CN109682979A - A kind of calprotectin joint lactoferrin antigen test strip and preparation method thereof - Google Patents
A kind of calprotectin joint lactoferrin antigen test strip and preparation method thereof Download PDFInfo
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- CN109682979A CN109682979A CN201910088992.0A CN201910088992A CN109682979A CN 109682979 A CN109682979 A CN 109682979A CN 201910088992 A CN201910088992 A CN 201910088992A CN 109682979 A CN109682979 A CN 109682979A
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- calprotectin
- lactoferrin
- test strip
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- 108010069316 Leukocyte L1 Antigen Complex Proteins 0.000 title claims abstract description 61
- 235000021242 lactoferrin Nutrition 0.000 title claims abstract description 56
- 108010063045 Lactoferrin Proteins 0.000 title claims abstract description 55
- CSSYQJWUGATIHM-IKGCZBKSSA-N l-phenylalanyl-l-lysyl-l-cysteinyl-l-arginyl-l-arginyl-l-tryptophyl-l-glutaminyl-l-tryptophyl-l-arginyl-l-methionyl-l-lysyl-l-lysyl-l-leucylglycyl-l-alanyl-l-prolyl-l-seryl-l-isoleucyl-l-threonyl-l-cysteinyl-l-valyl-l-arginyl-l-arginyl-l-alanyl-l-phenylal Chemical compound C([C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CS)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 CSSYQJWUGATIHM-IKGCZBKSSA-N 0.000 title claims abstract description 55
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Classifications
-
- 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
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
-
- 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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
-
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/06—Gastro-intestinal diseases
- G01N2800/065—Bowel diseases, e.g. Crohn, ulcerative colitis, IBS
Abstract
The invention discloses a kind of calprotectin joint lactoferrin antigen test strips and preparation method thereof, it is intended to mention that a kind of detection speed is fast, calprotectin easy to operate and high sensitivity joint lactoferrin test strip, a kind of simple process is also provided simultaneously, reproducible, finished product structure is stablized, the preparation method of the good calprotectin joint lactoferrin test strip of service performance.The calprotectin joint lactoferrin test strip includes bottom plate, sample pad, label pad, detecting pad, water absorption pad.The preparation method: preparing the sample pad, the label pad, the detecting pad first, then overlaps the sample pad, the label pad, the detecting pad, water absorption pad sequence on the bottom plate.The present invention is applied to the technical field of inflammatory bowel disease marker protein test strip.
Description
Technical field
The present invention relates to the technical field of inflammatory bowel disease marker protein test strip, in particular to a kind of calcium is defended
Albumen combines lactoferrin antigen test strip and preparation method thereof.
Background technique
Calprotectin (calprotectin) belongs to S-100 family, is calcium-zinc that a relative molecular mass is 36kDa
Binding protein, the heterotrimer being made of two MRP-8 chains and a MRP-14 chain, is mainly derived from neutrophil leucocyte, is
The important symbol object that neutrophil leucocyte updates.The appearance of calprotectin is stomach proliferative disease and intestinal inflammatory infection in excrement
The mark of property disease.Under normal circumstances, it is single be difficult to distinguish from disease symptoms uncomfortable illness caused by acute intestinal excitation with
Chronic infective enterogastric diseases are abused so as to cause unnecessary and excessive colonoscopy, and calprotectin is acute gastrointestinal sense
A kind of mark of dye, in infection and diseases associated with inflammation, since specifically (neutrophil leucocyte and macrophage are thin in inflammatory cell for it
Born of the same parents) in expression, the calprotectin in the case where infectiousness and inflammatory in serum can increase by 5 to 40 times.Calprotectin is in excrement
Just discovery in, its concentration in excrement are 6 times of normal serum concentration.It can be sent out in the excrement in the patient of inflammatory bowel disease
Now a large amount of calprotectin, and it is possible thereby to judge the degree of infection.
Lactoferrin (1actoferrin) is glycoprotein find in recent years, in conjunction with iron, and main distribution and expression is in neutrality
In granulocyte, epithelial cell and various tissues and body fluid, has and promote iron absorption, antibacterial, immunological regulation, anti-infective, antioxygen
The multiple biological functions such as change, antiviral, its content increases under many inflammatory conditions.Recently researches show that lactoferrins to exist
Content in inflammatory bowel disease (IBD) patient intestinal mucosa and excrement is apparently higher than Normal group, acts not only as evaluation disease
The index of sick activity, and can be used as predictive disease recurrence, detection therapeutic effect, the finger for evaluating the effects of curative effect of medication
Mark.
