CN110794047A - Method for predicting skin sensitization of complex test object - Google Patents
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Abstract
The invention discloses a method for predicting skin sensitization of a complex test object, which comprises the following steps: (1) a direct polypeptide binding assay improvement analysis and (2) a human cell line activation assay analysis, wherein when both the direct polypeptide binding assay improvement analysis and the human cell line activation assay analysis result are sensitization, the complex test object prediction result is sensitization, and when both the direct polypeptide binding assay improvement analysis and the human cell line activation assay result are non-sensitization, the complex test object prediction result is non-sensitization. The method improves the direct polypeptide binding test, combines the improved direct polypeptide binding test with the human cell activation test, is used for predicting the skin sensitization of the complex test object, has higher prediction accuracy than a single test method, is beneficial to finding a complex system with potential sensitization risk, realizes the rapid and effective screening of the complex test object, and can save time and labor cost.
Description
Technical Field
The invention belongs to the technical field of skin sensitization substance detection, and particularly relates to a method for predicting skin sensitization of a complex test object.
Background
Allergic Contact Dermatitis (ACD) is an immune-mediated skin reaction resulting from repeated exposure of the skin to some foreign substance. The skin is usually red, swollen, itchy, painful and the like, and the biological quality is affected in severe cases. Thus, substances that come into contact with the skin or that will come into contact with the skin multiple times, are necessary to undergo skin sensitization testing to ensure that skin-contacting substances, such as chemicals, daily chemicals and other skin-contacting products, have a complete risk assessment and toxicity assessment prior to use. Therefore, security is a very important link, which is also required by the consuming public.
The traditional methods for skin sensitization assessment are guinea pig trials and mouse regional lymph node experiments (LLNA). Since countries such as the european union have banned animal tests for cosmetics and cosmetic ingredients, there is an increasing demand for alternative tests. Meanwhile, the concept of Adverse Outcome Pathway (AOP) was proposed, and studies on toxicological effects using human-derived tissues or cells were started. The skin sensitizes the AOP pathway, the molecular initiation event of which is the covalent binding of a foreign substance to a skin protein to form a hapten, which subsequently triggers the critical events at the 2-cell level, namely the keratinocyte inflammatory response and the dendritic cell activation. Thereafter, activated dendritic cells migrate to regional lymph nodes, transferring a portion of the hapten chemical to naive T lymphocytes, ultimately leading to the development of a tissue level key event of proliferation and differentiation of memory T cells. The priming phase is entered when the skin is again exposed to sensitizers, resulting in a deleterious outcome of the ACD. Direct Polypeptide Reactivity Assay (DPRA) established based on the AOP framework and keratinocyte ARE-Nrf2 luciferase detection method (KeratinoSens)TM) And human cell line activation test (h-CLAT) have been recognized by OECD as a standard method.
The alternative method for skin sensitization mainly establishes an evaluation mode aiming at different key event points in an AOP (automated optical proximity) pathway, and a single method has limitations, and the prediction accuracy is possibly lower than that of an in-vivo LLNA (line-local Lane analysis) method. Therefore, trying to build a combined model of multiple alternative approaches to reduce the disadvantages of a single approach is a hot spot for further research, and at present, OECD has 12 skin-sensitizing combination schemes for chemicals, but no skin-sensitizing combination scheme for complex systems of complex subjects such as plant extracts. The plant extract inevitably has allergenicity due to various raw material acquirements, complex extraction process and components, and no allergenicity cannot be guaranteed by other substances with non-single components, so that a complex system can be preliminarily screened by adopting a combined model to improve the efficiency and accuracy of skin sensitization judgment. The simple combination of the two methods of DPRA and h-CLAT is convenient for the initial screening of the sensitization capacity of chemical substances, but DPRA is not suitable for a complex system and needs to be improved or improved.
Disclosure of Invention
The invention aims to provide a method for predicting skin sensitization of a complex test object, which improves a direct polypeptide binding assay (DPRA), combines the improved DPRA with a human cell activation assay (h-CLAT) and is used for predicting the skin sensitization of the complex test object.
The above object of the present invention is achieved by the following technical solutions: a method for predicting skin sensitization of a complex subject, comprising the steps of:
(1) improved assay for direct polypeptide binding assays
(1.1) sample preparation: establishing a control group and a complex test object group, wherein the control group comprises 750 mu L of cysteine polypeptide mother liquor, 200 mu L of acetonitrile and 50 mu L of complex test object dissolving solvent, the complex test object group comprises 750 mu L of phosphate buffer solution and cysteine polypeptide mother liquor, 200 mu L of acetonitrile and a series of complex test objects with different concentrations, and the concentration of the cysteine polypeptide mother liquor is 0.35-0.5 mg/mL;
(1.2) sample incubation: placing the control group and the complex test object group in a dark place at 25 +/-2.5 ℃ for incubation for 24 +/-2 hours for later use;
(1.3) sample detection: analyzing the sample incubated in the step (1.2) by adopting a liquid chromatograph;
(1.4) analysis of results: calculating the polypeptide consumption percentage of the sample and judging the sensitization grade;
(1.5) sensitization prediction model: the consumption percentage of the polypeptide is more than or equal to 0% and less than or equal to 13.89%, the complex test object is a non-sensitizer, the consumption percentage of the polypeptide is more than 13.89%, and the complex test object is a sensitizer;
(2) human cell line activation assay
(2.1) cell plating: selecting THP-1 cells, and pretreating for later use;
(2.2) Complex test substance Exposure: selecting a complex test object, diluting the complex test object according to a proportion to obtain a series of concentrations, adding the concentrations into the THP-1 cells in the step (2.1), and culturing the complex test object for later use;
(2.3) blocking: adding an FcR blocker into the cells cultured in the step (2.2) for blocking;
(2.4) detection of CD86/CD54 expression: adding an FITC-CD86 antibody, a PE-CD54 antibody, FITC-labeled mouse isotype control IgG1 and PE-labeled mouse isotype control IgG1 into the cells after the blocking in the step (2.3) to form a test object treatment group and a test object treatment group isotype control, and detecting a protein marker by using a flow cytometer after the incubation;
and (2.5) data processing: detecting a relative fluorescence intensity value RFI by using a flow cytometer;
(2.6) prediction model: on the premise that the cell viability is greater than 50%, for FITC-CD86, if RFIC 86 is greater than or equal to 150, the complex test object is an sensitizer; for PE-CD54, if RFICD54 is more than or equal to 200, the complex test substance is an sensitizer, namely when RFICD86 is more than or equal to 150 and/or RFICD54 is more than or equal to 200, the complex test substance is an sensitizer, and the rest cases are non-sensitizer;
(3) result prediction
And (3) when the results of the direct polypeptide binding assay improvement analysis in the step (1) and the human cell line activation assay analysis in the step (2) are both sensitization, the complex test object prediction result is sensitization, and when the results of the direct polypeptide binding assay improvement analysis in the step (1) and the human cell line activation assay analysis in the step (2) are both non-sensitization, the complex test object prediction result is non-sensitization.
