CN102323402A - Kit for in vitro detection of anti-cyclic citrullinated peptide (CCP) antibody and preparation method thereof - Google Patents
Kit for in vitro detection of anti-cyclic citrullinated peptide (CCP) antibody and preparation method thereof Download PDFInfo
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Abstract
The invention belongs to the field of biological engineering, and provides a kit for in vitro detection of an anti-cyclic citrullinated peptide (CCP) antibody, which comprises a detection plate, a CCP antigen, a colloidal gold-labeled recombinant gold staphylococcus aureus A protein conjugate, a confining liquid, a washing liquid, a positive reference product and a negative reference product. The invention also provides a preparation method of the kit for the in vitro detection of the anti-CCP antibody. The kit adopts an indirect method immunosorbent assay principle to detect the anti-CCP antibody in a human serum, the CCP antigen is coated on a nitrocellulose membrane to be made into a solid-phase antigen for capturing the anti-CCP antibody in the human serum, and then colloidal gold is labeled on staphylococcus aureus A protein (SPA) to form the colloidal gold conjugate as a tracer; and if the detected human serum contains the anti-CCP antibody, then a solid phase antigen-anti-CCP antibody-colloid gold conjugate is formed and has red spots. The invention can be applied to the auxiliary diagnosis of rheumatoid arthritis.
Description
Technical field
Bioengineering field of the present invention relates in particular to a kind of kit and preparation method thereof, qualitative vitro detection kit of particularly a kind of anti-cyclic citrulline peptide antibody and preparation method.
Background technology
Rheumatoid arthritis (Rheumatoid Arthritis; RA) be the systemic autoimmune disease that a kind of chronic inflammation, multi-joint are got involved; The criteria for classification of American Rheumatism Association (ARA) in 1987 revision is adopted in the diagnosis of RA at present, and wherein main lab index is rheumatoid factor (RF).External reported first had been synthesized cyclic citrullinated peptide (Cyclic Citrulinated Peptide according to the cDNA sequence of gathering keratin microfilament protein (Filaggrin) in 2000; CCP); Set up EUSA (ELISA) with this as antigen; And successfully in RA serum, detected anti-CCP antibody, find that this antibody has higher susceptibility and specificity in the diagnosis of RA.The rheumatoid arthritis patients prescription on individual diagnosis people quantity of small-middle hospital is few, and test sample is few, and the examining report cycle is long, incurs loss through delay the patient easily and clarifies a diagnosis and the medication treatment.
Summary of the invention
The object of the present invention is to provide a kind of anti-cyclic citrulline peptide antibody vitro detection kit and preparation method; Described this a kind of anti-cyclic citrulline peptide antibody vitro detection kit and preparation method adopt collaurum percolation and chromatography principle to detect the concentration of the anti-cyclic citrulline peptide antibody in the human serum; It is longer to have solved the time of detecting the anti-cyclic citrullinated peptide AC in the serum in the prior art, program complicated technology problem.
The invention provides a kind of anti-cyclic citrullinated peptide (CCP) antibody vitro detection kit, comprise check-out console, cyclic citrullinated peptide (CCP) antigen, collaurum bond, confining liquid, cleansing solution, positive with reference to article and negative with reference to article by recombination staphylococcus aureus A albumen (SPA) mark.
Further; Described check-out console by a plastic casing at the bottom of box; Be provided with one deck water accepting layer at the bottom of the described plastic casing in the box, described water accepting layer is provided with one deck nitrocellulose filter, and described nitrocellulose filter is provided with a plastic casing cover; Described plastic casing cover central authorities are provided with a detection reaction hole, and point adds cyclic citrullinated peptide (CCP) antigen on the corresponding nitrocellulose filter in detection reaction hole.
Further, described detection reaction hole is circular or other shape.
Further, described confining liquid is the phosphate solution that contains bovine serum albumin(BSA), and between the pH7.0 of phosphate solution～7.5, the percentage by weight of bovine serum albumin(BSA) in phosphate solution is 1.0%.
