CN112180091A - Colloidal gold kit for detecting infliximab in serum - Google Patents
Colloidal gold kit for detecting infliximab in serum Download PDFInfo
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- CN112180091A CN112180091A CN201910586707.8A CN201910586707A CN112180091A CN 112180091 A CN112180091 A CN 112180091A CN 201910586707 A CN201910586707 A CN 201910586707A CN 112180091 A CN112180091 A CN 112180091A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/715—Assays involving receptors, cell surface antigens or cell surface determinants for cytokines; for lymphokines; for interferons
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Abstract
The invention discloses a colloidal gold kit for detecting infliximab in serum. The colloidal gold kit comprises a colloidal gold detection test strip containing a colloidal gold pad for resisting the English fusiform idiotype antibody, and the concentration of the colloidal gold pad is 2.5-20 mu g/cm2The anti-infliximab idiotype antibody-colloidal gold complex of (a). The kit is simple and rapid to operate, and the treatment time is only 10-20 min; the result judgment is simple, clear and accurate; strong specificity, good reproducibility, strong stability of the kit and easy preservation.
Description
Technical Field
The invention belongs to the field of biotechnology detection, and particularly relates to a colloidal gold kit for detecting infliximab in serum.
Background
TNF- α, a cytokine with multiple biological activities, participates in resisting infection by bacteria, viruses and parasites, promotes tissue repair, causes apoptosis of tumor cells, etc. by binding to receptors. However, overexpression of TNF- α causes autoimmune diseases such as rheumatoid arthritis, myelitis ankylosis, and Inflammatory Bowel Disease (IBD). The anti-TNF-alpha monoclonal antibody becomes the first choice medicine for the diseases due to the advantages of strong specificity, long half life, small side effect and the like for the diseases. Currently, anti-TNF- α monoclonal antibody drugs available at home and abroad mainly include Infliximab (IFX), Adalimumab (ADA), Golimumab (Golimumab), cetuximab (Certolizumab pegol, CZP), ustenon monoclonal antibody (Ustekinumab), and the like.
The therapeutic effect of infliximab antibody drugs can be achieved mainly depending on the fact that the drugs reach and maintain sufficient therapeutic window concentration after entering a patient body, current researches show that the infliximab drug concentration is required to be maintained above 3 mu g/mL when the IBD patient maintains remission, and when the measured infliximab drug concentration of the IBD patient is lower than 3 mu g/mL, the mucosal healing rate of the patient can be remarkably improved by adopting dosage infusion or medication scheme adjustment for shortening the administration interval. Therefore, accurate and rapid evaluation of the in vivo concentration of infliximab drugs is of great significance for obtaining better curative effect of patients and treatment decision of clinicians.
At present, the detection methods of antibody drugs mainly comprise the following methods: enzyme-linked immunosorbent assay (ELISA), electrochemiluminescence immunoassay (ECL), Radioimmunoassay (RIA), and Surface Plasmon Resonance (SPR). The four methods all need expensive instruments and equipment, and have higher requirements on the professional skills of detection personnel. Meanwhile, the pretreatment of the sample is complicated and time-consuming, the detection cost is high, the requirements of high-throughput, quick and on-line detection are difficult to meet, and the method is not suitable for field operation of primary medical staff.
Although there are many patents on colloidal gold immunoassay test strips or kits in the prior art, there is no report on using colloidal gold test strips or kits to rapidly detect the concentration of a single-resistant drug in a sample, and rapidly determine the exposure of the drug in vivo.
Disclosure of Invention
The invention provides a colloidal gold kit for detecting infliximab in serum, aiming at solving the problem that a detection reagent for simply and quickly judging the in vivo exposure of a monoclonal antibody drug is lacked in the prior art.
Although there are many patents on colloidal gold immunoassay test strips or kits in the prior art, no previous report on the use of colloidal gold test strips or kits for detecting infliximab in human serum has been made. The use of biological drugs mainly depends on enzyme-linked immunosorbent assay (ELISA), electrochemiluminescence immunoassay (ECL), Radioimmunoassay (RIA), and the like.
The kit can detect infliximab in serum quickly, simply and low-cost, does not need to rely on complex chemical and biological analysis equipment, and is very suitable for primary medical staff to quickly detect the in-vivo drug exposure of a patient after receiving infliximab drug treatment, and adjust the injection dose and the administration period according to the in-vivo drug exposure.