In the method for predicting skin sensitization of the complex test object:
preferably, the complex test substance in step (1.1) is a non-single component system including, but not limited to, non-single component chemicals, cosmetic raw materials, cosmetic products, petroleum lubricants, plant extracts, household washing products, dermatologic drugs, bioactive raw materials, dust particles, air pollutant textiles, and textile leach solutions.
Further, the complex test subject is a non-single component system that may also include other skin contact products.
More preferably, the complex subject in step (1.1) is a plant extract.
Preferably, the complex test substance dissolving solvent in step (1.1) is one or more of acetonitrile, water, isopropanol, acetone and dimethyl sulfoxide.
More preferably, the complex test substance dissolving solvent in step (1.1) is acetonitrile.
Preferably, acetonitrile is selected as the solvent for the complex test substance in step (1.1), and if not dissolved, the complex test substance can be dissolved in water, 1:1 (volume ratio) water and acetonitrile, isopropanol, acetone, 1:1, or dissolving in dimethyl sulfoxide, diluting with acetonitrile, and optionally further treating with ultrasonic wave.
Preferably, the concentration of the series of different concentrations of the complex test substance in step (1.1) is 1000mg/mL, 500mg/mL, 250mg/mL, 100mg/mL, and 10 mg/mL.
Preferably, the cysteine polypeptide mother liquor in step (1.1) is prepared by using a phosphoric acid buffer solution or a mixed solution of a phosphoric acid buffer solution and acetonitrile in a volume ratio of 4: 1.
Preferably, the cysteine polypeptide has the structure of Ac-RFAACAA-COOH and the purity is 90-100%.
Preferably, the phosphate buffer solution may be a phosphate buffer solution having a pH of 7.5 ± 0.5 and a concentration of 100 mmol/L.
Further, the step (1.1) comprises a blank group, a positive control, a control group A, a control group B and a co-elution detection group, wherein the positive control is cinnamaldehyde, 4-ethoxymethylene-2-phenyloxazoline-5-ketone, 2, 4-dinitrochlorobenzene, formaldehyde or benzylidene acetone, the control group A comprises 750 mu L of cysteine polypeptide mother liquor and 250 mu L of acetonitrile, and the components of the control group B are the same as those of the control group, but the control group B is not subjected to incubation treatment.
Preferably, each sample group such as blank group, control group, positive control group, control group a, control group B, co-elution detection group, complex test object group, etc. is provided with parallel samples, preferably three parallel samples.
Preferably, in step (1.3), the sample incubated in step (1.2) is analyzed by liquid chromatography at the latest within 30 hours after the end of the reaction. If it exceeds 30 hours, the polypeptide itself is unstable and the results of the experiment are affected.
Preferably, the chromatographic column in the liquid chromatograph in the step (1.3) can be a C18 column with proper peak resolution.
Preferably, the flow rate of the eluent from the liquid chromatograph in step (1.3) is adjusted according to the actual situation, and generally, the flow rate of a 10cm column is set to about 0.35mL/min, and the flow rate of a 25cm column is set to about 1 mL/min.
Preferably, the sample amount of the liquid chromatograph in step (1.3) can be adjusted according to the peak shape, and if the peak is too wide, the sample amount should be reduced, and the automatic sample injector should be adjusted to avoid sucking samples from the bottom of the sample injection bottle.
Thus, in general, more preferably, the chromatographic conditions of the liquid chromatograph in step (1.3) are: a column, Zorbax SB-C182.1mm x 100mm x 3.5micron or Agilent Part Number 861753-902 or Phenomenex LunaC18(2)2.0mm x 100mm x 3micron particle; column temperature, 30 ℃ detector, 220nm and 258nm photodiode array detector or 220nm fixed wavelength absorption detector; the sample amount is 1-10 mu L; the flow rate is 0.2-1.0 mL/min; mobile phase, phase a: water phase, B phase: an organic phase; gradient, 0-10min, 90-75% A, 10-11min, 75-10% A, 11-13min, 10-10% A, 13-13.5min, 10-90% A, 13.5-20min and 90-90% A, wherein the phase A is trifluoroacetic acid aqueous solution with trifluoroacetic acid volume percentage of 0-0.1%, and the phase B is trifluoroacetic acid acetonitrile solution with trifluoroacetic acid volume percentage of 0-0.85%.
Specifically, the percentage of polypeptide consumption of the sample in step (1.4) is calculated using the following formula:
percent polypeptide consumption ═ 1- (complex test substance polypeptide peak area/control polypeptide peak area) ] × 100.
