CN106872694A - The detection method and hemoglobin detection kit of a kind of hemoglobin - Google Patents
The detection method and hemoglobin detection kit of a kind of hemoglobin Download PDFInfo
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- CN106872694A CN106872694A CN201710082997.3A CN201710082997A CN106872694A CN 106872694 A CN106872694 A CN 106872694A CN 201710082997 A CN201710082997 A CN 201710082997A CN 106872694 A CN106872694 A CN 106872694A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
- G01N33/559—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody through a gel, e.g. Ouchterlony technique
-
- 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
- G01N33/72—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood pigments, e.g. haemoglobin, bilirubin or other porphyrins; involving occult blood
- G01N33/721—Haemoglobin
Abstract
The invention provides the detection method and hemoglobin detection kit of a kind of hemoglobin, when reaching a certain amount of using HC in sample, HOOK effects can be formed, as concentration continues to increase, HOOK effects strengthen, form reverse depression effect, reverse depression effect is in regular increase in certain limit, by " hook-shaped " region for studying hemoglobin double-antibody sandwich HOOK effects, optimization Sample Dilution, strategic structural immunochromatography technique because of the HOOK effects cannot direct detection difficult point, so as to quickly and accurately detect the content of hemoglobin, make up the vacancy in the field, it is that Site Detection and family's detection provide convenient；The detection method is diluted by a step and cracks blood sample method, and the good content of hemoglobin in the range of human body can be obtained that controls is in good dose curve rule in HOOK effects area.
Description
Technical field
The present invention relates to the detection method and hemoglobin of biochemical analysis detection field, especially a kind of hemoglobin
Detection kit.
Background technology
At this stage, the detection method of hemoglobin includes：Copper sulphate titration carries out hemoglobinometry, or uses
The large-scale Biochemical Analyzer of mouth is measured.The former exist voluntarily prepare error it is big, it is easily influenced by ambient temperature the shortcomings of, Hou Zheyi
Device is expensive, and testing cost is high.The conventional method of testing of foreign countries is kit and Dry-type biochemical analytic approach, and RNA isolation kit needs
Large-scale Biochemical Analyzer, Dry-type biochemical analytic approach product mainly has the type whole blood Dry-type biochemical analyzers of Re-flotron IV of Germany
With the SWELAB blood analysers of Sweden, use is wavelength colorimetric method, and required blood sampling volume is larger, and the testing time is long, cost
It is high.
The advantage of immunochromatography colloidal gold technique is minimally invasive sampling, and sampling amount is few, low cost, and detection is fast, and the degree of accuracy is high, is fitted
For scene and family's detection, and do not occur the product that immunochromatography technique detects content of hemoglobin in the market, its skill
Art difficult point is that hemoglobin double-antibody sandwich has HOOK effects, therefore immunochromatography double-antibody technique or main collection at present
In in occult blood, the detection project occulted blood.
Hook effects refer to the high dose section of its dose-effect curve in two-site sandwich immunity test, are linearly walked
To be not in platform-like it is unlimited after prolong, but be turned under curved, this phenomenon is referred to as HOOK effects.At present for hemoglobin
Immunology detection, generally requiring multistep dilution can be just in the range of linearity, but the error for so causing will certainly be increased.
The content of the invention
The technical problems to be solved by the invention are to provide a kind of detection method of hemoglobin.
The technical problems to be solved by the invention are to provide a kind of hemoglobin detection examination for realizing above-mentioned detection method
Agent box.
In order to solve the above technical problems, the technical scheme is that：
A kind of detection method of hemoglobin, is detected using immuno-sandwich method, is comprised the following steps that：It is marked with colloid
After the hemoglobin discharged in the first antibody combination blood of gold grain, chromatographed forward by capillary force, chromatography to nitric acid
During the coated SA of cellulose membrane (i.e. solid phase carrier), the complex of " colloid gold particle-antibody-antigen-antibody " is formed
System, and be gathered on nitrocellulose filter, the signal of aubergine can be formed when colloid gold particle is largely assembled, by instrument energy
The intensity of enough accurate quantitative analysis interpretation detection signals, so as to realize quantitative determination, the first antibody is antihuman hemoglobin antibody,
SA is the hemoglobin antibodies of pairing, and the first antibody and SA can be in combination with the differences of target antigen
Epitope, so as to form compound system.
In the range of low dosage, as content of hemoglobin increases, detection signal enhancing, being proportionate property, when in sample
When hemoglobin exceedes a certain amount of, just inevitably there is HOOK effects, i.e., with the increase of HC, detection letter
Number successively decrease, in the negatively correlated property of certain limit.
