyl)(pyridin-2-yl)methyl)phenoxy)acetic acid-OVA(PC2-OVA) (see the synthetic route Figure 4) (see Example 4 for details).

2. Identification of Sodium Picosulfate Artificial Antigen

Take the PC1-LF synthesized above, carry out UV scanning, the result is shown in Figure 2.

Specifically, LF, PC1, and PC1-LF were identified by ultraviolet (200-350 nm) scanning respectively, and by comparing the highest absorbance values of each substance before and after coupling, it was found that the absorption curve of the sodium picosulfate immunogen PC1-LF was similar to that of the carrier. The protein LF is obviously different. PC1 has a characteristic peak at 240nm and 300nm. After the coupling reaction, the absorption peak of PC1-LF is significantly higher than that of LF at 240nm and 300nm. displacement. Since the unreacted drug and other small molecule components have been all removed by dialysis in the dialysis process after coupling, the drug characteristic peaks appearing in the coupling product are contributed by protein-bound drug molecules, indicating that the reaction product is the carrier protein and PC1 The complex was successfully coupled.

Take the PC3-LF synthesized above, carry out UV scanning, the result is shown in Figure 3.

Specifically, LF, PC3, and PC3-LF were identified by ultraviolet (200-350 nm) scanning respectively, and by comparing the highest absorbance values of each substance before and after coupling, it was found that the absorption curve of the sodium picosulfate immunogen PC3-LF was similar to that of the carrier. The protein LF is obviously different. PC3 has a characteristic peak at 240nm and 260nm. After the coupling reaction, the absorption peak of PC3-LF is significantly higher than that of LF at 240nm and 260nm. displacement. Since the unreacted drug and other small molecule components have been removed by dialysis in the dialysis process after coupling, the drug characteristic peaks appearing in the coupling product are contributed by protein-bound drug molecules, indicating that the reaction product is the carrier protein and PC3 The complex was successfully coupled.

Take the PC2-OVA synthesized above, carry out UV scanning, and the results are shown in Figure 5.

Specifically, OVA, PC2, and PC2-OVA were identified by ultraviolet (200-350 nm) scanning respectively, and by comparing the highest absorbance values of each substance before and after coupling, it was found that the absorption curve of the original PC2-OVA coated with sodium picosulfate was similar to that of the original PC2-OVA. The carrier protein OVA is obviously different. PC2 has a characteristic peak at 350nm, and the carrier protein OVA only has a characteristic peak at 280nm. After the coupling reaction, PC2-OVA has an obvious absorption peak at 350nm, and the curve of PC2 can be seen. significant displacement occurred. Since the unreacted drug and other small molecule components have been removed by dialysis in the dialysis process after coupling, the drug characteristic peaks in the coupling product are contributed by the protein-bound drug molecules, so the reaction product is the carrier protein and PC2. The complex was successfully coupled.

Take the PC4-OVA synthesized above and carry out UV scanning, and the results are shown in Figure 6.

Specifically, OVA, PC4, and PC4-OVA were identified by UV (200-350 nm) scanning respectively, and by comparing the highest absorbance values of each substance before and after coupling, it was found that the absorption curve of the original PC4-OVA coated with sodium picosulfate was the same as that of the original PC4-OVA. The carrier protein OVA is obviously different. PC4 has a characteristic peak at 210nm, and carrier protein OVA has characteristic peaks at 240nm and 280nm. After the coupling reaction, PC4-OVA has obvious absorption peaks at 220nm, 240nm and 260nm, and Comparing the curves for PC4 shows a significant shift. Since the unreacted drug and other small molecular components have been removed by dialysis in the dialysis process after coupling, the drug characteristic peaks in the coupling product are contributed by protein-bound drug molecules, indicating that the reaction product is the carrier protein and PC4 The complex was successfully coupled.

Example 3 Preparation of sodium picosulfate antibody

The prepared immunogen PC1-LF and immune adjuvant (incomplete Freund's adjuvant for the first immunization, and incomplete Freund's adjuvant for subsequent booster immunizations) were emulsified uniformly in a volume ratio of 1:1, and New Zealand white rabbit. The New Zealand white rabbit weighs 2.5-3 kg, and is injected subcutaneously at multiple points on the neck and back. The second immunization is performed after 4 weeks, and the booster immunization is performed every 3 weeks thereafter. One week after the third booster immunization, blood was collected from the marginal ear vein, and the serum titer was determined by indirect competitive ELISA. When the titer no longer rises, boost the immunization with the ear vein. One week later, blood was collected from the heart, and the collected blood was obtained by incubating the blood for 0.5 to 1 h at 37 °C, then standing at 4 °C overnight, and then sucking the precipitated serum with a pipette, and then at 4 °C for 3000 ~ Centrifuge at 5000 rpm for 10 min and take the supernatant. The antiserum was purified by ammonium sulfate precipitation to the polyclonal antibody and stored at -20°C for future use.