Specifically, in the step (1.5), the sensitization result is the highest among the five obtained sensitization results at different concentrations, and the sensitization is the final result, that is, the sensitization occurs at any concentration.
Specifically, the cell plating in step (2.1) comprises: pre-cultured THP-1 cells were centrifuged and the supernatant removed and the cells were resuspended in fresh complete medium at a concentration of 2 x 106At one/mL, 500. mu.L of the above resuspension was pipetted into a 24-well cell culture plate for exposure.
Specifically, the complex subject exposure in step (2.2) comprises: if the complex test substance is dissolved in physiological saline, the final concentration needs to be diluted by at least 100 times; the final concentration of complex test substance dissolved in DMSO requires at least 500-fold dilution to the highest concentration that gives rise to CV75, followed by 1:1.2 adding 5-7 concentrations including CV75, CV75/1.2, and CV75/1.22、CV75/1.23、CV75/1.24、CV75/1.25、CV75/1.26、CV75/1.27. Each experiment required 3 controls to be set up, a positive control for the medium control, DMSO control and DNCB respectively. The prepared test liquid was pipetted at 500. mu.L and added to the above-mentioned 24-well cell culture plate to be exposed. Placing in an incubator to incubate for 24 h.
Preferably, therefore, the highest concentration of the complex test substance in step (2.2) is the concentration that gives rise to CV75(Cell Viability, the highest concentration not being such that the Cell Viability is below 75%), followed by a 1:1.2 add 5-7 concentrations later for dilution ratio, i.e.: CV75, CV751.2、CV75/1.22、CV75/1.23、CV75/1.24、CV75/1.25、CV75/1.26、CV75/1.27。
Specifically, the blocking in step (2.3) includes: cells under the influence of complex test substances were transferred from 24-well cell culture plates into corresponding 1mLEP tubes, and the cells were collected by centrifugation and sedimentation. And washed once with FACS buffer (PBS + 0.1% BSA), and centrifuged again under the same conditions to collect cells. Cells obtained by centrifugation were then blocked by addition of FcR reagent.
Specifically, the detection of CD86/CD54 expression in step (2.4) comprises: the cells were evenly distributed into 4 1mL EP tubes at 30-50. mu.L/tube, approximately 2.5-3.0X 10/tube5(ii) individual cells; adding 50-100 μ L of antibody including FITC-CD86 antibody, PE-CD54 antibody, FITC labeled mouse isotype control IgG1 and PE labeled mouse isotype control IgG 1; dyeing for 30min at the temperature of 2-8 ℃ under the dark room condition; washing 2 times with FACS buffer; the cells were resuspended in 500. mu.L of FACS buffer and transferred to a 5mL flow tube and protein markers were detected using flow cytometry.
Specifically, the relative fluorescence intensity value RFI in step (2.5) is calculated using the following formula:
relative Fluorescence Intensity (RFI) ═ complex test article treated group MFI-complex test article treated group isotype control MFI)/(complex test article solvent treated group MFI-complex test article treated group isotype control; wherein the Mean Fluorescence Intensity (MFI) is the geometric mean fluorescence intensity.
Preferably, the result in step (2.6) is that the DMSO control expression in step (2.2) is not positive, the media control is not positive, and the DNCB control is positive, based on the results under the conditions acceptable.
Further, in step (3), when the result of the direct polypeptide binding assay improvement analysis in step (1) is not identical to the result of the human cell line activation assay analysis in step (2), it is determined by combining a third known method or a classical animal assay, and when the third known method or the classical animal assay is determined to be allergic, the test substance is sensitized, and when the third known method or the classical animal assay is determined to be non-sensitized, the test substance is non-sensitized.
The invention has the following beneficial effects:
(1) the method can be used for replacing a living animal (or human body) to predict the possible skin sensitization caused by a complex system;
(2) the method improves the DPRA according to the characteristics of a complex system (complex test object comprising non-single components), does not carry out experiments according to a molar ratio, but adopts a mass concentration ratio, so that the method is suitable for the complex system with unknown mass fraction, improves the accuracy of skin sensitization, greatly reduces the occurrence of false negative, combines the improved DPRA and H-CLAT methods, is used for screening the skin sensitization of the complex system, is shorter than that of a living animal experiment, has more objective results and is more convenient to operate;
(3) the two combined methods are relatively stable, the standardization procedure is high, and the preliminary judgment on the skin sensitization of a complex system can be realized;
(4) the method established by the invention can replace the skin of living animals and human beings, provides qualitative skin sensitization detection data, and is directly used for predicting the skin sensitization of products and samples such as raw materials, cosmetics, medicines, air pollutants and the like.
Detailed Description
The raw materials used in the following examples, such as reagents and culture media, are all commercially available products unless otherwise specified.
Example 1 radix astragali extract skin sensitization test based on DPRA improvement and H-CLAT
The method sequentially comprises the following steps:
1. improved direct polypeptide binding assay analysis;
1-1 preparation of a test article: the astragalus extract is prepared by the following steps of 1:1 (volume ratio) acetonitrile aqueous solution, and obtaining a group of astragalus extracts with the concentrations of 1000mg/mL, 500mg/mL, 250mg/mL, 100mg/mL and 10mg/mL respectively, a phosphate buffer solution with the pH value of 7.8 for standby, and cinnamaldehyde is adopted as a positive control. Establishing a blank group, a control group A, a control group B, a control group C (control group), a co-elution detection group and a group to be detected (complex test object group), and carrying out the following steps of 1: 15 ratio and recording the time of addition of the polypeptide to the sample. Covering tightly and mixing evenly. Wherein the composition of each experimental group is shown in table 1 below:
TABLE 1 composition of the experimental groups
| Reagent | 750μL | 200μL | 50μL |
| Blank group | Phosphate buffer acetonitrile (4: 1) | Phosphate buffer acetonitrile (4: 1) | Phosphate buffer acetonitrile (4: 1) |
| Control group A | 0.5mg/mL polypeptide mother liquor | Acetonitrile | Acetonitrile |
| Control group B | 0.5mg/mL polypeptide mother liquor | Acetonitrile | Acetonitrile water mixed solution |
| Control group C | 0.5mg/mL polypeptide mother liquor | Acetonitrile | Acetonitrile water mixed solution |
| Co-elution assay set | Phosphate buffer | Acetonitrile | Radix astragali extract with different concentrations |
| Group to be tested | 0.5mg/mL polypeptide mother liquor | Acetonitrile | Radix astragali extract with different concentrations |
Wherein:
a blank was used to equilibrate the column.
