CN107045062B - Detect colloidal gold immuno-chromatography test paper strip, the kit and preparation method thereof of human neutrophil genatinase associated lipocalin - Google Patents
Detect colloidal gold immuno-chromatography test paper strip, the kit and preparation method thereof of human neutrophil genatinase associated lipocalin Download PDFInfo
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Abstract
The invention discloses a kind of colloidal gold immuno-chromatography test paper strips for detecting human neutrophil genatinase associated lipocalin, kit and preparation method thereof, the test strips include bottom liner, and it is sequentially arranged at the sample pad on the bottom liner, gold-labelled pad, coated film and blotting paper, the chicken IgY antibody of anti-human NGAL the detection antibody and colloid gold label of colloid gold label is coated in the gold-labelled pad, the coated film includes being arranged in parallel, and the detection zone being spaced apart from each other and check plot, the detection zone is coated with anti-human NGAL coated antibody, the check plot is coated with the anti-chicken IgY antibody of goat, the anti-human corresponding epitope with the amino acid sequence as shown in SEQ ID No:5 of NGAL detection antibody.Colloidal gold immuno-chromatography test paper strip of the invention has easy, quick, practical, sensitive, efficient beneficial effect, can be mass, and is suitable for clinical quick diagnosis and field quick detection, is easy to save, is suitble to the popularization and use in basic hospital.
Description
Technical field
The invention belongs to field of biotechnology, specifically, the present invention relates to the neutrality in a kind of half-quantitative detection urine
The colloidal gold immuno-chromatography test paper strip of granulocyte gelatinase associated lipocalin (NGAL), kit and preparation method thereof.
Background technique
Neutrophil gelatinase-associated lipocalin (neutrophilgelatinase-associated
Lipocalin, NGAL) found in neutrophil leucocyte by Kjeldsen et al. in 1993, it is the glycoprotein of 25kD a kind of.
It has the typical beta sheet barrel structure of lipocalin family, with inflammation, embryonic development, immune response, chemotaxis, signal
Transduction and the processes such as the generation of kinds of tumors and development are closely related.NGAL may participate in cell differentiation and as a kind of survival because
Son inhibits Apoptosis；Such as visible NGAL high in adenocarcinoma of lung, the cancer of the esophagus, cancer of pancreas, colon cancer and breast cancer in human tumor
Expression, and NGAL albumen can be by protecting the activity of MMP-9 to promote the invasion of tumour cell.In recent years there are some researches prove,
NGAL directly takes part in the functions such as body iron metabolism and innate immune reaction (Goetz as small molecule iron complex binding protein
DH, 2002；Yang J, 2002), with renal ischaemia, and Nephrotoxicity and atherosclerosis (Forsblad J, 2002；
Hemdahl AL, 2006) etc. the occurrence and development of diseases are closely related, and help decide ischemic kidney as a kind of early sign object
Degree of injury (Mishra J, 2003；Mishra J, 2005).Therefore, function of the preparation and reorganization NGAL albumen to further investigation NGAL
The application of energy and its clinical diagnosis has great importance.
In the research for finding acute kidney injury (AKI) early diagnosis marker, it has been found that NGAL is one pre- well
Survey and diagnosis index.There is scholar in the AKI mouse model of acute ischemia reperfusion and cisplatin induction, in damage number occurs for discovery
The expression that can be detected NGAL within hour is obviously increased.Urine NGAL is an early stage of Ischemic kieney injury, sensitive finger
Mark, i.e. blood, urine NGAL level increase when AKI, and urine NGAL level increases more significant.And NGAI is increased to be damaged compared with early stage kidney
Other indexs such as Kidney injury molecule (kidneyinjurymolecular1, KIM-1), the Cyr61 of wound
(cysteinrichprotein61), 132- microglobulin etc. occurs early.Meanwhile NGAL level is in kidney injury degree
It is positively correlated, blood can be used as the early stage biological marker of the early stage of diagnosis AKI, sensitivity, specificity with urine NGAL, and in certain journey
It can reflect AKI severity and judging prognosis on degree.NGAL has been found to predict acute as after pediatric cardiac bypass surgery
The early stage reliability index of renal damage.
Up to 50% intensive care unit patient may suffer from a degree of acute renal failure.Acute renal declines
It exhausts the death rate and is in critical illness death rate forefront.Within past 40 years, there is no marked improvements for the prediction of acute renal failure.
Current diagnostic method only just has after renal function exacerbation expressed such as serum creatinine or serum Cys C, and this can
Can just it show at organ damage 1 day or more even still unobvious.NGAL is one kind of apolipoprotein, is initially to activate neutral grain
A kind of small-molecular-weight secreted protein being found in cell, modern research shows that NGAL is to diagnose most having for acute kidney injury
Imitate one of one of Biomarkers and promising tumor marker of early diabetic nephropathy.Human research has found after cardiac surgery patient
In the pathogenetic 2h of acute renal, blood, urine NGAL are significantly increased, and accordingly, NGAL is considered as the life of renal impairment early stage
Object marker.NGAL level is higher to generally occur within following situations: cardiovascular surgery patient, the personnel that are critically ill, septic or hemorrhagic
Shock, kidney transplant, the reaction of vein x-ray contrast agent and renal toxicity therapeutic response.