The goldstandard of evaluation enteritis is Sigmoidoscope biopsy at present.But it has invasive, belongs to traumatic examination, it cannot
It checks whole gastrointestinal tracts, and needs skilled operator and uncomfortable INTESTINAL CLEANSING.Calprotectin is commonly examined
Survey method is there are also enzyme linked immunosorbent assay and enzyme linked immunosorbent assay, enzyme linked immunosorbent assay and fluorescence ELISA
Method sensitivity is higher, is suitble to professional's detection, and operating time length needs professional to operate.
Summary of the invention
The technical problem to be solved by the present invention is to overcome the deficiencies of the prior art and provide, a kind of detection speed is fast, grasps
Make easy and high sensitivity calprotectin and combine lactoferrin test strip, while a kind of simple process, again being also provided
Renaturation is good, finished product structure is stable, the preparation method of the good calprotectin joint lactoferrin test strip of service performance.
The technical scheme adopted by the invention is that: the calprotectin joint lactoferrin test strip includes bottom plate,
Sequentially be overlapped with sample pad, label pad, detecting pad, water absorption pad on the bottom plate, detecting pad be equipped with calprotectin detection line T1,
Lactoferrin detection line T2 and nature controlling line C is coated with calprotectin antigen, the cream iron egg in the calprotectin detection line T1
It is coated with lactoferrin antigen on white detection line T2, is coated with sheep anti mouse or goat anti-rabbit igg on the nature controlling line C.
Further, the calprotectin detection line T1 and the lactoferrin detection line T2 are described close to the label pad
Nature controlling line C is close to the water absorption pad.
Further, the material of the detecting pad is nitrocellulose filter, and the material of the label pad is glass fibre element.
A kind of preparation method of calprotectin joint lactoferrin antigen test strip includes the following steps:
A, the sample pad is prepared；
B, the label pad is prepared；
C, it prepares the detecting pad: taking nitrocellulose filter, the calprotectin detection line T1, the lactoferrin are marked in interval
The detection line T2 and nature controlling line C is coated with calprotectin antigen, the lactoferrin detection in the calprotectin detection line T1
It is coated with lactoferrin antigen on line T2, is coated with sheep anti mouse or goat anti-rabbit igg, the cellulose nitrate that will be coated on the nature controlling line C
Plain film is dry, spare；
D, the sample pad, the label pad, the detecting pad, water absorption pad sequence are overlapped on the bottom plate.
In step, the sample pad prepare it is as follows: by the sample pad be placed in sample pad treatment fluid impregnate or will
Then the sample pad containing sample pad treatment fluid is placed in temperature in the sample pad by sample pad treatment fluid even application
It is spare to dry 3~5h in 37 DEG C, environment of the relative humidity less than 20%；Wherein, sample pad treatment fluid is containing 0.5% tween-
20, the pH of 1%BSA is the 1mol/L borate buffer solution or Tris-HCl buffer of 8 .0.
In stepb, the preparation of the label pad includes the following steps:
A, color latex microballoon is prepared；
B, color latex microballoon marks calprotectin monoclonal antibody and lactoferrin monoclonal antibody respectively, micro- in color latex
Calprotectin monoclonal antibody and lactoferrin monoclonal antibody are separately added by 0.6~1.0mg/ml in ball solution；
C, the mixed liquor of color latex microballoon dilution and color latex microspheres solution is added after agitated in the solution again；
D, the mixed liquor in step c is removed into supernatant through centrifugation, obtains sediment；
E, the sediment is dissolved in buffer by 4~10mg/ml and obtains gold labeling antibody solution, contain its weight in the buffer
1%~10% animal serum albumin and 0.01%~0.06% Sodium azide；
F, the gold labeling antibody solution is leached with glass fibre, it, then will be described until the gold labeling antibody solution starts exudation
Glass fibre is dried to form the label pad.
The beneficial effects of the present invention are: when judging intestinal mucosal injury, it usually needs determination is acute gastrointestinal sense first
Dye or chronic infective enterogastric diseases, test strips of the invention can be with the calprotectins and newborn iron egg in joint-detection excrement
It is white, it can quickly judge whether to be acute gastrointestinal infection, be of great significance for the diagnosis of intestinal mucosal injury disease.Pass through
Realize joint inspection, it is primary to be loaded using general sample buffer, two can be obtained simultaneously as a result, joint-detection can be improved
The sensitivity and specificity of detection.Therefore, the calprotectin of the invention joint lactoferrin antigen test strip is sensitive
Degree height, high specificity, and speed is fast, easy to operate, is not necessarily to specialized facilities, is suitable for a variety of fields such as clinical detection or generaI investigation
It closes.