Example 4 Optimization of the combination of sodium picosulfate immunogen and coating original

Sodium picosulfate artificial antigen: 4-((4-(carboxymethoxy)phenyl)(pyridin-2-yl)methyl)phenylsodium sulfate-LF(PC1-LF), 2-(4 -((4-hydroxyphenyl)(pyridin-2-yl)methyl)phenoxy)acetic acid-LF(PC2-LF), 4-((4-((5-carboxypentyl)oxy)benzene yl)(pyridin-2-yl)methyl)phenyl sulfate-LF(PC3-LF), 2-(4-((4-(benzyloxy)phenyl)(pyridin-2-yl)methyl ) phenoxy) acetic acid-LF (PC4-LF) was used to immunize New Zealand white rabbits, and the obtained antibodies were screened for the coating of all structures. The titers and inhibition rates detected by ELISA were shown in Table 1.

The specific operation steps are as follows:

(1) Dilute sodium picosulfate antibody with PBST to 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, 1:256000, and set blank control wells at the same time (replaced with PBST);

(2) Sodium picosulfate artificial antigen 4-((4-(carboxymethoxy)phenyl)(pyridin-2-yl)methyl)phenylsulfate-OVA (PC1-OVA), 2-( 4-((4-Hydroxyphenyl)(pyridin-2-yl)methyl)phenoxy)acetic acid-OVA(PC2-OVA), 4-((4-((5-carboxypentyl)oxy) Phenyl)(pyridin-2-yl)methyl)phenylsulfate-OVA(PC3-OVA), 2-(4-((4-(benzyloxy)phenyl)(pyridin-2-yl)methyl Base)phenoxy)acetic acid-OVA (PC4-OVA) were diluted with coating solution (0.05M carbonate buffer solution, pH 9.6) to a concentration of 125ng/mL, coated with 96-well microtiter plate, and added to each well. 100 μL, incubate overnight at 37°C in a constant temperature water bath, discard the coating solution, and wash twice with PBST (0.01M PBS, 0.06% Tween-20 (v/v));

(3) Add 120 μL of blocking solution (1% isinglass) to each well, block at 37°C for 3 hours, discard the blocking solution, clasp the plate, and dry it in a drying oven at 37°C for later use;

(4) Dilute 1 mg/mL sodium picosulfate 1000 times with PBST to 1 μg/mL;

(5) Add 50 μL sodium picosulfate diluent to each row (three groups in parallel), add 50 μL PBST diluent per well, incubate at 37°C for 40 min, and wash 5 times;

(6) Add goat anti-rabbit secondary antibody IgG-HRP (5000-fold dilution), incubate at 37°C for 30 minutes, wash 5 times, and pat plate;

(7) Add color developing solution, and warm bath for color development at 37°C for 10min;

(8) Add 50 μL of 10% H ₂ SO ₄ to stop the reaction, and read the OD value at 450 nm;

Experimental results: The test results of the titer inhibition rate of the antiserum obtained by immunizing New Zealand white rabbits are shown in Table 1. The antiserum produced by the rabbits immunized with different sodium picosulfate artificial antigens as immunogens has a certain titer. , the antiserum obtained has different degrees of inhibitory effect on the target analyte sodium picosulfate. Among them, the antiserum titer of 1:32000 and the inhibition rate of 75.0% shown by the combination of the immunogen and coating original structure number 1 is the best combination. Under this combination, the sodium picosulfate antibody can not only specifically recognize the target analysis Sodium picosulfate, and the antibody sensitivity is good. Both the antiserum titers and inhibition rates were higher than those of the immunogen and coat structure combinations numbered 2, 3, and 4, although the inhibition rates shown by the immunogen and coat structure combinations of numbers 5 and 6 were higher than those of the immunogen combination number 1. The inhibition rate shown by the combination of the original and the original coating structure is similar, but the antiserum titer of these two combinations is much lower than the antiserum titer of the combination of No. 1, so the combination of the immunogen of No. 1 and the original coating structure is the best. best combination.