It is earliest enzyme-linked immunosorbent assay (ELISA) or immunoblotting analysis to the NGAL detection method in blood, Urine specimens
Method detects.But these are all manually to operate, and can not be standardized, and detection cycle is longer and somewhat expensive, therefore does not push away
It recommends for clinical practice, is used only for scientific research.The ELISA kit of detection NGAL used at present can be not only used for craft
Detection, it can also be used to which automated analysis provides laboratory testing efficiency.Meanwhile there are multiple producers to detect using immunoturbidimetry
The kit of NGAL in blood or urine obtains certificate of registry.Turbidimetry is also referred to as nephelometry, belong to scattering spectrum analysis, be
Grow up on the basis of colorimetric, many aspects such as measurement theory, method, calculating are similar with colorimetric method, but substantially again
It has any different.For various turbidity all without special (absorption spectrum), only the particle in suspension (blocks scatter incident light), causes
Luminosity weakens.The just immune turbid determination techniques of transmittance of nephelometry point and immune scattering turbidimetric assay technology are current detection people's blood
The common method of specific protein in clear.It is general in field of medical examination by the specific protein analyzer of principle of scattering turbidimetry at present
All over use.After there is skeptophylaxis precipitation assay, i.e., ratio is immunized in immune transmission turbidimetric assay and immune scattering turbidimetry since then
Turbid measurement is gradually widely used in the field that protein content detects in clinical body fluid.In recent years, the turbid survey of transmittance is immunized
Determine technology to be widely used on automatic clinical chemistry analyzer, realizes high speed, sensitive and automation.
NGAL detection method has at present:
1, double antibodies sandwich immunochemiluminescence method --- the method is easy to operate, high specificity, and sensibility is high.But there is operation
It is cumbersome, the disadvantages of sample needs to pre-process, and detection time is long.
2, Gold standard --- the method has the characteristics of fast and convenient, easily to observe.
3, immune turbidimetry is transmitted --- the measuring method is easy, quick, can automate, be suitable for batch detection, still
Immune transmittance turbid methodology and clinical application are verified it is still necessary to further.
Summary of the invention
Based on this, in order to overcome the defects of the prior art described above, the present invention provides a kind of colloidal golds for detecting human neutrophil genatinase associated lipocalin
Immuno-chromatographic test paper strip, kit and preparation method thereof, which, which has, is able to achieve half-quantitative detection, detection time
It is short, specificity is good, the advantages such as economic cost is low.
In order to achieve the above-mentioned object of the invention, this invention takes following technical schemes:
A kind of colloidal gold immuno-chromatography test paper strip detecting human neutrophil genatinase associated lipocalin, including bottom liner and be sequentially arranged on the bottom liner
Sample pad, gold-labelled pad, coated film and blotting paper, the anti-human NGAL detection antibody of colloid gold label is coated in the gold-labelled pad
With the chicken IgY antibody of colloid gold label, the coated film includes being arranged in parallel and the detection zone being spaced apart from each other and check plot, institute
It states detection zone and is coated with anti-human NGAL coated antibody, the check plot is coated with the anti-chicken IgY antibody of goat, the anti-human NGAL inspection
Survey the corresponding epitope with any shown amino acid sequence of such as SEQ ID No:1~SEQ ID No:7 of antibody.
In wherein some embodiments, the chicken of anti-human NGAL the detection antibody and colloid gold label of the colloid gold label
The concentration of IgY antibody is respectively 0.2-0.5mg/ml and 0.2mg/ml, and the package amount in the gold-labelled pad is 1.2ul/cm.
In wherein some embodiments, the colloid gold particle diameter in the gold-labelled pad is 35~45nm.
In wherein some embodiments, the detection zone close to the gold-labelled pad, the check plot close to the blotting paper,
0.4cm is divided between the detection zone and the check plot.
In wherein some embodiments, the concentration of the anti-human NGAL coated antibody is 2mg/ml, in the detection zone
Package amount is 1.2ul/cm.
In wherein some embodiments, the concentration of the anti-chicken IgY antibody of goat is 0.7mg/ml, in the check plot
Package amount is 1.2ul/cm.
The present invention also provides the preparation method of the colloidal gold immuno-chromatography test paper strip of above-mentioned detection human neutrophil genatinase associated lipocalin, including it is following
Step:
(1), anti-human NGAL coated antibody is prepared, fixes anti-human NGAL coated antibody and the anti-chicken of goat respectively on coated film
IgY antibody forms detection zone and check plot；
(2), anti-human NGAL is detected antibody and chicken IgY antibody to be marked with colloidal gold, and be sprayed in gold-labelled pad；Institute
Stating anti-human NGAL detection antibody has the epitope such as any shown amino acid sequence of SEQ ID No:1~SEQ ID No:7；
(3), on bottom liner overlap joint paste sample pad, gold-labelled pad, coated film and blotting paper to get.