The invention provides a colloidal gold kit for detecting infliximab in serum, which comprises a colloidal gold detection test strip containing colloidal gold pads and anti-infliximab idiotype antibodies and a sample diluent; the colloidal gold test strip for the infliximab comprises a substrate, a sample pad, a colloidal gold pad, a coating film and a water absorption pad; the content of the colloidal gold pad is 2.5-20 mu g/cm2The anti-infliximab idiotype antibody-colloidal gold complex of (a).
Preferably, the concentration of the colloidal gold pad is 5-10 mug/cm2The anti-infliximab idiotype antibody-colloidal gold complex of (a).
Wherein the preparation of said anti-infliximab idiotype antibody-colloidal gold complex preferably comprises the steps of:
(1) adjusting the pH value of the colloidal gold solution to 6.8-7.4;
(2) adding 10-20 mu g of anti-infliximab idiotype antibody HCA212 into every 1mL of the colloidal gold solution obtained in the step (1), and keeping stirring for 10-15 min;
(3) adding 100-200 mu L of 1% BSA solution into every 1mL of the colloidal gold solution obtained in the step (2), continuously stirring for 1-2 h, and sealing the exposed gold particles;
(4) after centrifugation, resuspending the mixture by using 0.1-0.3M phosphate buffer, then centrifuging the mixture, and repeatedly washing the mixture for 1-3 times;
(5) resuspending the precipitate in the step (4) by using 100-200 mu L of buffer solution, wherein the buffer solution contains 1-3% of surfactant or stabilizer;
(6) the colloidal gold compound is added at a concentration of 50-100 μ L/cm2Spraying onto the colloidal gold pad, and drying.
In the step (3), the final concentration of BSA is preferably 0.2%;
in the step (4), the temperature of the centrifugation is preferably 4 ℃, the centrifugation speed of the centrifugation is preferably 3000 to 5000r/min, more preferably 4000r/min, and the centrifugation time is preferably 30 min.
In step (4), the concentration of the phosphate buffer is preferably 0.2M.
In the step (5), the surfactant is preferably tween-80, tween-60 or poloxamer; the stabilizer is preferably polyvinyl alcohol or polyethylene glycol.
In the step (6), the drying is vacuum drying.
Preferably, the sample diluent is phosphate buffer. The concentration of the phosphate buffer solution may be conventional in the art; preferably, the concentration of the phosphate buffer solution is preferably 0.2M.
Wherein the coating film preferably comprises a detection region and a quality control region, the detection region comprises a recessive detection line prepared from an anti-infliximab idiotype antibody HCA213 solution, and the quality control region comprises a recessive quality control line prepared from an anti-IgG polyclonal antibody solution;
the concentration of the anti-infliximab idiotype antibody HCA213 solution is preferably 0.5-1.0 mg/mL, and/or the concentration of the anti-IgG polyclonal antibody solution is preferably 0.5-1.0 mg/mL;
preferably, the anti-IgG polyclonal antibody is a goat anti-mouse IgG antibody or a goat anti-rabbit IgG antibody.
In a preferred embodiment of the present invention, the preparation method of the coating film comprises the following steps:
(1) spraying the anti-infliximab idiotype antibody HCA213 solution on a detection area of a coating film by using a film spraying instrument;
(2) spraying the anti-IgG polyclonal antibody solution on a quality control area of the coating film;
(3) and (3) drying the coating film obtained in the step to obtain the coating film.
In a preferred embodiment, the substrate is a PVC plate, the coating film is a nitrocellulose film, the colloidal gold pad is made of polyester, and/or the absorbent pad is made of cellulose.
Preferably, the sample pad, the colloidal gold pad, the coating film and the absorbent pad are sequentially lapped on the substrate.
The reagents and starting materials used in the present invention are commercially available.
Wherein the anti-inflixi idiotype antibodies HCA213 and HCA212 are purchased from BioRad, and the order of use of HCA213 and HCA212 can be reversed in the present invention. In addition, other similar counterparts in the art may also be used
The positive progress effects of the invention are as follows:
(1) the kit is simple and convenient to operate and rapid. The treatment time can be as low as 10-20 min; the result judgment is simple, clear and accurate; the kit has strong stability and is easy to store.
(2) The kit can simply and inexpensively detect the infliximab in serum without depending on complex chemical and biological analysis equipment, is very suitable for primary medical staff to quickly detect the in-vivo drug exposure of a patient after receiving the infliximab drug treatment, and adjusts the injection dose and the administration period according to the in-vivo drug exposure.