Inhibition rate=(OD value of titer-OD value of inhibition)/OD value of inhibition*100%;

Table 1 The titer and inhibition rate data of 6 groups of immunogen and coating original combination of sodium picosulfate

Numbering	immunogen	original coating	potency		Inhibition rate
1	PC1-LF	PC2-OVA	1:32000	75%
2	PC2-LF	PC1-OVA	1:2000	36.5%
3	PC1-LF	PC4-OVA	1:8000	50.5%
4	PC3-LF	PC1-OVA	1:16000	44.5%

5	PC1-LF	PC4-OVA	1:1000	70%
6	PC3-LF	PC4-OVA	1:1000	60%

Example 5 Sensitivity and specificity determination of sodium picosulfate antibody

1. Sodium Picosulfate Antibody Sensitivity Determination

The determination of the sensitivity of sodium picosulfate antibody is expressed by establishing a standard curve of sodium picosulfate antibody (ELISA) and calculating the half-inhibitory concentration as _IC50 .

The specific steps of standard curve establishment include:

(1) The sodium picosulfate antibody prepared in Example 3 was diluted to 1:8000 with PBST, and a blank control well (replaced with PBST) was set at the same time;

(2) Dilute the sodium picosulfate artificial antigen PC2-OVA with coating solution to a concentration of 250ng/mL, coat a 96-well microtiter plate, add 100 μL to each well, incubate in a constant temperature water bath at 37°C for 12h, discard the package The quilt was washed twice with PBST (0.01M PBS, 0.06% Tween-20 (v/v)) and patted dry;

(3) Add 120 μL of blocking solution (1% isinglass solution) to each well, block at 37°C for 3 hours, discard the blocking solution, clasp the plate, and dry it in a drying oven at 37°C for later use;

(4) Dilute sodium picosulfate to 100000.00, 10000.00, 1000.00, 100.00, 10.00, 0.10, 0.01, 0ng/mL with PBST;

(5) Add 50 μL of sodium picosulfate diluent to each row, the concentrations are 100000.00, 10000.00, 1000.00, 100.00, 10.00, 0.10, 0.01ng/mL (three parallel groups), and add 50 μL/well to the wells with a concentration of 0ng/mL PBST dilution solution, then add the sodium picosulfate antibody dilution solution described in step (1), and add 50 μL to each well. After 40min incubation at 37°C, the liquid in the wells was discarded, washed 5 times with PBST (0.01M PBS, 0.06% Tween-20 (v/v)), and patted dry;

(6) Add goat anti-rabbit secondary antibody IgG-HRP (5000-fold dilution), incubate at 37°C for 30min, discard the liquid in the well, add PBST (0.01M PBS, 0.06% Tween-20 (v/v) ) washed 5 times and patted dry;

(7) Add 100 μL of chromogenic solution to each well, and incubate for 10 min at 37°C;

(8) Add 50 μL of stop solution (10% H ₂ SO ₄ ) to each well to stop the reaction, and read the OD value at 450 nm with a microplate reader;

The formula of PBST is: Na ₂ HPO ₄ ·12H ₂ O 14.50 g, NaCl 42.50 g, KCl 1.00 g, KH ₂ PO ₄ 1.00 g, Tween-20 3.0 mL, and distilled water was added to make up to 5000 mL.

1% isinglass solution preparation: such as dissolving 0.01g of isinglass powder in 1mL of PBST, the specific calculation is based on the actual amount.

Taking the OD value as the ordinate and the logarithmic value of the corresponding standard concentration as the abscissa, the four parameters of the origin software are used to perform curve fitting on the function: y=(AD)/[1+(X/C)B]+D, where , A and D represent the minimum and maximum absorbance values (OD) of the drug concentration respectively, C is the midpoint concentration, when the standard concentration is equal to C, the OD value is (A+D)/2, which is at the inflection point of the curve, The half-inhibitory concentration is IC ₅₀ , and B represents the steepness of the curve, which is called the slope factor: IC ₁₀ is the detection limit, and IC ₂₀ to IC ₈₀ is the detection range.

The standard curve of ELISA was established with sodium picosulfate as the standard, the lowest detection limit was 0.10ng/mL, and the half inhibitory concentration was 5ng/mL. Combining with Fig. 7, it can be seen that the standard curve established with sodium picosulfate as the standard has a typical S-shaped curve, and the detection sensitivity is good.