In wherein some embodiments, the detection antibody of anti-human NGAL described in step (1) the preparation method comprises the following steps: according to NGAL
Holoprotein sequence design difference peptide fragment is immunized animal and obtains specific antibody；Specific antibody is matched, screening obtains spirit
The highest antibody of sensitivity to get.
The present invention also provides a kind of colloidal gold immunochromatographykit kits for detecting human neutrophil genatinase associated lipocalin, including above-mentioned detection human neutrophil genatinase associated lipocalin
Colloidal gold immuno-chromatography test paper strip.
Compared with prior art, the invention has the following advantages:
1, clinical verification is carried out using colloidal gold immuno-chromatography test paper strip of the invention, detects 135 (its of urine specimen altogether
It is middle positive 33,102 negative), positive coincidence rate 100%, negative match-rate 100%, total coincidence rate is 100%, about
Mounting index is that 1, Kappa value is 1 (Kappa is examined, P < 0.05), and testing result coincidence rate is 100%, with existing control stripes item
There is good consistency, the results showed that test strips stability of the invention is good, as a result more accurate and reliable；Test strips are easy to operate
It is convenient, and cost is cheap compared with import test strips, has good market application value；
2, the colour developing power of colloidal gold immuno-chromatography test paper strip of the invention is directly proportional to NGAL concentration in urine specimen, can
The concentration range of NGAL in sample is detected according to the colorimetric card interpretation of offer, realizes half-quantitative detection, is had easy, quick, real
With, sensitive, efficient beneficial effect；
3, colloidal gold immuno-chromatography test paper strip of the invention can be mass, and is suitable for clinical quick diagnosis and scene is quick
Detection, is easy to save, is suitble to the popularization and use in basic hospital.
Detailed description of the invention
Fig. 1 is the structural schematic diagram of the colloidal gold immuno-chromatography test paper strip of the embodiment of the present invention 1；
Fig. 2 is the relevance verification result in test example 1 of the present invention using anti-NGAL-5 antibody as detection antibody；
Fig. 3 is the HOOK effect test result of the colloidal gold immuno-chromatography test paper strip of three batches of embodiment of the present invention 1；
Appended drawing reference: 1, sample pad；2, gold-labelled pad；3, coated film；4, detection zone；5, check plot；6, blotting paper；7, bottom
Plate.
Specific embodiment
Below by way of the drawings and specific embodiments, the present invention will be described in detail.
Raw material used in following embodiment unless otherwise specified, derives from commercially available.
The colloidal gold immuno-chromatography test paper strip and preparation method thereof of the detection human neutrophil genatinase associated lipocalin of embodiment 1
Referring to Fig. 1, the structural schematic diagram of the colloidal gold immuno-chromatography test paper strip for detection human neutrophil genatinase associated lipocalin of the invention, this reality
The colloidal gold immuno-chromatography test paper strip for applying the detection human neutrophil genatinase associated lipocalin of example, including bottom liner 7 and the sample being sequentially arranged on the bottom liner 7
Product pad 1, gold-labelled pad 2, coated film 3 and blotting paper 6, the anti-human NGAL detection that colloid gold label is coated in the gold-labelled pad 2 are anti-
The chicken IgY antibody of body and colloid gold label, the coated film 3 include being arranged in parallel and the detection zone 4 being spaced apart from each other and check plot
5, the detection zone 4 is coated with anti-human NGAL coated antibody, and the check plot 5 is coated with the anti-chicken IgY antibody of goat, described anti-human
NGAL detects the amino acid sequence that antibody has such as SEQ ID No:5.
In the present embodiment, the chicken IgY antibody of anti-human NGAL the detection antibody and colloid gold label of the colloid gold label
Concentration be respectively 0.2mg/ml and 0.2mg/ml, the package amount in the gold-labelled pad 2 is 1.2ul/cm.The gold-labelled pad
Colloid gold particle diameter on 2 is about 40nm.The detection zone 4 is close to the gold-labelled pad 2, and the check plot 5 is close to the suction
Water paper 6 is divided into 0.4cm between the detection zone 4 and the check plot 5.The concentration of the anti-human NGAL coated antibody is 2mg/
Ml is 1.2ul/cm in the package amount of the detection zone 4.The concentration of the anti-chicken IgY antibody of goat is 0.7mg/ml, described
The package amount of check plot 5 is 1.2ul/cm.