Further, described cleansing solution is the Tris-HCl solution that contains Tween-20, and between the pH7.0 of Tris-HCl solution～7.5, the percentage by weight of Tween-20 in Tris-HCl solution is 0.05%.
Further, in every milliliter of collaurum bond, the quality of recombination staphylococcus aureus A albumen is 0.025 milligram.
Further, described positive reference article are the anti-CCP antibody of concentration greater than 6.0RU/mL.
Further, described negative reference article are the anti-CCP antibody of concentration less than 6.0RU/mL.
The present invention also provides the preparation method of above-mentioned a kind of anti-cyclic citrullinated peptide (CCP) antibody vitro detection kit; The step that comprises a preparation check-out console; The step of preparation cyclic citrullinated peptide (CCP) antigenic solution, the step of the recombination staphylococcus aureus A protein conjugates of a preparation colloid gold label, the step of a preparation confining liquid; The step of a preparation cleansing solution, preparation positive a step and the negative step of preparation with reference to article with reference to article.
Principle of work of the present invention is: this kit adopts indirect method immunoabsorption principle in order to detect the anti-CCP antibody in the human serum.CCP is antigen coated in nitrocellulose filter, process solid phase antigen, in order to catch the anti-CCP antibody that possibly exist in the human serum.Again SPA is marked on collaurum, forms the collaurum bond as tracer.As containing anti-CCP antibody in tested person's serum, then form solid phase antigen-anti-CCP antibody-collaurum bond, the spot reaction that takes on a red color, the concentration of the anti-CCP antibody that contains in its shade and the tested serum is relevant, but is not proportionate relationship.
The present invention compares with prior art, and its technical progress is significant.Anti-cyclic citrullinated peptide of the present invention (CCP) antibody indirect prize law principle and collaurum diafiltration or the qualitative vitro detection kit of colloidal gold chromatographic detection technique; In order to detect the anti-CCP antibody in the human serum; Citrulline peptide wherein is made up of 18 polypeptide, inanimate object toxicity.The present invention can be used for detecting the anti-cyclic citrullinated peptide of human serum sample (CCP) AC, uses as rheumatoid arthritis (RA) auxiliary diagnosis.Adopt kit of the present invention can receive examining report the same day, testing result is accurate and convenient.
Description of drawings
Fig. 1 has shown that check-out console reacting hole center check point has punctation, positive result.
Fig. 2 has shown check-out console reacting hole center check point redfree spot, negative result.
Embodiment
Embodiment 1 Preparation of Colloidal Gold condition is selected
1.1 the trisodium citrate consumption is selected
The A group: 0.05% chlorogold solution adds 2.0 milliliters of 5% trisodium citrates for 100 milliliters.
The B group: 0.05% chlorogold solution adds 1.5 milliliters of 5% trisodium citrates for 100 milliliters.
The C group: 0.05% chlorogold solution adds 1.0 milliliters of 5% trisodium citrates for 100 milliliters.
The D group: 0.05% chlorogold solution adds 0.5 milliliter of 5% trisodium citrate for 100 milliliters.
Experimental result:
Wavelength X: 540 535 530 525 520 510nm.
ABS value: A organizes 0.471 0.608 0.791 1.044 1.426 1.063
B organizes 0.601 0.746 1.093 1.411 0.942 0.711
C organizes 0.623 0.803 1.318 0.952 0.714 0.635
D organizes 0.998 1.431 1.091 0.801 0.678 0.600
Range estimation colloidal gold solution color: A group: orange red.B group: redness.C group: red, D group: aubergine.
Reach visual observations colloidal gold solution color according to colourimetry collaurum peakedness ratio, prepare the colloid gold particle about 40 nanometers, 0.05% chlorogold solution is intended for 100 milliliters and is added 1.2 milliliters of 5.0% trisodium citrates.
Embodiment 2 collaurum bond preparation conditions are selected
2.1 the pH value of colloid gold label SPA is selected
Select the pH value of colloid gold label SPA, use 1.0% Carbon Dioxide sodium solution to regulate colloid gold label pH value, compare the suitable pH value scope of colloid gold label SPA through detection sensitivity.