2. Sodium picosulfate antibody specificity assay

The specificity of the sodium picosulfate antibody was determined by the cross-reaction experiment between sodium picosulfate and its analogs, and the specificity of the antibody was expressed by the cross-reactivity ratio (CR). The smaller the cross-reaction, the better the specificity.

Sodium _picosulfate and its analogs were used as competing antigens, and serial dilutions were made respectively, and the indirect competitive ELISA method was used for determination. The cross-reactivity ratio (CR) of sodium picosulfate and each analog was calculated using the following formula:

The results of the cross-reaction experiment between sodium picosulfate and its analogs are shown in Table 1. It was found that the cross-reaction rate of sodium picosulfate antibody to sodium picosulfate was 100%, and the IC ₅₀ value was 5ng/mL. There is no reaction or the cross-reaction rate is less than 10%; it shows that the sodium picosulfate antibody has extremely high specificity for sodium picosulfate, which can effectively eliminate the interference of its analogs, and can be used exclusively for sodium picosulfate. detection.

The above results show that the sodium picosulfate antibody prepared by the present invention has strong detection specificity for sodium picosulfate.

Table 2 Experimental results of cross-reaction between sodium picosulfate and its analogs

Note: NR means no response.

Example 6 Development of Sodium Picosulfate ELISA Kit

1. Enzyme conjugate

The sodium picosulfate antibody prepared in Example 3 was labeled with horseradish peroxidase.

2. Preparation of ELISA plate

Dilute PC2-OVA to 1 μg/mL with coating buffer, add 100 μL to each well, incubate overnight at 37°C in the dark, pour off the liquid in the well, wash twice with washing solution for 30 s each time, pat dry, and then incubate in each well. Add 120 μL of blocking solution to the wells, incubate at 25°C in the dark for 2 h, pour off the liquid in the wells and pat dry, and store them in vacuum sealed with aluminum film after drying.

3. The establishment of an enzyme-linked immunosorbent assay kit for the detection of sodium picosulfate

An ELISA kit for the detection of sodium picosulfate was constructed, including the following components:

(1) ELISA plate coated with original PC2-OVA;

(2) 6 bottles of sodium picosulfate standard solution, the concentrations are 0μg/L, 0.1μg/L, 1μg/L, 10μg/L, 100μg/L, 1000μg/L.

(3) Enzyme conjugate: horseradish peroxidase-labeled sodium picosulfate antibody.

(4) The substrate color developing solution is composed of A solution and B solution, A solution is carbamide peroxide, and B solution is tetramethylbenzidine;

(5) The stop solution is 10% sulfuric acid;

(6) The washing solution is pH 7.4, containing 0.5%～1.0% Tween-20, 0.01‰～0.03‰ sodium azide preservative, 0.1～0.3mol/L phosphate buffer, and the percentage is weight volume percentage;

4. Actual sample testing

Number the corresponding microwells of the samples and standards in sequence, make two parallel wells for each sample and standard, and record the positions of the standard wells and the sample wells. Dilute the enzyme conjugate concentrate with the enzyme conjugate diluent according to the required amount at a volume ratio of 1:11 (i.e. 1 part of the enzyme conjugate concentrate is added to 11 parts of the enzyme conjugate diluent, which is prepared and used now). Add 50 μL of standard/sample to the corresponding microwells, then add 50 μL/well of enzyme conjugate working solution, gently shake and mix, cover the plate with a cover film, and place it in a dark environment at 25°C to react for 35 minutes. Drain the liquid in the wells, add 300 μL/well of washing working solution, wash thoroughly 4 to 5 times, every 10s interval, pour off the washing solution in the wells of the plate, and pat dry with absorbent paper (air bubbles that are not removed after patting dry can be used punctured by an unused tip). Add 50 μL/well of Substrate Solution A, and then add 50 μL/well of Substrate Solution B, gently shake to mix, cover the plate with a cover plate, and place it in a dark environment at 25°C for 10 minutes. Add 50 μL/well of stop solution, shake and mix gently, set the microplate reader at 450 nm, and measure the OD value of each well.

5. Analysis of test results

The percent absorbance of the standard or sample is equal to the average of the absorbance values of the standard or sample (double well) divided by the average of the absorbance values of the first standard (0 standard), and then multiplied by 100% to obtain the standard The percent absorbance value of the product or sample. Take the percentage absorbance of the standard product as the ordinate and the logarithm of the standard concentration of sodium picosulfate (μg/L) as the abscissa to draw the standard curve. Substitute the percent absorbance of the sample into the standard curve, read the concentration corresponding to the sample from the standard curve, and multiply the corresponding dilution factor to obtain the actual concentration of sodium picosulfate in the sample.