The preparation method of the colloidal gold immuno-chromatography test paper strip of the detection human neutrophil genatinase associated lipocalin of the present embodiment, comprising the following steps:
(1), prepared by colloidal gold
It takes 0.5-1.2% trisodium citrate aqueous solution 3ml in 297ml tri-distilled water, and shakes up the round bottom burning that 500ml is added
In bottle；Round-bottomed flask is placed in heating mantle to the stirrer stirring that suitable size is added；It is heated to boiling, and starts to stir, and
Timing at once；Boil 3 minutes, is rapidly added the 0.5-0.8% aqueous solution of chloraurate of 3ml, blender stir 10 minutes it
Mixing speed is turned down afterwards；Round-bottomed flask is taken out from heating mantle, is put to the station for being placed with hot pad；The colloid prepared
Gold solution is placed at room temperature for after temperature lowers, and sealing is kept in dark place in 4 DEG C of refrigerators.The colloidal gold that magnetic heating stirrer is fired
Grain size is uniform, and diameter is about 40nm；
(2), prepared by gold-labelled pad
Gold-labelled pad is layered in metal working tool, the gold-labelled pad treatment fluid of 12ml is added in the gold-labelled pad of 15*20cm；37 DEG C of drums
4 hours are dried in wind drying box to be completely dried up to gold-labelled pad；Dried gold-labelled pad will be handled to take out, it is close with aluminium foil bag
Envelope.Gold-labelled pad treatment fluid: 20-100mM Tris-Hcl, 5% sucrose, 5%BSA, 0.5%-1.0% trisodium citrate and
Logical -100 composition of 0.1%-0.15% Qula.
By the amount of package amount 1.2ul/cm, using spray film instrument by concentration be 0.2mg/ml colloid gold label it is anti-human
The chicken IgY antibody of NGAL detection antibody and colloid gold label is sprayed in the gold-labelled pad handled well and vacuum freeze drying is spare；
(3), prepared by sample pad
Sample pad is lain in paving metal working tool；The sample pad treatment fluid of 25ml is added in the sample pad of 15*20cm；With paving
Gold coating is uniform；4 hours are dried in 37 DEG C of air dry ovens to be completely dried up to sample pad.The formula of sample pad treatment fluid
Are as follows: 20-100mM Tris-HCl, 5% sucrose, 5%BSA, 0.5%-1.0% trisodium citrate and 0.1%-0.15% tween-
20。
(4), the preparation of coated film
By the amount of package amount 1.2ul/cm, the anti-human NGAL coated antibody that concentration is 2mg/ml is sprayed on packet using spray film instrument
The detection zone of envelope, by the amount of package amount 1.2ul/cm, the anti-chicken IgY antibody of goat for the use of spray film instrument being 0.7mg/ml by concentration
It is sprayed on the check plot of coated film, detection zone and check plot are arranged in parallel, are spaced apart from each other 0.4cm.
(5), the assembling of test strips
Blotting paper is pasted and pastes sample pad on the left of the right side of bottom plate, bottom plate, gold-labelled pad is pasted in sample pad and packet
Above envelope junction, film adhesive then is sticked on gold-labelled pad surface, is packed.
The preparation of the anti-NGAL specific antibody of test example 1
According to NGAL holoprotein sequence design difference peptide fragment, animal is immunized and obtains specific antibody, the specific steps are as follows:
(1), the preparation of NGAL native protein
According to the DNA sequence dna of the human neutrophil genatinase associated lipocalin provided in Genbank, pair of primers is designed, ' is distinguished at end by the 5 of two primers
NdeI+XhoI restriction enzyme site is introduced, the target gene of NGAL is obtained by PCR amplification, by carrier pET-28a and passes through agarose
The NGAL target gene fragment of gel-purified, carries out double digestion processing with NdeI+XhoI, will after purification with T4DNA ligase
Digestion products connection, obtains recombinant plasmid pET-28a-NGAL, recombinant plasmid transformed is entered bacillus coli DH 5 alpha, containing ammonia
Selected clone on the LB plate of parasiticin, prepares plasmid in a small amount, goes out positive colony by double digestion/PCR evaluation and screening, sequencing
The result shows that the NGAL segment of recombination and the sequence of design are completely the same.
For recombinant plasmid pET-28a-NGAL after sequence verification, conversion enters Escherichia coli (BL21), green containing ammonia benzyl
It is cultivated in the LB culture medium of mycin, positive colony can be selected on LB plate and carries out plasmid enzyme restriction identification, prepare plasmid in a small amount,
Go out positive colony with double digestion PCR evaluation and screening, it is final to obtain the recombinant plasmid engineering bacteria containing NGAL.
In expression, the recombinant plasmid engineering bacteria of NGAL is trained in the LB culture medium containing 100 μ g/ml ampicillins
It supports, A600Reach between 0.5-0.6, the Isopropyl β-D-1- of final concentration of 0.5mM is then added
Thiogalactopyranoside (IPTG) is in 37 DEG C of induction 4h, bacterium solution 4 after the completion of inducing, and 000rpm is centrifuged 10min, receives
Collect thallus, and washs precipitating with PBS；PBS is resuspended precipitating and is placed in ice bath, and 12000rpm is centrifuged 20min after carrying out ultrasonic bacteria breaking, on
Cleer and peaceful precipitating carries out SDS-PAGE electrophoresis respectively, the results showed that the NGAL recombinant protein of expression is the expression of endochylema insolubility, will
The recombinant protein is named as BL21 (DE3)-NGAL.