Control a was used to verify HPLC stability.
Control B was used to verify the stability of the blank control throughout the analysis phase, unlike control C, which was not incubated.
Control C was used to verify the effect of solvent on the percent polypeptide consumed.
The co-elution test set was used to verify the effect of solvent on the percent polypeptide consumption.
The positive control group is used for proving the effectiveness of the experiment.
1-2 sample incubation: the sample bottles were incubated in the dark at 26 ℃ for 24 h.
1-3, sample detection:
the chromatographic conditions are as follows: chromatography column, Zorbax SB-C182.1mm x 100mm x 3.5 micron; column temperature, 30 ℃; a detector, a fixed wavelength (220nm) absorption detector; sample size, 7 μ L; flow rate, 0.35 mL/min; mobile phase, phase a: aqueous phase (0.085% aqueous trifluoroacetic acid), phase B: organic phase (0.085% trifluoroacetic acid in acetonitrile); gradient, 0-10min, 90-75% A, 10-11min, 75-10% A, 11-13min, 10-10% A, 13-13.5min, 10-90% A, 13.5-20min, 90-90% A.
1-4 analysis of results: the peak areas of 5 concentrations of the astragalus extract and the control C were collated and the percent polypeptide consumption of the samples was calculated according to the following formula. And used for judging sensitization grade.
Percent polypeptide consumption ═ 1- (complex test substance polypeptide peak area/control polypeptide peak area) ] × 100.
The complex test substance polypeptide peak area is the peak area of the group to be tested, and the control group is the control group C above.
1-5 sensitization prediction model:
the percentage of polypeptide consumption is more than or equal to 0% and less than or equal to 13.89%, the radix astragali extract is a non-sensitizer, the percentage of polypeptide consumption is more than 13.89%, the radix astragali extract is a sensitizer, and the detailed results are shown in the following table 2.
TABLE 2 DPRA assay results for Scutellaria baicalensis Georgi extract
| Name (R) | Elimination rate of cysteine% | Predicted results |
| Scutellaria baicalensis extract (1000mg/mL) | 96.11±7.99 | Sensitizers |
| Scutellaria baicalensis extract (500mg/mL) | 65.39±6.82 | Sensitizers |
| Scutellaria baicalensis extract (250mg/mL) | 53.23±7.52 | Sensitizers |
| Scutellaria baicalensis extract (100mg/mL) | 17.36±8.02 | Sensitizers |
| Scutellaria baicalensis extract (10mg/mL) | 11.34±3.56 | Non-sensitizers |
As is clear from Table 2, the analyte was a sensitizer when the concentration was 100-1000mg/mL and a non-sensitizer when the concentration was 10mg/mL, but the analyte was judged to be a sensitizer when any one of the concentrations was a sensitizer, and therefore, the result of the analysis by the modified direct polypeptide binding assay in this example was a sensitizer.
2. Human cell line activation assay analysis;
2-1 cell plating: pre-cultured THP-1 cells were centrifuged and the supernatant removed and the cells were resuspended in fresh complete medium at a concentration of 2 x 106At one/mL, 500. mu.L of the above resuspension was pipetted into a 24-well cell culture plate for exposure.
2-2 subject exposure: diluting radix astragali extract 500 times. The highest concentration was the concentration that caused CV75, followed by a 1:1.2 add 5 concentrations later for dilution ratio, i.e.: CV75, CV75/1.2, CV75/1.22、CV75/1.23、CV75/1.24、CV75/1.25. Each experiment required 3 controls to be set up, a positive control for the medium control, DMSO control and DNCB respectively. The prepared test liquid was pipetted at 500. mu.L and added to the above-mentioned 24-well cell culture plate to be exposed. Placing in an incubator to incubate for 24 h.
2-3FcR blocking: transferring the cells under the action of the astragalus extract from 24-hole cell culture plates into corresponding 1mLEP tubes respectively, and collecting the cells by centrifugal precipitation. And washed once with FACS buffer (PBS + 0.1% BSA), and centrifuged again under the same conditions to collect cells. The cells obtained by centrifugation were subsequently blocked with FcR.
2-4CD86/CD54 expression assay: the cells were evenly distributed in 4 1mL EP tubes at 30. mu.L/tube, about 3.0X 10/tube5(ii) individual cells; adding 100 μ L of antibody including FITC-CD86 antibody, PE-CD54 antibody, FITC labeled mouse isotype control IgG1 and PE labeled mouse isotype control IgG 1; dyeing for 30min at the temperature of 3 ℃ under the condition of a dark room; washing 2 times with FACS buffer; the cells were resuspended in 500. mu.L of FACS buffer and transferred to a 5mL flow tube and protein markers were detected using flow cytometry.