Method due to preparing the test strip are as follows: prepare the sample pad, the label pad, the detection first
Then the sample pad, the label pad, the detecting pad, water absorption pad sequence are overlapped on the bottom plate, that is, are made by pad
The calprotectin combines lactoferrin antigen test strip, easy to operate, therefore, described to prepare the test strip
Method is simple, reproducible, and the test strip stable structure prepared with this method, service performance are good.
Detailed description of the invention
Fig. 1 is the structural schematic diagram of the calprotectin joint lactoferrin test strip.
Specific embodiment
Embodiment one
As shown in Figure 1, in the present embodiment, the calprotectin joint lactoferrin test strip includes bottom plate 1, the bottom
Sequentially be overlapped with sample pad 2, label pad 3, detecting pad 4, water absorption pad 5 on plate 1, detecting pad 4 be equipped with calprotectin detection line T1,
Lactoferrin detection line T2 and nature controlling line C is coated with calprotectin antigen, the cream iron egg in the calprotectin detection line T1
It is coated with lactoferrin antigen on white detection line T2, is coated with sheep anti mouse or goat anti-rabbit igg on the nature controlling line C.
The calprotectin detection line T1 and the lactoferrin detection line T2 are close to the label pad 3, the nature controlling line C
Close to the water absorption pad 5.
The material of the detecting pad 4 is nitrocellulose filter, and the material of the label pad 3 is glass fibre element.
A kind of preparation method of calprotectin joint lactoferrin antigen test strip includes the following steps:
A, the sample pad 2 is prepared；
B, the label pad 3 is prepared；
C, prepare the detecting pad 4: by concentration be 1.0~2.0mg/ml calprotectin and lactoferrin antibody in be added 1%~
3% fixative recycles point film machine to put the calprotectin and lactoferrin antibody on nitrocellulose membrane respectively, puts film
It is put into 37~39 DEG C of environment and dries afterwards, then the PBS buffer solution for containing 0.5% polysorbas20 with 0.01 mole of pH value for 9.0,37
It closes 30 minutes and dries at DEG C.In the present embodiment, calprotectin is taken to combine lactoferrin monoclonal antibody, heregulin concentration to 1.5mg/
Ml is added and draws film dilution, sprays the calprotectin detection line T1, the lactoferrin in nitrocellulose membrane middle section with Membrane jetter
Detection line T2；It takes goat anti-rabbit igg antibody heregulin concentration to 1mg/ml again, is added and draws film dilution, with Membrane jetter in nitric acid fibre
Spray film amount, antibody coating is arranged by 0.036ul/l0cm away from the nature controlling line C is sprayed at detection line a distance in the middle section for tieing up film
Film drying, cellulose acetate film can replace the nitrocellulose filter；
D, the sample pad 2, the label pad 3, the detecting pad 4, the water absorption pad 5 are sequentially overlapped on the bottom plate 1.
In step, the sample pad 2 prepare it is as follows: by the sample pad 2 be placed in sample pad treatment fluid impregnate or
By sample pad treatment fluid even application in the sample pad 2, then the sample pad 2 containing sample pad treatment fluid is placed in
Temperature is 37 DEG C, dries 3~5h in environment of the relative humidity less than 20%, spare；Wherein, sample pad treatment fluid is containing 0.5%
Tween-20,1%BSA pH be 8 .0 1mol/L borate buffer solution or Tris-HCl buffer.
In stepb, the preparation of the label pad 3 includes the following steps:
A, color latex microballoon is prepared；
B, color latex microballoon marks calprotectin monoclonal antibody and lactoferrin monoclonal antibody respectively, micro- in color latex
Calprotectin monoclonal antibody and lactoferrin monoclonal antibody are separately added by 0.6~1.0mg/ml in ball solution；
C, the mixed liquor of color latex microballoon dilution and color latex microspheres solution is added after agitated in the solution again；
D, the mixed liquor in step c is removed into supernatant through centrifugation, obtains sediment；
E, the sediment is dissolved in buffer by 4~10mg/ml and obtains gold labeling antibody solution, contain its weight in the buffer
1%~10% animal serum albumin and 0.01%~0.06% Sodium azide；
F, the gold labeling antibody solution is leached with glass fibre, it, then will be described until the gold labeling antibody solution starts exudation
Glass fibre is dried to form the label pad 3.
Calprotectin combines lactoferrin antigen detection method: sample to be measured is taken, in the present embodiment, the sample to be measured
For stool sample, the sample to be measured is after thaw at RT, and drop is marked in color latex microballoon after taking about 50~150 mg to dissolve
Calprotectin and lactoferrin antibody carrier on, if any calprotectin or lactoferrin antibody on the detection carrier shape
Then it is the positive at the calprotectin detection line T1, the lactoferrin detection line T2 and the nature controlling line C, is otherwise feminine gender.