The thallus that great expression is obtained, is centrifuged after ultrasonication, then carries out inclusion body washing, and GE is used after the completion of washing
The His Trap FF purification column of Healthcare company albumen is purified (according to product description carry out preparation of reagents and
Purifying).The albumen finally obtained is analyzed with SDS-PAGE electrophoresis, is measured its concentration with BCA protein quantification kit and is
0.23mg/ml。
(2), the preparation of NGAL peptide fragment
The NGAL recombinant protein sequence that step 1 is obtained first imports online epitope design software, will be located at albumen
Conformation extra amino acid sequences come out.Full NGAL protein sequence is imported into DNASTAR software again, passes through Epitope prediction
Tool counts peptide fragment combination.Two groups of data are compared and show that most possible is 7 groups of peptide fragments of epitope.Pass through Shang Haiji
Your biochemical biotech firm synthesizes 7 groups of peptide fragments, and particular sequence is as follows:
Peptide fragment 1:NH2-CQDSTSDLIPAPPLSKVPLQ(SEQ ID No:1)
Peptide fragment 2:NH2-CILREDKDPQKMYATIYEL(SEQ ID No:2)
Peptide fragment 3:NH2-CTFVPGCQPGEFTLGNIKSY(SEQ ID No:3)
Peptide fragment 4:NH2-CSQNREYFKITLYGRTKELTS(SEQ ID No:4)
Peptide fragment 5:NH2-CAPPLSKVPLQQNFQDNQF(SEQ ID No:5)
Peptide fragment 6:NH2-CRTKELTSELKENFIRFSKS(SEQ ID No:6)
Peptide fragment 7:NH2-CLPENHIVFPVPIDQCIDG(SEQ ID No:7)
(3), the preparation and purification of specific antibody
Using NGAL native protein and above-mentioned 7 groups of peptide fragments be immunized the female BAl BIc of 8 week old, weight 18g or so and health/
After c mouse each 2, adaptive feeding 1 week, acquires negative blood and used as control；Using intermediate range immunization protocol (0.3ml/ only, 2
Week/time), (50 μ g/ are only) is by immunogene and isometric Freund's complete adjuvant stirring and emulsifying, dorsal sc multiple spot when first immunisation
Hereafter injection carries out routine immunization by immunogene and isometric incomplete Freund's adjuvant stirring and emulsifying；3 times it is immune when, generally
50 μ g antigen+TiterMax mixed in equal amounts emulsify back part multi-point injection, survey potency after 7 days.Mouse titers obviously reach certain
Booster immunization after it is required that, booster immunization are not added adjuvant, and booster immunization dosage is 50 μ g, 3 days after booster immunization, pluck eyeball blood sampling,
Separate serum keeping.Spleen is taken to be merged simultaneously.When cell fusion, splenocyte and myeloma cell are carried out by 4:1 or so
Mixing, and merged in the case where the rush of polyethylene glycol (PEG, molecular weight 1450) melts effect, HAT is selectively trained fused cell again
It is cultivated in nutrient solution, the positive hybridoma that can be reacted with NGAL native protein is filtered out by indirect ELISA method after 10 days
Cell, and the positive hybridoma cell that primary dcreening operation is obtained expands culture, with limiting dilution assay by the positive hybridoma cell of acquisition
Continuous to be subcloned more than at least twice, subclone is cultivated with HT selective medium every time, and subclone carries out after 8-10 days
ELISA screening obtains energy stably excreting and is directed to NGAL native protein and peptide until monoclonal cell positive rate is 100%
The monoclonal cell strain of the specific antibody of section.
The female sex-health BALB/c mouse of 8-12 week old is selected, norphytane is injected intraperitoneally, 0.5ml/ is only；After 7-10 days, to every
Mouse peritoneal injection 1 × 106~5 × 106A monoclonal hybridoma, pay attention to blowing down cell or diluting cells need to PBS or
Serum free medium；By ascites 10,000r/min is centrifuged 15min, removes cell component and other sediments, fat and oil
Layer etc., collect middle layer, measure antibody titer, packing, set -70 DEG C freeze it is spare.Saturated ammonium sulphate: 5ml processing is drawn
Good ascites moves into small beaker, under stiring, the PBS 5.0ml of 0.22 μm of filter membrane is added dropwise；After mixing, then by
It is added dropwise to 10ml saturated ammonium sulfate solution (pH7.4), continues to be slowly stirred 30min；10,000r/min is centrifuged 15 points after standing 2h
Clock discards supernatant, and the PBS of the used 0.22 μm of filter membrane of sediment is resuspended, and the re-suspension liquid is then crossed 0.22 μm of filter membrane again；According to anti-
Body different subtype selectes the purification column of different GE Healthcare companies, collects antibody peak；With PBS buffer solution by antibody into
Row dialysis with BCA protein quantification kit measurement antibody concentration, and antibody is dispensed and is saved.
(4) pairing experiment obtains and is directed to the natural highest antibody pair of NGAL albumen potency
By natural NGAL albumen as antigen, the antibody of native protein preparation is as capture antibody, and peptide fragment preparation is anti-
Body is as detection antibody.By matching experiment (the capture antibody of 100ng/100ul is coated in 96 orifice plates and is incubated for；Successively plus
Enter by degree of passing diluted natural NGAL protein 10 0ng, 50ng, 25ng, 12.5ng, 6.25ng, 3.125ng, 1.5625ng, and
Blank control PBS；7 groups of different detection antibody are added to verify its potency) detect the antibody pair matched.Experimental result such as table
Shown in 1.