2-5, data processing: relative Fluorescence Intensity (RFI) was calculated using fluorescence intensity values (MFI) as detected by flow cytometry:
the relative fluorescence intensity value RFI is calculated using the following formula: relative Fluorescence Intensity (RFI) ═ complex test article treated group MFI-complex test article treated group isotype control MFI)/(complex test article solvent treated group MFI-complex test article treated group isotype control; wherein the fluorescence intensity value is calculated by the following formula: mean Fluorescence Intensity (MFI) is the geometric mean of fluorescence intensities.
2-6 prediction model:
the detailed results are shown in Table 3 below.
TABLE 3 Scutellaria baicalensis Georgi extract h-CLAT test results
| Name (R) | Cell viability% | RFI CD86 | RFI CD54 | Predicted results |
| Scutellaria baicalensis Georgi extract | 85.8 | 232 | 134 | Sensitizers |
3. Result integration and prediction
The prediction results are evaluated as follows:
| NO. | DPRA improvement method | h-CLAT | Predicted results |
| Scutellaria baicalensis Georgi extract | Sensitizers | Sensitizers | Sensitizers |
4. Conclusion
The test adopts a combination method of direct polypeptide binding test and human cell line activation test to detect the skin sensitization of the scutellaria baicalensis extract. The direct polypeptide combination test result shows that the scutellaria baicalensis extract is strongly combined with cysteine polypeptide from 100mg/mL to 1000mg/mL and is taken as a sensitizer, and the human cell line activation test result predicts that the scutellaria baicalensis extract is taken as the sensitizer. Comprehensively predicting that the scutellaria baicalensis extract is a skin sensitizing substance.
Example 2 certain cosmetic skin sensitization test based on DPRA improvement and h-CLAT
The method sequentially comprises the following steps:
1. improved direct polypeptide binding assay analysis;
1-1 preparation of a test article: a complex sample (a certain cosmetic) was dissolved in pure water to obtain a set of cosmetic solutions with concentrations of 1000mg/mL, 500mg/mL, 250mg/mL, 100mg/mL, and 10mg/mL, respectively, in a phosphate buffer at pH 7.5 for use, using 4-ethoxymethylene-2-phenyloxazoline-5-one as a positive control. Establishing a blank group, a control group A, a control group B, a control group C, a co-elution detection group and a group to be detected (a complex test object group), and carrying out the following steps of 1: 15 ratio and recording the time of addition of the polypeptide to the sample. Covering tightly and mixing evenly. The composition of each experimental group is shown in table 4:
TABLE 4 composition of the experimental groups
| Reagent | 750μL | 200μL | 50μL |
| Blank group | Phosphate buffer: acetonitrile (4: 1) | Phosphate buffer: acetonitrile (4: 1) | Phosphate buffer: acetonitrile (4: 1) |
| Control group A | 0.5mg/mL polypeptide mother liquor | Acetonitrile | Acetonitrile |
| Control group B | 0.5mg/mL polypeptide mother liquor | Acetonitrile | Pure water |
| Control group C | 0.5mg/mL polypeptide mother liquor | Acetonitrile | Pure water |
| Co-elution assay set | Phosphate buffer | Acetonitrile | Certain cosmetic with different concentrations |
| Group to be tested | 0.5mg/mL polypeptide mother liquor | Acetonitrile | Certain cosmetic with different concentrations |
A blank was used to equilibrate the column.
Control a was used to verify HPLC stability.
Control B was used to verify the stability of the blank control throughout the analysis phase, unlike control C, which was not incubated.
Control C was used to verify the effect of solvent on the percent polypeptide consumed.
The co-elution test set was used to verify the effect of solvent on the percent polypeptide consumption.
The positive control group is used for proving the effectiveness of the experiment.
1-2 sample incubation: the sample bottles were incubated in the dark at 25 ℃ for 22 h.
1-3, sample detection:
the chromatographic conditions are as follows: chromatography column, Agilent 2.1mm x 250mm x 3.5micron plus Phenomenex LunaC18(2)2.0mm x 100mm x 3micron particle; column temperature, 30 ℃; detectors, photodiode array detectors (220nm and 258nm) absorption detectors; sample size, 10 μ L; flow rate, 1.0 mL/min; mobile phase, phase a: water, phase B: acetonitrile; gradient, 0-10min, 90-75% A, 10-11min, 75-10% A, 11-13min, 10-10% A, 13-13.5min, 10-90% A, 13.5-20min, 90-90% A.
1-4 analysis of results: the peak areas of the 5 concentrations of a cosmetic and the control C were collated and the percent polypeptide consumption of the samples was calculated according to the following formula. And used for judging sensitization grade.
Percent polypeptide consumption ═ 1- (complex test article parallel polypeptide peak area/control C polypeptide peak area) x 100.
The peak area of the complex test substance parallel sample polypeptide is the peak area of the parallel sample to be tested (namely the peak area of the complex test substance).
1-5 sensitization prediction model:
the consumption percentage of the polypeptide is more than or equal to 0% and less than or equal to 13.89%, the certain cosmetic is a non-sensitizer, the consumption percentage of the polypeptide is more than 13.89%, and the certain cosmetic is a sensitizer; the detailed results are shown in Table 5.
TABLE 5 DPRA improvement test results for certain cosmetic products
| Name (R) | Elimination rate of cysteine% | Predicted results |
| Certain cosmetic (1000mg/mL) | 2.23±0.11 | Non-sensitizers |
| Certain cosmetic (500mg/mL) | 5.23±1.35 | Non-sensitizers |
| Certain cosmetic (250mg/mL) | 3.84±0.75 | Non-sensitizers |
| Certain cosmetic (100mg/mL) | 1.96±0.85 | Non-sensitizers |
| Certain cosmetic (10mg/mL) | 1.03±0.56 | Non-sensitizers |
2. Human cell line activation assay analysis;
2-1 cell plating: pre-cultured THP-1 cells were centrifuged and the supernatant removed and the cells were resuspended in fresh complete medium at a concentration of 2 x 106At one/mL, 500. mu.L of the above resuspension was pipetted into a 24-well cell culture plate for exposure.