The calprotectin and lactoferrin antibody are monoclonal antibody, perhaps mostly anti-or be the monoclonal antibody and described how anti-
Mixture, in the present embodiment, the calprotectin and lactoferrin antibody are mouse anti-calprotectin and lactoferrin monoclonal
Antibody.
Calprotectin joint lactoferrin antigen detection method is one-step method, species specificity with height, very
It is few the clinical false negative of puzzlement, false positive issue occur, there is the sensitivity of height, and easy to operate, is not necessarily to specialized facilities.
The calprotectin joint lactoferrin antigen test strip detection sensitivity is high, detection is rapid, quick, side
Just, seldom there is the problem of false negative and false positive, be suitable for clinical or generaI investigation.
Embodiment two
The present embodiment and one difference of embodiment: in the present embodiment, the control line 232 is formed by sheep anti-mouse igg antibody
Composition.
The present invention is applied to the technical field of inflammatory bowel disease marker protein test strip.
Although the embodiment of the present invention is described with practical solution, the limit to meaning of the present invention is not constituted
It makes, for those skilled in the art, is all to the modification of its embodiment and with the combination of other schemes according to this specification
Obviously.
Claims (6)
1. a kind of calprotectin combines lactoferrin antigen test strip, including bottom plate (1), it is characterised in that: the bottom plate
(1) sample pad (2), label pad (3), detecting pad (4), water absorption pad (5) are sequentially overlapped on, detecting pad (4) is equipped with calcium and defends egg
White detection line (T1), lactoferrin detection line (T2) and nature controlling line (C) are coated with calcium on the calprotectin detection line (T1) and defend
Proteantigen is coated with lactoferrin antigen on the lactoferrin detection line (T2), is coated with goat-anti on the nature controlling line (C)
Mouse or goat anti-rabbit igg.
2. a kind of calprotectin according to claim 1 combines lactoferrin antigen test strip, it is characterised in that: institute
Calprotectin detection line (T1) and the lactoferrin detection line (T2) are stated close to the label pad (3), the nature controlling line (C) is leaned on
The nearly water absorption pad (5).
3. a kind of calprotectin according to claim 1 or 2 combines lactoferrin antigen test strip, feature exists
In: the material of the detecting pad (4) is nitrocellulose filter, and the material of the label pad (3) is glass fibre element.
4. a kind of preparation method of calprotectin joint lactoferrin antigen test strip as described in claim 1, special
Sign is: the preparation method includes the following steps:
A, the sample pad (2) are prepared；
B, the label pad (3) are prepared；
C, the detecting pad (4) are prepared: taking nitrocellulose filter, the calprotectin detection line (T1), the cream are marked in interval
Ferritin detection line (T2) and the nature controlling line (C) are coated with calprotectin antigen on the calprotectin detection line (T1), described
It is coated with lactoferrin antigen in lactoferrin detection line (T2), is coated with sheep anti mouse or goat anti-rabbit igg on the nature controlling line (C), it will
The nitrocellulose filter being coated with is dry, spare；
D, the sample pad (2), the label pad (3), the detecting pad (4), the water absorption pad (5) are sequentially overlapped into the bottom
On plate (1).
5. the preparation method according to claim 4, it is characterised in that: in step, the preparation of the sample pad (2) is such as
Under: the sample pad (2) is placed in sample pad treatment fluid and is impregnated or by sample pad treatment fluid even application in the sample pad
(2) on, it is 37 DEG C, relative humidity less than 20% that the sample pad (2) containing sample pad treatment fluid, which is then placed in temperature,
3~5h is dried in environment, it is spare；Wherein, it is 8 .0 that sample pad treatment fluid, which is the pH containing 0.5% Tween-20,1%BSA,
1mol/L borate buffer solution or Tris-HCl buffer.
6. the preparation method according to claim 4, it is characterised in that: in stepb, the preparation packet of the label pad (3)
Include following steps:
A, color latex microballoon is prepared；
B, color latex microballoon marks calprotectin monoclonal antibody and lactoferrin monoclonal antibody respectively, micro- in color latex
Calprotectin monoclonal antibody and lactoferrin monoclonal antibody are separately added by 0.6~1.0mg/ml in ball solution；
C, the mixed liquor of color latex microballoon dilution and color latex microspheres solution is added after agitated in the solution again；
D, the mixed liquor in step c is removed into supernatant through centrifugation, obtains sediment；
E, the sediment is dissolved in buffer by 4~10mg/ml and obtains gold labeling antibody solution, contain its weight in the buffer
1%~10% animal serum albumin and 0.01%~0.06% Sodium azide；
F, the gold labeling antibody solution is leached with glass fibre, it, then will be described until the gold labeling antibody solution starts exudation
Glass fibre is dried to form the label pad (3).
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