Drawings
FIG. 1 is a schematic structural diagram of a colloidal gold test strip for detecting adalimumab antibody of the present invention; the device comprises a PVC base plate, 2 sample pads, 3 colloidal gold pads, 4 detection lines, 5 quality control lines, 6 water absorption pads and 7 nitrocellulose membranes.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.
Materials: the PVC base plate is purchased from Shanghai gold-labeled Biotech Co., Ltd; the sample pad was purchased from Shanghai gold-labeled Biotech, Inc.; the colloidal gold pad was purchased from Shanghai gold-labeled Biotech Co., Ltd; the absorbent pad is purchased from Shanghai gold-labeled Biotech limited; nitrocellulose membranes were purchased from Millipore, usa.
The anti-inflixi idiotype antibody (HCA213) and anti-inflixi idiotype antibody (HCA212) used in the following examples are both commercially available products, purchased from BioRad; other similar counterparts in the art may also be used.
Example 1 preparation of colloidal gold test paper for infliximab
(1) Preparing a colloidal gold pad:
preparation of anti-inflixi idiotype antibody-colloidal gold complex: adjusting the pH value of the colloidal gold solution to 6.8, adding an anti-Invisu idiotype antibody (HCA213) diluted by PBS according to the proportion of adding 10 mu g of antibody into every 1mL of colloidal gold solution, keeping stirring for 10-15 min, adding 100 mu L of 1% BSA, sealing naked gold particles, continuously stirring for 1-2 h, centrifuging for 30min at 4 ℃, 4000r/min, re-suspending with 0.2M phosphate buffer, centrifuging again, repeatedly washing for 2 times, re-dissolving the precipitate by adopting 100-200 mu L of buffer in every 1mL of colloidal gold solution in the original volume, wherein the buffer contains 1% of Tween-80. Besides tween-80, tween-60 or some other milder surfactant, such as poloxamer, etc., or stabilizer: such as polyvinyl alcohol (PVA), polyethylene glycol (PEG), may also be used in the present invention. Spraying anti-unique antibody of infliximab (HCA212) -colloidal gold compound on the gold label pad by using a film spraying instrument, wherein the spraying amount is 50 mu L/cm2And drying in vacuum for later use. The concentration of the antibody in the colloidal gold pad is 2.5-5 mug/cm2。
(2) Preparation of coating film: preparing an anti-infliximab idiotypic antibody (HCA213) into a solution with the concentration of 1.0mg/mL by using a phosphate buffer solution, and scratching the solution on a detection area of a nitrocellulose membrane; preparing goat anti-mouse IgG into a solution with the concentration of 1.0mg/mL by using a phosphate buffer solution, and scratching the solution on a quality control area of a nitrocellulose membrane; drying the nitrocellulose membrane for later use.
(3) Assembling the test strip: the sample pad, the colloidal gold pad coated with anti-infliximab idiotypic antibody (HCA212) -colloidal gold complex, the nitrocellulose membrane coated with anti-infliximab idiotypic antibody (HCA213) and goat anti-mouse IgG, and the absorbent pad were sequentially lapped on a PVC pad, cut, and packaged.
(4) Preparing a sample diluent: 0.2M phosphate buffer.
EXAMPLE 2 preparation of colloidal gold test paper for adalimumab anti-drug antibodies
(1) Preparing a colloidal gold pad:
preparation of anti-infliximab idiotypic antibody (HCA212) -colloidal gold complex: adjusting the pH of the colloidal gold solution to 7.4, adding 20 mu g of antibody into 1mL of colloidal gold solution, adding PBS (phosphate buffer solution) diluted anti-Invisulfie idiotypic antibody (HCA212), keeping stirring for 10-15 min, adding 200 mu L of 1% BSA, sealing naked gold particles, continuously stirring for 1-2 h, centrifuging for 30min at 4 ℃, 4000r/min, re-suspending with 0.2M phosphate buffer solution, centrifuging again, repeatedly washing for 2 times, re-dissolving the precipitate with 100-200 uL of buffer solution in 1mL of colloidal gold solution per original volume, wherein the buffer solution contains 1% of polyethylene glycol (PEG). Spraying anti-unique antibody of infliximab (HCA212) -colloidal gold compound on the gold label pad by using a film spraying instrument, wherein the spraying amount is 100 mu L/cm2And drying in vacuum for later use.