Experimental result:
Experimental result shows; O'clock critical value sample is cloudy partially in pH6.0～6.4 for the pH value scope of colloid gold label SPA; The effect of pH >=6.4 o'clock colloid gold label SPA is all similar, and no significant difference advises that in view of the above the pH value scope of colloid gold label SPA is controlled at pH6.4～7.2; That is per 100 milliliters of collaurums, add 1.2 milliliters of 1.0% Carbon Dioxide sodium solutions.
2.2.2 colloid gold label SPA concentration is selected
Get collaurum and add 1.2 milliliters of 1.0% Carbon Dioxide sodium solutions according to per 100 milliliters, SPA concentration is selected in the according to the form below operation again:
Static 2 hours of room temperature, collaurum the 3rd pipe, taking on a red color of 0.025 milligram of every milliliter of colloid gold label SPA is transparent, and the 4th pipe promptly is little aubergine.Show that every milliliter of collaurum needs 0.025 milligram of mark SPA.
Embodiment 3 check-out console preparation conditions are selected
3.3.1 check point encapsulates the comparison of medium pH condition
Experiment material:
(1) nitrocellulose filter, aperture are 0.45 μ m.
(2) encapsulate damping fluid: 1.0mol/L NaHCO
3
NaHCO
3(analyzing pure) 8.401 gram is put into container, measures process water with graduated cylinder and pours container into, waits to dissolve, and the taking technique water is supplied volume to 100 milliliter, mixing again.
(3) check point antigen: cyclic citrullinated peptide (CCP) antigen (1.0mg/mL)
Experimentation:
(1) check point antigen is handled:
Cyclic citrullinated peptide (CCP) antigen (1.0mg/mL) stoste, pH=7.0～7.5.
Cyclic citrullinated peptide (CCP) antigen (1.0mg/mL) stoste adds 1.0mol/L NaHCO for 0.05 milliliter
30.005 milliliter, mixing, pH>=8.0.
(2) encapsulate: get check point antigen 2.0uL, point is added in processes check-out console on the nitrocellulose filter.
(3) negative, each 10 times comparisons of the positive sample check point colour generation degree of test.
Experimental result:
It is even more ideal with pH>=8.0 to show that according to our experimental result check point encapsulates the pH condition of medium, and adopts 1.0mol/L NaHCO
3Adjust pH and have the stable advantage of the convenient use of preparation.Draft and encapsulate medium and be employed in and add 1/10th 1.0M NaHCO in the envelope antigen
3Adjustment pH>=8.0.
3.3.2 check point antigen working concentration is selected
Experiment material:
(1) nitrocellulose filter, aperture are 0.45 μ m.
(2) check point antigen: cyclic citrullinated peptide (CCP) antigen (2.0mg/mL) stoste adds 1.0mol/L NaHCO for 0.1 milliliter
30.01 milliliter, mixing, this antigen concentration are 2.0mg/mL, use 100mmol/L NaHCO again
3Becoming antigen concentration as gradient dilution is 1.0mg/mL, 0.5mg/mL, 0.25mg/mL.
Experimentation:
(1) encapsulate: press antigen concentration and divide into groups, get check point antigen 2.0uL, point is added in processes check-out console on the nitrocellulose filter.
(2) test feminine gender, the positive, sensitivity are with reference to each 5 times comparisons of article check point colour generation degree.