Table 1 matches experimental result
Table 1 the result shows that, by anti-NGAL-1 antibody, anti-NGAL-2 antibody, anti-NGAL-3 antibody and anti-NGAL-6 antibody (peptide
The antibody of 1,2,3,6 preparation of section) as detection antibody, anti-NGAL antibody can get as capture antibody and be directed to natural NGAL albumen
The higher antibody pair of potency.
(5), serology verifies specific antibody pair
Using anti-NGAL antibody as capture antibody, anti-NGAL-1 antibody, anti-NGAL-2 antibody, anti-NGAL-3 antibody, resist
NGAL-4 antibody, anti-NGAL-5 antibody, anti-NGAL-6 antibody and anti-NGAL-7 antibody determine 30 as detection antibody respectively
Sample has simultaneously done correlation analysis, as a result proves anti-NGAL-1 antibody, anti-NGAL-2 antibody, anti-NGAL-3 antibody, anti-NGAL-6
Antibody is weak to the NGAL identification in serum, and correlation is lower, more lower than sample true value of universal measured value；
After cross match, screening has obtained a pair of to the preferable antibody pair of serum identification: anti-NGAL antibody and anti-NGAL-5 antibody,
Therefore, illustrate peptide fragment 5 (SEQ ID No:5) for the higher epitope of specificity.
Using anti-NGAL-5 antibody as the relevance verification result of detection antibody as shown in Fig. 2, as can be seen from Figure 2, using
The combine detection of anti-NGAL-5 antibody and anti-human NGAL antibody, R consistent with the correlation of the detected value of hospital2Reach 0.9853.
The performance detection of the colloidal gold immuno-chromatography test paper strip of 2 embodiment 1 of test example
1, negative reference product coincidence rate
1.1 test method
The test strip of the embodiment of the present invention 1 of each batch is respectively taken into 5 person-portions respectively, each batch detects 5 parts of feminine genders
Reference material, each negative reference product sample (mixing Healthy Human Serum) measurement 1 time, guarantees the correct of result, sees in 5~10min
It examines as a result, counting the negative reference product coincidence rate of each test strip.
1.2 test results are as shown in table 2.
2 negative reference product coincidence rate test result of table
| Lot number | 5 parts of negative reference product colour developing situations | Negative reference product coincidence rate |
| 2015001 | Without colour developing | 100% |
| 2015002 | Without colour developing | 100% |
| 2016001 | Without colour developing | 100% |
1.3 conclusion (of pressure testing)
By 2 result of table as it can be seen that test strip negative reference product are without colour developing, coincidence rate 100%.
2, positive reference product coincidence rate
2.1 test method
The test strip of the embodiment of the present invention 1 of each batch is respectively taken into 3 person-portions respectively, it is (calibrated to positive reference product
NGAL recombinant protein) detected, each positive reference product sample measures 1 time, guarantee the correct of result, seen in 5~10min
It examines as a result, each batch reference material colour developing situation is compared with reference to colorimetric card with respective batch, statistics colour developing result consistency,
To count the positive reference product coincidence rate of each test strip.
2.2 test results are as shown in table 3.
3 positive reference product coincidence rate test result of table
2.3 conclusion (of pressure testing)
By 3 result of table as it can be seen that the positive reference product of the test strip of the embodiment of the present invention 1 develop the color and refer to colorimetric card
Unanimously, coincidence rate 100%.
3, minimum detection limit
3.1 verifying minimum detection limit test methods
Randomly select 10 person-portion test strips from 2015001 products, take positive reference product (calibrated NGAL recombinant protein,
100ng/mL), positive reference product (100ng/mL) are diluted to as dilution by 60ng/ using negative newborn bovine serum step by step
ML, 30ng/mL, 15ng/mL, 0 (dilution).With the test strip of embodiment 1 to the above sample (60ng/mL, 30ng/mL,
15ng/mL, 0 (dilution) are detected, and the above experiment is repeated 2 times, and minimum detection limit can show result with test strips detection line
NGAL minimum concentration value indicate.
3.2 minimum detection limits repeat test method
The test strip of the embodiment of the present invention 1 of each batch takes 20 person-portions respectively, detects to minimum detection limit, 5
Observation in~10min should can develop the color, and it is consistent to develop the color as a result, colour developing situation is compared with respective batch with reference to colorimetric card.
3.3 test results are as shown in table 4 and table 5.
4 minimum detection limit testing result of table
5 minimum detection limit of table repeats testing result
3.4 conclusion (of pressure testing)
Minimum detection limit testing result through the above table 4 and table 5, the minimum inspection of the test strip of the embodiment of the present invention 1
Survey is limited to 30ng/mL.
4, interfering substance is tested
4.1 interfering substances detect test method
Use negative newborn bovine serum by three kinds of interfering substance bilirubin, hemoglobin and triglycerides be configured to concentration for
The solution of 1mg/mL is detected with the test strip of the embodiment of the present invention 1, and observation three of the above interfering substance addition is in yin
Influence in property sample to negative sample testing result.