It can be seen from Figure 7 that the half inhibitory concentration (IC ₅₀ ) of the sodium picosulfate antibody is 5 ng/mL, and the minimum detection limit is 0.10 ng/mL; it shows that the sodium picosulfate antibody prepared by the present invention can meet the detection requirements, and is suitable for the detection of sodium picosulfate. Sodium picosulfate has a high-sensitivity identification ability, and the detection sensitivity of sodium picosulfate is high.

6. Add recovery experiment

Enzyme foods (including jelly, candy, preserves and beverages) were selected as spiked samples, spiked with three concentrations of 2, 10, and 20 μg/kg, respectively. Unspiked samples were set and verified to be free of sodium picosulfate. The samples were pretreated according to the sample extraction method in "Determination of Sodium Picosulfate in Food-BJS 201911".

Calculate according to the following recovery rate formula: recovery rate=(detected concentration of sodium picosulfate in spiked sample-detected concentration of sodium picosulfate in unspiked sample)/spiked concentration×100%.

Table 3 Sample addition recovery test results

Embodiment 7 The rapid detection method of sodium picosulfate colloidal gold

1. Preparation of gold-labeled antibodies and gold-labeled conjugate pads

A colloidal gold suspension with an average diameter of 40 nm was prepared by reducing chloroauric acid with trisodium citrate. Under reflux conditions, heat 100 mL of 0.01% chloroauric acid solution to boiling, keep stirring, and quickly add 1.1 mL of 1% trisodium citrate. When the color of the reaction solution became grape red, heating and stirring were continued for 5 min. After cooling to room temperature, 0.05% sodium azide was added and stored at 4°C.

Before the colloidal gold was labeled with the antibody prepared in Example 3, the pH was adjusted to about 8.2 with 0.2 mol of K ₂ CO ₃ solution, and 30 μg of the antibody was labeled with 1 mL of colloidal gold solution by the classical NaCl titration method. Then, label according to the optimal labeling amount. After labeling for 1 hour, add 10% BSA under stirring (to make the final BSA concentration 1%), incubate for 1 hour, centrifuge at 10000 rpm at 4°C for 25 minutes, and remove the supernatant. Add 5% BSA solution with the same volume of colloidal gold solution to resuspend, centrifuge at 10000rpm for 25min at 4°C, repeat twice. Finally, resuspend with 1/5 volume of colloidal gold solution in TB solution (containing 3% BSA, 3% sucrose, 0.01 mol/L sodium borate and 0.05% sodium azide), and store at 4°C. Use XYZ-3000 three-dimensional film sprayer to spray 4% BSA solution on glass wool at a volume of 8 μL/cm, use a drying oven to dry at 42°C for 50 min, and then spray the gold-labeled antibody at a volume of 6 μL/cm on the glass wool. , dried in a drying oven at 42 °C for 50 min, and stored under vacuum.

2. Coated cellulose membrane with conjugated antigen goat anti-rabbit

The coated antigen with a concentration of 1 mg/mL was sprayed on the lower side of the cellulose membrane at a concentration of 1.2 μL/cm using the XYZ-3000 three-dimensional spray film instrument as the detection line. Goat anti-rabbit IgG with a concentration of 120 μg/L was sprayed on the upper side of the cellulose membrane at a concentration of 1.2 μL/cm using an XYZ-3000 three-dimensional film sprayer, as a control line, with an interval of 8 mm between the two lines.

3. Assembly of the quick test strip

As shown in FIG. 8 , the cellulose film 4 was pasted on the middle part of the backing plate 1 , and the water-absorbing pad 7 was pasted on the upper side of the cellulose film 4 and overlapped with the cellulose film 4 by 1 mm. The gold-labeled conjugate pad 3 is pasted under the cellulose film 4 and overlapped by 1 mm. The sample pad 2 is pasted under the gold-labeled conjugate pad 3 and overlapped by 2mm. The assembled test board was cut into 3.05mm wide test strips with a cutter.

4. Preparation of test sample solution

1) Solid sample

Weigh 1g (accurate to 0.001g) of the sample into a 50mL centrifuge tube, add 10mL of water, water bath at 80°C for 10min, centrifuge at 8000r/min for 5min, collect the extract, add 20mL of water to wash the residue, vortex for 30s, centrifuge at 8000r/min For 5 min, the two extracts were combined. If the extract was turbid, an appropriate amount of the extract could be centrifuged at 8000 r/min for 5 min, and the supernatant was taken for testing.