Experimental result:
(1) envelope antigen concentration: cyclic citrullinated peptide (CCP) antigen 2.0mg/mL.
| Sample | Negative with reference to article | Sensitivity is with reference to article | Positive in article |
| Sequence number | Check point | Check point | Check point |
| 1 | ± | + | ++ |
| 2 | ± | + | ++ |
| 3 | ± | + | ++ |
| 4 | ± | + | ++ |
| 5 | ± | + | ++ |
(2) envelope antigen concentration: cyclic citrullinated peptide (CCP) antigen 1 .0mg/mL.
| Sample | Negative with reference to article | Sensitivity is with reference to article | Positive in article |
| Sequence number | Check point | Check point | Check point |
| 1 | - | ± | + |
| 2 | - | ± | + |
| 3 | - | ± | + |
| 4 | - | ± | + |
| 5 | - | ± | + |
(3) envelope antigen concentration: cyclic citrullinated peptide (CCP) antigen 0.5mg/mL.
| Sample | Negative with reference to article | Sensitivity is with reference to article | Positive in article |
| Sequence number | Check point | Check point | Check point |
| 1 | - | - | ± |
| 2 | - | - | ± |
| 3 | - | - | ± |
| 4 | - | - | ± |
| 5 | - | - | ± |
(4) envelope antigen concentration: cyclic citrullinated peptide (CCP) antigen 0.25mg/mL.
| Sample | Negative with reference to article | Sensitivity is with reference to article | Positive in article |
| Sequence number | Check point | Check point | Check point |
| 1 | - | - | ± |
| 2 | - | - | ± |
| 3 | - | - | ± |
| 4 | - | - | ± |
| 5 | - | - | ± |
(1) experimental result shows that the concentration that encapsulates of check point cyclic citrullinated peptide (CCP) antigen is advisable with 1.0mg/mL.When encapsulating concentration 2.0mg/mL, negative with reference to article sun partially, and when encapsulating concentration 0.5mg/mL, sensitivity is with reference to article, positive reference article the moon partially.
3.3.3 check point encapsulates liquid volume added relatively
Experiment material:
(1) check point antigen: cyclic citrullinated peptide (CCP) antigen (1.0mg/mL) is put into container for 0.25 milliliter, adds 1.0mol/L NaHCO
30.025 milliliter, mixing.
Experimentation:
Get and encapsulate liquid volume added on the nitrocellulose filter that check point antigen point is added in check-out console reacting hole center and be respectively: 1.0uL, 1.5uL, 2.0uL, 2.5uL, 3.0uL.The test positive sample, repeatability and the spot size and the spot consistent degree of the colour generation degree of positive spots are observed in every group of repeated test 20 times.
Experimental result:
Though can save material when encapsulating dosage 1.0uL, but the colour generation degree of positive spots is repeated relatively poor, inadvisable, and reason is the wayward suction amount of the too little micro sample adding appliance of 1.0uL sampling amount.And the consistent degree of colour generation spot is also undesirable.The colour generation spot is too little in addition can influence the effect of estimating.
The repeated result of the colour generation degree of test positive spots is more satisfactory when encapsulating dosage 1.5～3.0uL.The consistent degree of spot size is also more satisfactory in each group.
Along with the increase that encapsulates dosage, the diameter of positive spots is also big more.The diameter of positive spots is too big when encapsulating dosage 3.5uL, and causes raw-material waste.
Should recommend to encapsulate dosage 1.5～2.5uL, it is comparatively desirable that this encapsulates the visual effect that adds positive spots size in the weight range, and the repeatability of the colour generation degree of positive spots is also consistent.
Embodiment 4 production technologies
4.1 collaurum bond preparation technology
4.1.1 preparation collaurum
4.1.1.10.05% chlorogold solution is 300 milliliters, is heated to and boils.
4.1.1.2 add 3.6 milliliters of 5% trisodium citrates.
4.1.1.3 continued heated and boiled 15 minutes, stop heating, room temperature to be chilled to returns to original volume with distilled water.
4.1.2 colloid gold label
4.1.2.1 collaurum 100mL.
4.1.2.2 add 1.2 milliliters of 1.0% Carbon Dioxide sodium solutions, school pH 6.4～7.2.
4.1.2.3 add 2.5 milliliters of SPA (1.0mg/mL), static 30 minutes of room temperature.
Add 20 milliliters of regulation and control of 10X Tris-HCl pH7.2～7.5 4.1.2.4 stir.
Add BSA2 gram, PEG200001 gram, ProClin3000.2 milliliter 4.1.2.5 stir.