4.2 interfering substance checking test methods
400 μ g/mL of bilirubin concentration, 100 μ g/mL of hemoglobin concentration, 100 μ g/mL of triglyceride concentration are prepared, with three
Test strips detection is criticized, observation colour developing situation in 5~10min.
4.3 test results are as shown in table 6~9.
6 bilirubin interference detection results of table
7 hemoglobin interference detection results of table
8 triglycerides interference detection results of table
The interference verifying testing result of table 9
4.4 conclusion (of pressure testing)
Above 6~9 interference testing inspection of table the result shows that, bilirubin be lower than 400 μ g/mL when do not influence testing result；
Hemoglobin concentration will not influence testing result when being lower than 100 μ g/mL；Triglyceride concentration will not influence when being lower than 100 μ g/mL
Testing result.
5, HOOK effect
5.1 test method
NGAL calibration antigen (Raybiotech company of the U.S.) is added separately in negative newborn bovine serum, is obtained following
The solution of concentration: 10ng/mL, 25ng/mL, 50ng/mL, 100ng/mL, 200ng/mL, 400ng/mL, 800ng/mL,
1000ng/mL, 1200ng/mL, 1500ng/mL are detected with the test strip of the embodiment of the present invention 1, will test calibration
For the concentration of antigen as X-axis, the intensity to be developed the color in 5~10min using test strips (numerical value is used to indicate) does curve as Y-axis, sees
Examine the detectable concentration for generating high dose Hook effect.
5.2 test results are as shown in Figure 3.
5.3 conclusion (of pressure testing)
By Fig. 3 result as it can be seen that test strip of the invention would not observe that height when being no more than 1000ng/mL
Dosage Hook effect.
6 repeatability
6.1 test method
Random sampling 20 from every batch of test strips are separately added into high concentration accuracy reference material (300ng/mL), repeat
Measurement 10 times；It is added low concentration accuracy reference material (100ng/mL), replication 10 times, guarantees the correct of result, 5~
Result is observed in 10min.By the high and low concentration essence of each batch high and low concentration accuracy reference material colour developing situation and respective batch
Close property is compared with reference to colorimetric card, and statistics colour developing result consistency is to count the repeatability of test strips in each batch.
6.2 test results are as shown in table 10.
The repeated testing result of table 10
6.3 conclusion (of pressure testing)
Low, the high concentration accuracy reference material of the test strip of 3 batches repeats detection 10 times, all with reference colorimetric card
The band colour developing that develops the color is consistent.
The clinical performance of the test strip of 3 embodiment of the present invention 1 of test example is verified
This test example collects urine specimen 135 altogether, wherein positive sample (contrast agents detect > 100ng/mL) 33,
Feminine gender detection (contrast agents detect < 30ng/mL) 102.With the test strip of the embodiment of the present invention 1 and list
Property granulocyte gelatinase associated lipocalin measurement test strips 135 parts of urine specimens of collection are detected respectively, converge
Total testing result.Yin and yang attribute coincidence rate, the youden index of 135 parts of data are analyzed with four fold table, and carries out Kappa inspection, are evaluated
The test strip of the embodiment of the present invention 1 and the consistency of control stripes item.As a result as shown in table 11.
11 clinical performance verification result of table
From table 11, calculated result is as follows:
Positive sample coincidence rate (true positive rate)=33/33 × 100%=100%
Negative sample coincidence rate (true negative rate)=102/102 × 100%=100%
False positive rate=0/76 × 100%=0
False negative rate=0/34 × 100%=0
Total coincidence rate=135/135 × 100%=100%
Youden index=33/33+102/102-1=1
Value=1 kappa, > 0.75, illustrate that the test strip of the embodiment of the present invention 1 and control stripes consistency are fine.
Testing result coincidence rate=(total number of samples-difference sample number)/total number of samples × 100%=(135-0)/135 ×
100%=100%
As can be seen from the above data, egg is delivered using the neutrophil leucocyte gelatinase related lipid of the embodiment of the present invention 1
White (NGAL) test strip (colloidal gold method) carries out clinical verification, with the neutrophil leucocyte gelatinase related lipid fortune listed
Carrying protein determination test strips (latex enhancing immune turbidimetry) is control, detect altogether urine specimen 135 (wherein positive 33,
Negative 102), arrange and analysis detection as a result, kits for evaluation clinical performance, positive coincidence rate 100%, feminine gender meets
Rate is 100%, and total coincidence rate is 100%, youden index 1.Kappa value is 1 (Kappa is examined, P < 0.05), testing result symbol
Conjunction rate is 100%, and the test strip and control stripes item of the embodiment of the present invention 1 have good consistency.The result of appraisal show
The test strip of the embodiment of the present invention 1 and control stripes detection performance are similar, and stability is good, as a result more accurate and reliable.
Test strips are simple to operate, and cost is cheap compared with import test strips, have good market application value.