2-2 subject exposure: dissolving a certain cosmetic in normal saline, and diluting by 100 times at the final concentration; the highest concentration was the concentration that caused CV75, followed by a 1:1.2 dilution ratio, adding 7 concentrations, i.e. CV75, CV75/1.2, CV75/1.22、CV75/1.23、CV75/1.24、CV75/1.25、CV75/1.26、CV75/1.27. Each experiment required 3 controls to be set up, a positive control for the medium control, DMSO control and DNCB respectively. The prepared test liquid was pipetted at 500uL and added to the 24-well cell culture plate to be exposed as described above. Placing in an incubator to incubate for 24 h.
2-3FcR blocking: cells under the action of certain cosmetics with different concentrations are respectively transferred into corresponding 1mLEP tubes from 24-well cell culture plates, and the cells are collected by centrifugal precipitation. And washed once with FACS buffer (PBS + 0.1% BSA), and centrifuged again under the same conditions to collect cells. The cells obtained by centrifugation were subsequently blocked with FcR.
2-4CD86/CD54 expression assay: the cells were evenly distributed in 4 1mL EP tubes at 50. mu.L/tube, approximately 2.5X 10 cells/tube5(ii) individual cells; adding 70 μ L of antibody including FITC-CD86 antibody, PE-CD54 antibody, FITC labeled mouse isotype control IgG1 and PE labeled mouse isotype control IgG 1; dyeing for 30min at 5 ℃ in a dark room; washing 2 times with FACS buffer; the cells were resuspended in 500. mu. LFACS buffer and transferred to a 5mL flow tube and protein markers were detected using a flow cytometer.
2-5, data processing: the Relative Fluorescence Intensity (RFI) was calculated from the fluorescence intensity value (MFI) at the time of detection by flow cytometry, and the formula was the same as in example 1.
2-6 prediction model:
the detailed results are shown in Table 6.
TABLE 6 detection results of h-CLAT for certain cosmetics
| Name (R) | Cell viability% | RFI CD86 | RFI CD54 | Predicted results |
| Certain cosmetic | 85.8 | 102 | 129 | Non-sensitizers |
3. Result integration and prediction
The prediction results are evaluated as follows:
| NO. | DPRA improvement method | h-CLAT | Predicted results |
| Certain cosmetic | Non-sensitizers | Non-sensitizers | Non-sensitizers |
4. Conclusion
The test employs a combination of direct polypeptide binding assays and human cell line activation assays to detect skin sensitization in a cosmetic. The direct polypeptide combination test result shows that a certain cosmetic and cysteine polypeptide are not combined and are non-sensitizers, and the human cell line activation test result predicts that a certain cosmetic is a negative sensitizer. Comprehensively predicting that a certain cosmetic is a skin non-sensitizing substance.
Example 3 certain efficacy raw material skin sensitization test based on DPRA improvement and h-CLAT
The method sequentially comprises the following steps:
1. direct polypeptide binding assay analysis;
1-1 preparation of a test article: the functional raw materials are dissolved by acetone to obtain a group of functional raw material test groups (to-be-tested groups) with the concentrations of 1000mg/mL, 500mg/mL, 250mg/mL, 100mg/mL and 10mg/mL respectively, a phosphate buffer solution with the pH of 7.0 is used for standby, and 4-ethoxymethylene-2-phenyloxazoline-5-ketone is used as a positive control. Establishing a blank group, a control group A, a control group B, a control group C (control group), a co-elution detection group and a group to be detected (complex test object group), and carrying out the following steps of 1: 15 ratio (volume ratio) cysteine polypeptide test samples were prepared and the time of addition of the polypeptide to the samples was recorded. Covering tightly and mixing evenly. The composition of each experimental group is shown in table 7:
TABLE 7 composition of the experimental groups
1-2 sample incubation: the sample bottles were incubated for 26h at 23 ℃ in the dark.
1-3, sample detection:
the chromatographic conditions are as follows: chromatography column, Zorbax SB-C182.1mm x 100mm x 3.5 micron; column temperature, 30 ℃; a detector, a fixed wavelength (220nm) absorption detector; sample size, 2 μ l; flow rate, 0.5 ml/min; mobile phase, phase a: water phase, B phase: an organic phase; gradient, 0-10min, 90-75% A, 10-11min, 75-10% A, 11-13min, 10-10% A, 13-13.5min, 10-90% A, 13.5-20min, 90-90% A.
1-4 analysis of results: the peak areas of the 5 concentrations of the functional materials and the control group are collated, and the consumption percentage of the polypeptide in the sample is calculated according to the following formula. And used for judging sensitization grade.
Percent polypeptide consumption ═ 1- (complex test substance polypeptide peak area/control polypeptide peak area) ] × 100.
The complex test substance polypeptide peak area is the peak area of the group to be tested, and the control group is the control group C.
1-5 sensitization prediction model:
the consumption percentage of the polypeptide is more than or equal to 0 percent and less than or equal to 13.89 percent, the functional raw material is a non-sensitizer, the consumption percentage of the polypeptide is more than 13.89 percent, and the functional raw material is a sensitizer; the detailed results are shown in Table 8.
TABLE 8 DPRA improvement method for certain efficacy raw materials
| Name (R) | Elimination rate of cysteine% | Predicted results |
| Raw material with certain effect (1000mg/mL) | 56.23±3.67 | Sensitizers |
| Raw material with certain effect (500mg/mL) | 42.33±5.31 | Sensitizers |
| Raw material with certain effect (250mg/mL) | 12.62±5.27 | Non-sensitizers |
| Raw material with certain effect (100mg/mL) | 3.98±0.54 | Non-sensitizers |
| Raw material with certain effect (10mg/mL) | 3.03±0.82 | Non-sensitizers |
2. Human cell line activation assay
2-1 cell plating: pre-cultured THP-1 cells were centrifuged and the supernatant removed and the cells were resuspended in fresh complete medium at a concentration of 2 x 106At one/mL, 500. mu.L of the above resuspension was pipetted into a 24-well cell culture plate for exposure.