(2) Preparation of coating film: preparing an anti-infliximab idiotypic antibody (HCA213) into a solution with the concentration of 0.5mg/mL by using a phosphate buffer solution, and scratching the solution on a detection area of a nitrocellulose membrane; preparing goat anti-mouse IgG into a solution with the concentration of 0.5mg/mL by using a phosphate buffer solution, and scratching the solution on a quality control area of a nitrocellulose membrane; drying the nitrocellulose membrane for later use.
(3) Assembling the test strip: the sample pad, the colloidal gold pad coated with anti-infliximab idiotypic antibody (HCA212) -colloidal gold complex, the nitrocellulose membrane coated with anti-infliximab idiotypic antibody (HCA213) and goat anti-mouse IgG, and the absorbent pad were sequentially lapped on a PVC pad, cut, and packaged.
(4) Preparing a sample diluent: phosphate buffer.
| Example 1 | Example 2 | |
| Amount of antibody in colloidal gold (. mu.g) | 10 | 20 |
| Spray amount (μ L/cm)2) | 50 | 100 |
| Coated primary antibody and secondary antibody (mg/ml) | 1.0 | 0.5 |
Effect example 1
The embodiment provides an application of the colloidal gold test paper for infliximab in embodiments 1-2.
The patient collected 1mL of peripheral venous blood, coagulated at room temperature for 30min, and centrifuged at 1600g and 4 ℃ for 10min to separate serum. Sucking 10 mu L of serum, uniformly mixing with 190 mu L of sample diluent, immediately sucking 100 mu L of serum, dropwise adding the serum on a sample pad, and observing a detection line and a quality control line within 5-10 min. If the detection line and the quality control line are colored, the concentration of the infliximab in the serum sample of the patient is within an effective window, otherwise, the concentration of the infliximab is not within the effective window. No matter what the result is, the quality control line should be a line, and if the quality control line is not a line, the test paper is invalid.
The present effect example shows that the kits of examples 1 and 2 can each measure an effect consistent with the detection using the ELISA technique. The specific steps of measuring infliximab in serum by ELISA method are as follows:
(1) mu.l of 1.000. mu.g/ml anti-Invitrogen idiotypic antibody (HCA212) was added to each well in 96-well plates and incubated overnight (about 14-18 h) in a refrigerator (2-8 ℃).
(2) The plate was spun off, washed three times with 0.05% tween 20 in PBS and patted dry on paper. Mu.l of 1% (w/v) BSA-PBS solution was added to each well for blocking, and incubated at 25. + -. 1 ℃ for 2 to 5 hours.
(3) And (4) throwing off liquid in the plate, washing the plate for three times, and patting the plate dry on paper. Mu.l of standard curve, quality control sample and individual sample are added into corresponding wells, and incubated at 25 + -1 deg.C for 1h + -5 min.
(4) And (4) throwing off liquid in the plate, washing the plate for three times, and patting the plate dry on paper. Mu.l of HRP-labeled anti-Invitrogen idiotypic antibody (HCA213) solution (10 fold dilution in 1% BSA-PBS) was added to each well, and the plates were sealed with membrane plates and incubated at 25. + -. 1 ℃ for 1 h. + -. 5 mins.
(5) The liquid in the plate is thrown off, the plate is washed for four times, and the paper is patted dry. Adding 100 mul of TMB single-component color developing solution into each well, and incubating for 15mins +/-3 mins at 25 +/-1 ℃ in a dark place.
(6) The reaction was stopped by adding 100. mu.L of stop solution to each well. The plate was shaken and mixed for 5s using a microplate reader, and the plate was read at a wavelength of 450-. And obtaining the concentration of the individual sample through four-parameter fitting, and judging whether the concentration of the infliximab in the individual sample is in an effective window or not according to the concentration.
Serum and plasma were separated from peripheral blood of 5 patients using inflixine as a drug, respectively, using colloidal gold test paper and ELISA described in examples 1 to 2, and the test results are shown in table 1 below. The data in Table 1 show that the detection results obtained in examples 1 and 2 are 100% identical to the ELISA method, and no false positive or false negative occurs.
TABLE 1 comparison of the results of ELISA assays in examples 1-2
Effect example 2 sensitivity test
The test paper prepared in the embodiment 1-2 is tested for sensitivity: dissolving positive infliximab in negative blank serum to prepare a solution of 100 mu g/mL, and performing gradient dilution by 2 times to obtain a series of serum samples containing infliximab with different concentrations. The series of samples are adopted to detect the detection sensitivity of the colloidal gold detection test paper. The test results showed that the sensitivity of the colloidal gold test strips prepared according to the embodiments of example 1 and example 2 was 3.125. mu.g/mL (Table 2).