Each technical characteristic of embodiment described above can be combined arbitrarily, for simplicity of description, not to above-mentioned reality
It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, all should be considered as described in this specification.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that coming for those of ordinary skill in the art
It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to protection of the invention
Range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
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Claims (9)
1. it is a kind of detect human neutrophil genatinase associated lipocalin colloidal gold immuno-chromatography test paper strip, which is characterized in that the test strips include bottom liner and
Sample pad, gold-labelled pad, coated film and the blotting paper being sequentially arranged on the bottom liner are coated with colloid gold label in the gold-labelled pad
Anti-human NGAL detection antibody and colloid gold label chicken IgY antibody, the coated film includes being arranged in parallel and being spaced apart from each other
Detection zone and check plot, the detection zone are coated with anti-human NGAL coated antibody, and it is anti-that the check plot is coated with the anti-chicken IgY of goat
Body, the anti-human corresponding epitope with the amino acid sequence as shown in SEQ ID No:5 of NGAL detection antibody.
2. the colloidal gold immuno-chromatography test paper strip of detection human neutrophil genatinase associated lipocalin according to claim 1, which is characterized in that the colloid
Gold label anti-human NGAL detection antibody and colloid gold label chicken IgY antibody concentration be respectively 0.2~0.5mg/ml and
0.2mg/ml, the package amount in the gold-labelled pad are 1.2 μ l/cm.
3. the colloidal gold immuno-chromatography test paper strip of detection human neutrophil genatinase associated lipocalin according to claim 1, which is characterized in that the gold mark
Colloid gold particle diameter on pad is 35~45nm.
4. the colloidal gold immuno-chromatography test paper strip of detection human neutrophil genatinase associated lipocalin according to claim 1, which is characterized in that the detection
Area is divided between the blotting paper, the detection zone and the check plot close to the gold-labelled pad, the check plot
0.4cm。
5. the colloidal gold immuno-chromatography test paper strip of detection human neutrophil genatinase associated lipocalin according to claim 1, which is characterized in that described anti-human
The concentration of NGAL coated antibody is 2mg/ml, is 1.2 μ l/cm in the package amount of the detection zone.
6. the colloidal gold immuno-chromatography test paper strip of detection human neutrophil genatinase associated lipocalin according to claim 1, which is characterized in that the goat
The concentration of anti-chicken IgY antibody is 0.7mg/ml, and the package amount in the check plot is 1.2 μ l/cm.
7. the preparation method of the colloidal gold immuno-chromatography test paper strip of the described in any item detection human neutrophil genatinase associated lipocalins of claim 1~6, special
Sign is, comprising the following steps:
(1), anti-human NGAL coated antibody is prepared, fixes anti-human NGAL coated antibody and the anti-chicken IgY of goat respectively on coated film
Antibody forms detection zone and check plot；
(2), anti-human NGAL is detected antibody and chicken IgY antibody to be marked with colloidal gold, and be sprayed in gold-labelled pad；It is described anti-
Human neutrophil genatinase associated lipocalin detects the corresponding epitope having such as SEQ ID No:5 amino acid sequence of antibody；
(3), on bottom liner overlap joint paste sample pad, gold-labelled pad, coated film and blotting paper to get.
8. the preparation method of the colloidal gold immuno-chromatography test paper strip of detection human neutrophil genatinase associated lipocalin according to claim 7, feature exist
In, the detection antibody of anti-human NGAL described in step (2) the preparation method comprises the following steps: according to NGAL holoprotein sequence design difference peptide fragment,
Immune animal obtains specific antibody；Specific antibody is matched, screening obtains anti-human NGAL detection antibody.
9. a kind of colloidal gold immunochromatographykit kit for detecting human neutrophil genatinase associated lipocalin, which is characterized in that the kit includes claim 1
The colloidal gold immuno-chromatography test paper strip of~6 described in any item detection human neutrophil genatinase associated lipocalins.
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| CN109709340B (en) * | 2018-12-29 | 2022-02-11 | 江苏众红生物工程创药研究院有限公司 | Human neutrophil gelatinase-associated lipocalin quantitative detection card and clinical application thereof |
| CN109608542B (en) * | 2018-12-29 | 2022-04-05 | 江苏众红生物工程创药研究院有限公司 | Anti-human NGAL antibody and application thereof in detection test paper card |
| CN110003321A (en) * | 2019-04-02 | 2019-07-12 | 青岛中科爱博生物科技有限公司 | A kind of soluble recombination NGAL Argine Monohydrochloride, gene order and protein preparation method |
| CN110794145B (en) * | 2019-10-30 | 2023-06-23 | 中国药科大学 | Rapid detection system for early kidney injury urine marker neutrophil gelatinase related lipocalin, preparation method and application |
| CN116990512B (en) * | 2023-09-25 | 2023-12-08 | 聚诚(北京)生物科技有限责任公司 | Matrix metalloproteinase-9 detection kit |
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Address after: No.79, Ruihe Road, Science City, Guangzhou hi tech Industrial Development Zone, Guangdong 510000 Patentee after: Reboo (Guangzhou) Biotechnology Co.,Ltd. Address before: 510530 No. 79 Ruihe Road, Luogang District, Guangzhou City, Guangdong Province Patentee before: RAYBIOTECH, Inc. |