2) Liquid samples

Weigh 1g (accurate to 0.001g) of the sample into a 50mL centrifuge tube, add 5mL of water accurately, bath at 80°C for 10min, let it cool and centrifuge at 8000r/min for 5min, and take the supernatant for testing.

5. Rapid detection test strip detection and judgment

Figure 9 shows the results of the rapid detection of the test strips. The specific detection and judgment method is as follows: when the sample solution to be tested is added to the test strip or the test end of 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 the cellulose by siphoning The membrane 4 diffuses and eventually penetrates the end of the absorbent pad 7 . During the diffusion process, if there is an analyte in the sample, the analyte binds to the gold-labeled antibody, which in turn occupies the antigen binding point on the gold-labeled antibody, preventing the gold-labeled antibody from interacting with the invisible detection line 5 on the cellulose membrane 4 ( The combination of the hapten and the carrier protein) makes the invisible detection line 5 show no color or very weak color, which means that the test sample is positive or weakly positive; if there is no sample to be tested in the sample, the gold-labeled antibody is in the process of moving up. , when the invisible detection line 5 shows a clear red line, it means that the test sample is negative. Likewise, the gold-labeled antibody also binds to the invisible control line 6 (goat anti-rabbit IgG) on the cellulose membrane 4, making the invisible control line 6 appear red. The presence or absence of the color of the invisible control line 6 respectively indicates the validity or invalidity of the test strip.

6. Determination of detection limit

The detection limits of sodium picosulfate colloidal gold immunochromatographic test strips for different samples are shown in Table 4. A series of concentrations of standard drugs were added to the blank solid and liquid samples. After sample pretreatment, the samples were detected with the above-mentioned colloidal gold test strips, and the visual detection limit was determined by qualitative judgment with the naked eye.

Table 4 Detection limits of sodium picosulfate colloidal gold immunochromatographic test strips for different samples

name	Solid sample detection limit (μg/g)	Liquid sample detection limit (μg/g)
Sodium Picosulfate	0.05	0.2

Claims (10)

1. A kind of sodium picosulfate hapten, is characterized in that, the structural formula of described sodium picosulfate hapten is as shown in formula (I) or formula (III):

   
2. A kind of sodium picosulfate hapten, is characterized in that, the structural formula of described sodium picosulfate hapten is as shown in formula (II) or formula (IV):

   
3. A kind of sodium picosulfate artificial antigen, it is characterised in that the sodium picosulfate artificial antigen is obtained for the sodium picosulfate hapten coupled carrier protein described in claim 1, and its structural formula is such as formula (V) or formula (VI) ) as shown:

   
4. A kind of sodium picosulfate artificial antigen, it is characterized in that, described sodium picosulfate artificial antigen is to obtain in claim 2 described sodium picosulfate hapten coupled carrier protein, and its structural formula is such as formula (VII) or formula ( VIII) shows:

   
5. The artificial antigen of sodium picosulfate according to claim 3 or 4, wherein the carrier protein is any one or more of bovine serum albumin, keyhole limpet hemocyanin, lactoferrin or chicken ovalbumin .
6. A kind of sodium picosulfate antibody is characterized in that, utilizes the artificial antigen of sodium picosulfate described in claim 3 and/or 4 to be prepared.
7. The sodium picosulfate antibody according to claim 6, wherein the antibody is any one or more of a monoclonal antibody and a polyclonal antibody.
8. A kind of artificial antigen group for immunodetection of sodium picosulfate, it is characterized in that, contain the artificial antigen of sodium picosulfate described in claim 3 as immunogen and the sodium picosulfate described in claim 4 as coating original artificial antigens.
9. A sodium picosulfate immunoassay kit, characterized in that, prepared by using the artificial antigen group of claim 8;

   The sodium picosulfate immunoassay kit is an enzyme-linked immunosorbent assay kit and/or a colloidal gold rapid detection kit.
10. Any one or more of the sodium picosulfate artificial antigen described in claim 3 or the sodium picosulfate artificial antigen described in claim 4 or the sodium picosulfate antibody described in claim 6 in food and/or containing health care products Rapid immunodetection of sodium picosulfate, and/or application in the preparation of sodium picosulfate immunoassay products.

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