TABLE 2 sensitivity comparison
| Example 1 | Example 2 | |
| Sensitivity (μ g/mL) | 3.125 | 3.125 |
Claims (10)
1. A colloidal gold kit for detecting infliximab in serum is characterized in that the colloidal gold kit comprises a colloidal gold detection test strip containing a colloidal gold pad and resisting an infliximab idiotype antibodyAnd a sample diluent; the colloidal gold test strip for the infliximab comprises a substrate, a sample pad, a colloidal gold pad, a coating film and a water absorption pad; the content of the colloidal gold pad is 2.5-20 mu g/cm2The anti-infliximab idiotype antibody-colloidal gold complex of (a).
2. The colloidal gold kit according to claim 1, wherein the colloidal gold pad has a concentration of 5 to 10 μ g/cm2The anti-infliximab idiotype antibody-colloidal gold complex of (a).
3. The colloidal gold kit of claim 1 or 2, wherein the anti-infliximab idiotype antibody-colloidal gold complex is prepared by the steps of:
(1) adjusting the pH value of the colloidal gold solution to 6.8-7.4;
(2) adding 10-20 mu g of anti-inflixie idiotype antibody HCA212 into 1mL of the colloidal gold solution obtained in the step (1), and keeping stirring for 10-15 min;
(3) adding 100-200 mu L of 1% BSA solution into every 1mL of the colloidal gold solution obtained in the step (2), continuously stirring for 1-2 h, and sealing the exposed gold particles;
(4) after centrifugation, resuspending the mixture by using 0.1-0.3M phosphate buffer, then centrifuging the mixture, and repeatedly washing the mixture for 1-3 times;
(5) resuspending the precipitate in the step (4) by using 100-200 mu L of buffer solution, wherein the buffer solution contains 1-3% of surfactant or stabilizer;
(6) the colloidal gold compound is added at a concentration of 50-100 μ L/cm2Spraying onto the colloidal gold pad, and drying.
4. The colloidal gold kit according to claim 3,
the final concentration of BSA in step (3) is 0.2%;
the centrifugation speed in the step (4) is 3000-5000 r/min, preferably 4000r/min, and the centrifugation time is 30 min;
the concentration of the phosphate buffer solution in the step (4) is 0.2M;
the surfactant in the step (5) is Tween-80, Tween-60 or poloxamer, and the stabilizer is polyvinyl alcohol or polyethylene glycol;
and/or, the drying in the step (6) is vacuum drying.
5. A colloidal gold kit as claimed in any one of claims 1 to 4 wherein the sample diluent is phosphate buffered saline.
6. The colloidal gold kit of claim 5, wherein the phosphate buffer is at a concentration of 0.2M.
7. The colloidal gold kit of any one of claims 1 to 6, wherein the coating film comprises a detection region and a quality control region, wherein the detection region comprises a recessive detection line prepared from an anti-infliximab idiotype antibody HCA213 solution, and the quality control region comprises a recessive quality control line prepared from an anti-IgG polyclonal antibody solution;
the concentration of the anti-infliximab idiotype antibody HCA213 solution is preferably 0.5-1.0 mg/mL, and/or the concentration of the anti-IgG polyclonal antibody solution is preferably 0.5-1.0 mg/mL;
preferably, the anti-IgG polyclonal antibody is a goat anti-mouse IgG antibody or a goat anti-rabbit IgG antibody.
8. The colloidal gold kit according to claim 7, wherein the coating film is prepared by a method comprising the steps of:
(1) spraying the anti-infliximab idiotype antibody HCA213 solution on a detection area of a coating film by using a film spraying instrument;
(2) spraying the anti-IgG polyclonal antibody solution on a quality control area of the coating film;
(3) and (3) drying the coating film obtained in the step to obtain the coating film.
9. The colloidal gold kit according to claim 1, wherein the substrate is a PVC plate, the coating film is a nitrocellulose film, the colloidal gold pad is made of polyester, and/or the absorbent pad is made of cellulose.
10. The colloidal gold kit according to claim 9, wherein the sample pad, the colloidal gold pad, the envelope, and the absorbent pad are sequentially attached to a substrate.
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| CN201910586707.8A CN112180091A (en) | 2019-07-01 | 2019-07-01 | Colloidal gold kit for detecting infliximab in serum |
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| CN201910586707.8A CN112180091A (en) | 2019-07-01 | 2019-07-01 | Colloidal gold kit for detecting infliximab in serum |
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Application publication date: 20210105 |