2-2 subject exposure: the functional raw materials are diluted by 500 times. The highest concentration is the concentration that causes CV75, and 6 concentrations, CV75, CV75/1.2, CV75/1.2, are added later at a dilution ratio of 1:1.22、CV75/1.23、CV75/1.24、CV75/1.25、CV75/1.26. Each experiment required 3 controls to be set up, a positive control for the medium control, DMSO control and DNCB respectively. The prepared test liquid was pipetted at 500uL and added to the lower 24 well cell culture plate to be exposed as described above. Placing in an incubator to incubate for 24 h.
2-3FcR blocking: cells with different concentrations under the action of certain functional raw materials are respectively transferred into corresponding 1mLEP tubes from 24-hole cell culture plates, and the cells are collected by centrifugal precipitation. And washed once with FACS buffer (PBS + 0.1% BSA), and centrifuged again under the same conditions to collect cells. The cells obtained by centrifugation were subsequently blocked with FcR.
2-4CD86/CD54 expression assay: the cells were evenly distributed in 4 1mL EP tubes at 30. mu.L/tube, approximately 2.8X 10 cells/tube5(ii) individual cells; adding 50 μ L of antibody including FITC-CD86 antibody, PE-CD54 antibody, FITC labeled mouse isotype control IgG1 and PE labeled mouse isotype control IgG 1; dyeing for 30min at 7 ℃ in a dark room; washing 2 times with FACS buffer; the cells were resuspended in 500. mu.L of FACS buffer and transferred to a 5mL flow tube and protein markers were detected using flow cytometry.
2-5, data processing: the Relative Fluorescence Intensity (RFI) was calculated using the fluorescence intensity value (MFI) at the time of detection by flow cytometry, and the calculation formula was the same as in example 1.
2-6 prediction model: the detailed results are shown in Table 9.
TABLE 9 detection results of h-CLAT for certain efficacy materials
| Name (R) | Cell viability% | RFI CD86 | RFI CD54 | Predicted results |
| Raw materials with certain effects | 72.3 | 102 | 93 | Non-sensitizers |
3. Result integration and prediction
The prediction results are evaluated as follows:
| NO. | DPRA improvement method | h-CLAT |
| Raw materials with certain effects | Sensitizers | Non-sensitizers |
And (4) continuing judging by combining the third KeratinoSens method, if the detection result of the KeratinoSens method is sensitization, the functional raw material is a sensitizer, and if the detection result of the KeratinoSens method is non-sensitization, the functional raw material is a non-sensitizer.
4. Conclusion
The test employs a combination of direct polypeptide binding assays and human cell line activation assays to detect skin sensitization of a functional material. The direct polypeptide combination test result shows that a certain functional raw material is combined with cysteine polypeptide and is a sensitizer, and the human cell line activation test result predicts that the functional raw material is a non-sensitizer. And then, combining a third alternative method (KeratinoSens) to confirm, wherein if the detection result of the KeratinoSens method is sensitization, the efficacy raw material is a sensitizer, and if the detection result of the KeratinoSens method is non-sensitization, the efficacy raw material is a non-sensitizer.
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 and are included in the scope of the present invention. For example, the present invention only includes some cosmetic materials, products, and plant extracts. However, other non-single component systems mentioned in the present invention, such as personal care products (shampoos, conditioners, hair dyes, skin care products, sunscreens, foundations, BB creams, CC lotions, eye shadows, lip gloss, oils, body washes, body lotions, etc.), household washing products (laundry detergents, washing powders, soaps, detergents, bleaches, disinfectants, softeners, etc.), pharmaceuticals (penicillins, sulfonamides, etc.), plant extracts (crude extracts, fine extracts, and mixtures thereof), bioactive materials (polypeptides, polymers, genetically engineered products), dust and air pollutants, and other skin contact products (textile extracts such as clothing, underwear), etc., can also be tested for skin sensitization using the methods of the present invention. This is not further enumerated here.