4.2 check-out console preparation technology
4.2.1 the assembling check-out console is got the plastic casing bottom, places water accepting layer, on water accepting layer, places nitrocellulose filter, covers the plastic casing cover, compresses.
4.2.2 check point antigen: cyclic citrullinated peptide (CCP) antigen (1.0mg/mL) adds 1.0mol/LNaHCO for 1.0 milliliters
30.1 milliliter, mixing.
4.2.3 get check point antigen 2.0uL, put on the nitrocellulose filter that is added in check-out console reacting hole center and process check-out console.
4.3 preparation confining liquid and cleansing solution
4.3.1 the preparation confining liquid, 1.0% bovine serum albumin(BSA), 0.02M pH7.0～7.5 phosphate solutions.
4.3.2 the preparation cleansing solution, 0.05%Tween-20,0.05M pH7.0～7.5 Tris-HCl solution.
4.4 prepare anti-CCP antibody with reference to article.
4.4.1 positive in the article preparation, confining liquid is added anti-CCP antibody (200RU/mL) 5.0 milliliters for 15 milliliters, mixing is about anti-CCP antibody 50RU/mL.
4.4.2 negative with reference to the article preparation, 13.5 milliliters of confining liquids are added the positive with reference to 1.5 milliliters of article, mixing is anti-CCP antibody＜6.0RU/mL.
4.4.3 sensitivity prepares with reference to article: 14.5 milliliters of confining liquids are added 2.0 milliliters of positive reference article, and mixing is about anti-CCP antibody 6.0RU/mL.
4.4.4 yin, yang and sensitivity are foundation with the testing result of anti-cyclic citrullinated peptide (CCP) TPPA kit (ELISA) Shanghai food medicine prison tool (standard) word 2010 all with reference to article CCP antigen concentration.
4.5 normal reference range is confirmed; Anti-CCP antibody＜6.0RU/mL is a foundation with the normal reference range of anti-cyclic citrullinated peptide (CCP) TPPA kit (ELISA); Because this law is a qualitative detection; The anti-CCP antibody content of sample check point in normal reference range does not develop the color, so select to do sensitivity with reference to article with the sample that anti-CCP antibody is about 6.0RU/mL.
Embodiment 5 methods of inspection
1, uses preceding elder generation that reagent and sample are taken out, placed at room temperature 15-30 minute, make it restore to room temperature.
2, take out check-out console.
3, add 2 of confining liquids at check-out console reacting hole center, treat that confining liquid infiltrates fully.
4, add testing sample 40 microlitres at check-out console reacting hole center with pipettor, treat that sample infiltrates fully.
5, add 4 of cleansing solutions at check-out console reacting hole center, treat that cleansing solution infiltrates fully.
6, add 3 of collaurum bonds at check-out console reacting hole center, treat that the collaurum bond infiltrates fully.
7, add 4 of cleansing solutions at check-out console reacting hole center, treat that cleansing solution infiltrates fully, visual observation.
[assay interpretation]
The user following situation may occur when carrying out sample detection:
A. check-out console reacting hole center check point has punctation, positive result (as shown in Figure 1).
B. check-out console reacting hole center check point redfree spot, negative result (as shown in Figure 2).
[reference value (term of reference)]
Anti-CCP antibody＜6.0RU/mL is a foundation with the normal reference range of anti-cyclic citrullinated peptide (CCP) TPPA kit (ELISA); The anti-CCP antibody content of sample is check-out console reacting hole center check point redfree spot in normal reference range, shows negative findings.
Normal reference range: show negative findings (pointing out anti-CCP antibody content) less than 6.0RU/mL.
[explanation of assay]
1, negative findings, then pointing out the anti-CCP antibody content＜6.0RU/mL of this sample is normal range.
2, positive findings, then pointing out the anti-CCP antibody content >=6.0RU/mL of this sample is undesired scope.
[limitation of the method for inspection]
1, this method can only qualitative determination human serum sample in anti-cyclic citrullinated peptide (CCP) antibody, only use as auxiliary diagnosis.