Claims (10)
1. A method for predicting skin sensitization of a complex test object is characterized by comprising the following steps:
(1) improved assay for direct polypeptide binding assays
(1.1) sample preparation: establishing a control group and a complex test object group, wherein the control group comprises 750 mu L of cysteine polypeptide mother liquor, 200 mu L of acetonitrile and 50 mu L of complex test object dissolving solvent, the complex test object group comprises 750 mu L of phosphate buffer solution and cysteine polypeptide mother liquor, 200 mu L of acetonitrile and a series of complex test objects with different concentrations, and the concentration of the cysteine polypeptide mother liquor is 0.35-0.5 mg/mL;
(1.2) sample incubation: placing the control group and the complex test object group in a dark place at 25 +/-2.5 ℃ for incubation for 24 +/-2 hours for later use;
(1.3) sample detection: analyzing the sample incubated in the step (1.2) by adopting a liquid chromatograph;
(1.4) analysis of results: calculating the polypeptide consumption percentage of the sample and judging the sensitization grade;
(1.5) sensitization prediction model: the consumption percentage of the polypeptide is more than or equal to 0% and less than or equal to 13.89%, the complex test object is a non-sensitizer, the consumption percentage of the polypeptide is more than 13.89%, and the complex test object is a sensitizer;
(2) human cell line activation assay
(2.1) cell plating: selecting THP-1 cells, and pretreating for later use;
(2.2) Complex test substance Exposure: selecting a complex test object, diluting the complex test object according to a proportion to obtain a series of concentrations, adding the concentrations into the THP-1 cells in the step (2.1), and culturing the complex test object for later use;
(2.3) blocking: adding an FcR blocker into the cells cultured in the step (2.2) for blocking;
(2.4) detection of CD86/CD54 expression: adding an FITC-CD86 antibody, a PE-CD54 antibody, FITC-labeled mouse isotype control IgG1 and PE-labeled mouse isotype control IgG1 into the cells after the blocking in the step (2.3) to form a test object treatment group and a test object treatment group isotype control, and detecting a protein marker by using a flow cytometer after the incubation;
and (2.5) data processing: detecting a relative fluorescence intensity value RFI by using a flow cytometer;
(2.6) prediction model: on the premise that the cell viability is greater than 50%, for FITC-CD86, if RFIC 86 is greater than or equal to 150, the complex test object is an sensitizer; for PE-CD54, if RFICD54 is more than or equal to 200, the complex test substance is an sensitizer, namely when RFICD86 is more than or equal to 150 and/or RFICD54 is more than or equal to 200, the complex test substance is an sensitizer, and the rest cases are non-sensitizer;
(3) result prediction
And (3) when the results of the direct polypeptide binding assay improvement analysis in the step (1) and the human cell line activation assay analysis in the step (2) are both sensitization, the complex test object prediction result is sensitization, and when the results of the direct polypeptide binding assay improvement analysis in the step (1) and the human cell line activation assay analysis in the step (2) are both non-sensitization, the complex test object prediction result is non-sensitization.
2. The method of claim 1, wherein the method comprises: the complex test substance in step (1.1) is a non-single component system, and comprises non-single component chemicals, cosmetic raw materials, cosmetic products, petroleum lubricants, plant extracts, household washing products, dermatologic medicines, bioactive raw materials, dust particles, air pollutant textiles and textile leaching liquor.
3. The method of claim 1, wherein the method comprises: in the step (1.1), the complex test object dissolving solvent is one or more of acetonitrile, water, isopropanol, acetone and dimethyl sulfoxide; the concentration of a series of complex test substances with different concentrations in the step (1.1) is 1000mg/mL, 500mg/mL, 250mg/mL, 100mg/mL and 10 mg/mL; the cysteine polypeptide mother liquor in the step (1.1) is prepared by adopting a phosphoric acid buffer solution or a solution with the volume ratio of 4:1 phosphoric acid buffer solution and acetonitrile.
4. The method of claim 1, wherein the method comprises: the step (1.1) also comprises a blank group, a positive control, a control group A, a control group B and a coelution detection group, wherein the positive control is cinnamaldehyde, 4-ethoxymethylene-2-phenyloxazoline-5-ketone, 2, 4-dinitrochlorobenzene, formaldehyde or benzylidene acetone, the control group A comprises 750 mu L of cysteine polypeptide mother liquor and 250 mu L of acetonitrile, and the components of the control group B are the same as those of the control group but are not incubated.
5. The method of claim 1, wherein the method comprises: the chromatographic conditions of the liquid chromatograph in the step (1.3) are as follows: a column, Zorbax SB-C182.1mm x 100mm x 3.5micron or Agilent Part Number 861753-902 or Phenomenex Luna C18(2)2.0mm x 100mm x 3 micron; column temperature, 30 ℃; a detector, 220nm and 258nm photodiode array detectors or 220nm fixed wavelength absorption detectors; the sample amount is 1-10 mu L; the flow rate is 0.2-1.0 mL/min; mobile phase, phase a: an aqueous phase; phase B: an organic phase; gradient, 0-10min, 90-75% of A, 10-11min, 75-10% of A, 11-13min, 10-10% of A, 13-13.5min, 10-90% of A, 13.5-20min, 90-90% of A, and the balance of phase B, wherein the phase A is trifluoroacetic acid aqueous solution with trifluoroacetic acid volume percentage of 0-0.1%, and the phase B is trifluoroacetic acid acetonitrile solution with trifluoroacetic acid volume percentage of 0-0.85%.
6. The method of claim 1, wherein the method comprises: the percent polypeptide consumption of the sample in step (1.4) was calculated using the following formula: percent polypeptide consumption ═ 1- (complex test substance polypeptide peak area/control polypeptide peak area) ] × 100.
7. The method of claim 1, wherein the method comprises: in the step (1.5), according to a series of sensitization results with different concentrations, the final result is the result with the highest sensitization result, namely, the sensitization is the sensitizers as long as any concentration appears.
8. The method of claim 1 for predicting skin sensitization of a complex subjectThe method is characterized by comprising the following steps: the highest concentration of complex test substance in step (2.2) is the concentration that gives rise to CV75, followed by a 1:1.2 adding 5-7 concentrations including CV75, CV75/1.2, and CV75/1.22、CV75/1.23、CV75/1.24、CV75/1.25、CV75/1.26、CV75/1.27。
9. The method of claim 1, wherein the method comprises: the relative fluorescence intensity value RFI in step (2.5) is calculated using the following formula: relative Fluorescence Intensity (RFI) ═ complex test article treated group MFI-complex test article treated group isotype control MFI)/(complex test article solvent treated group MFI-complex test article treated group isotype control; wherein the mean value of the fluorescence intensity is calculated by adopting the following formula: mean Fluorescence Intensity (MFI) is the geometric mean of fluorescence intensities.
10. The method of claim 1, wherein the method comprises: in step (3), when the analysis result of the direct polypeptide binding assay improvement in step (1) is not consistent with the analysis result of the human cell line activation assay in step (2), a third known method or a classical animal assay is combined for confirmation, when the third known method or the classical animal assay confirms sensitization, the test substance is sensitized, and when the third known method or the classical animal assay confirms non-sensitization, the test substance is non-sensitized.
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