2, the stability of reaction spot: positive spots can not faded in 30 minutes, and negative findings is no change still.
[product performance index]
1, physical behavior
Outward appearance: clean and tidy, sign (title, lot number, the term of validity etc.) is complete, and literal is clear.Each liquid reagent should clear, no insolubles.Confining liquid is a light yellow transparent liquid, and cleansing solution is a colourless transparent liquid, and the collaurum bond is red transparency liquid, and positive reference article are light yellow transparent liquid, and negative reference article are light yellow transparent liquid.The kit content should be accurate.
2, specificity
Detect the sample of 300 routine anti-CCP antibody contents less than 6.0RU/mL, the range estimation testing result, check-out console reacting hole center check point is the exhibit red spot not, and reaction result is negative.
3, sensitivity for analysis
The sample of getting the about 6.0RU/mL of anti-CCP antibody content detects, the range estimation testing result, and check-out console reacting hole center check point is answered the exhibit red spot.
Claims (8)
1. an anti-cyclic citrulline peptide antibody vitro detection kit is characterized in that: comprise check-out console, cyclic citrullinated peptide antigen, the recombination staphylococcus aureus A protein conjugates by colloid gold label, confining liquid, cleansing solution, positive in article and negative with reference to article.
2. a kind of anti-cyclic citrulline peptide antibody vitro detection kit as claimed in claim 1; It is characterized in that: described check-out console comprises box at the bottom of the plastic casing; Be provided with one deck water accepting layer at the bottom of the described plastic casing in the box; Described water accepting layer is provided with one deck nitrocellulose filter; Described nitrocellulose filter is provided with a plastic casing cover, and described plastic casing cover cover central authorities are provided with a detection reaction hole, and point adds cyclic citrullinated peptide antigen on the corresponding nitrocellulose filter in detection reaction hole.
3. a kind of anti-cyclic citrulline peptide antibody vitro detection kit as claimed in claim 1; It is characterized in that: described confining liquid is the phosphate solution that contains bovine serum albumin(BSA); Between the pH 7.0～7.5 of phosphate solution, the percentage by weight of bovine serum albumin(BSA) in phosphate solution is 1.0%.
4. a kind of anti-cyclic citrulline peptide antibody vitro detection kit as claimed in claim 1; It is characterized in that: described cleansing solution is the Tris-HCl solution that contains Tween-20; Between the pH7.0 of Tris-HCl solution～7.5, the percentage by weight of Tween-20 in Tris-HCl solution is 0.05%.
5. a kind of anti-cyclic citrulline peptide antibody vitro detection kit as claimed in claim 1, it is characterized in that: in every milliliter of collaurum bond, the quality of recombination staphylococcus aureus A albumen is 0.025 milligram.
6. a kind of anti-cyclic citrulline peptide antibody vitro detection kit as claimed in claim 1 is characterized in that: described positive reference article are the anti-CCP antibody of concentration greater than 6.0 RU/mL.
7. a kind of anti-cyclic citrulline peptide antibody vitro detection kit as claimed in claim 1 is characterized in that: described negative reference article are the anti-CCP antibody of concentration less than 6.0 RU/mL.
8. the preparation method of the described a kind of anti-cyclic citrulline peptide antibody vitro detection kit of claim 1; It is characterized in that: the step that comprises a preparation check-out console; The step of a preparation cyclic citrullinated peptide antigenic solution, the step of the recombination staphylococcus aureus A protein conjugates of a preparation colloid gold label, the step of a preparation confining liquid; The step of a preparation cleansing solution, preparation positive a step and the negative step of preparation with reference to article with reference to article.
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| CN108948153A (en) * | 2018-05-22 | 2018-12-07 | 北京蛋白质组研究中心 | A kind of citrulling modified peptides antigen combination and its application |
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| CN110161247A (en) * | 2018-02-11 | 2019-08-23 | 北京科美生物技术有限公司 | Detect homogeneous immunological detection reagent box and its application of cyclic citrullinated peptid |
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