A kind of hemoglobin detection kit for realizing above-mentioned detection method, including by hemoglobin Test paper, external
Get stuck, sample lysate and the interior IC-card for having set respective standard curve and decision method, wherein,
The hemoglobin Test paper, width is 3-5mm, and length is 7-8cm, and the Test paper is by containing collaurum
The colloidal gold pad of the antihuman hemoglobin antibody of mark, be coated with T lines and C lines (be coated with pairing hemoglobin antibodies and
Anti- mouse IgG) nitrocellulose filter (NC films, are loose structure film, and aperture is 8-15um), sample pad, sample suction pad and plastic bottom
Plate is constituted, and the T lines are the hemoglobin antibodies of pairing, and C lines are anti-mouse IgG, and the antihuman hemoglobin antibody is used as first
Antibody labeling has colloid gold particle, and sample pad, colloidal gold pad, nitrocellulose filter and sample suction pad are arranged successively from plastic bottom board one end
Arrange to the other end；
It is described it is external get stuck, comprising upper lid and lower cover, two hemoglobin Test papers be placed in parallel in it is described under
Lid is internal, and the upper lid counter sample pad part is provided with sample well, for adding sample and dilution, upper lid correspondence nitric acid
Cellulose membrane medium position is provided with viewing window, for observing the C lines on nitrocellulose filter, T lines, result of determination.
The sample lysate, main formula is：0.02M Tris-HCL, 0.15molNaCL, 1%Triton100 regulations
PH to 7.4.
The IC-card, built-in standard curve and decision method, standard curve is drawn by test of many times result and obtained, should
IC-card supports the use NS3001B type immune chromatography result interpretations recorder (Newscen Coast Bio-Pharmaceutical Co., Ltd.'s product)
Use, for the quantitative judgement to testing result, specifically, the interpretation of hemoglobin test paper is determined and obtains signal value, using IC
Card is changed so as to obtain concentration value signal value and concentration value, is 50-250g/L to hemoglobin detection range.
Preferably, above-mentioned hemoglobin detection kit, the sample suction pad is absorbent filter.
Preferably, above-mentioned hemoglobin detection kit, the preparation for being coated with the nitrocellulose filter with antagonist
Method is：The hemoglobin antibodies of pairing are configured to by final concentration 1-2mg/ with the phosphate buffers of 0.01M pH 7.4 (PBS)
ML, spray film instrument is rule on nitrocellulose filter with the parameter of 1.2-1.4ul/cm, is coated with T lines, while fine in nitric acid
The plain film top of dimension is coated with the anti-mouse IgG of 1-2mg/ml, as C lines, dries, standby.
Preferably, above-mentioned hemoglobin detection kit, the preparation method of the colloidal gold pad is：Take 100ml collaurums
Liquid is placed in beaker, uses 0.2M K2CO3PH7.0-8.0 is adjusted to, 1-2mg mark hemoglobin antibodies are added, 1 is stirred at room temperature
Hour, closing, 12000rpm is centrifuged 30 minutes, abandons supernatant, is redissolved to 60ml with working solution, is then distinguished equably with metal spraying machine
It is sprayed on non-woven fabrics or glass, drying for standby.
Paper box assembly method is：It is less than under conditions of 30% in relative humidity, takes plastic bottom board, blood red egg will be coated with
The white NC films with antagonist are pasted onto the middle part of base plate, and colloidal gold pad is pasted in NC film T lines side, viscous in colloidal gold pad opposite side
Patch sample pad；Sample suction pad is pasted in NC film C lines side；Each paste composition interface mutually laminates 1-2mm, and the big plate for pasting is cut into
Test strips 3-5mm wide.Then by test strips be respectively placed in plastics get stuck lower cover groove in, then cover lid, compress, shape
Into paper box.
The operating method of above-mentioned hemoglobin detection kit is：The blood sample of 10ul is added to 1ml samples during detection
In this lysate, mix, wait 2 minutes, the sample-adding during 100 μ L add hemoglobin detection reagent card is taken from sample lysate
End, in 15-20 minutes, is judged testing result with NS3001B type immune chromatography result interpretation recorders.
The beneficial effects of the invention are as follows：
The hemoglobin detection kit and method, for detecting the content of hemoglobin in people's whole blood sample, have
Easy to operate, reaction is quick, result is accurate, credible, the advantages of be adapted to Site Detection.Reached using HC in sample
When a certain amount of (1g/L), HOOK effects can be formed, as concentration continues to increase, the enhancing of HOOK effects forms reverse depression effect,
Reverse depression effect is in regular increase in certain limit, by " hook-shaped " area for studying hemoglobin double-antibody sandwich HOOK effects
Domain, optimizes Sample Dilution, strategic structural immunochromatography technique because of the HOOK effects cannot direct detection difficult point so that accurately
Quickly the content of detection hemoglobin, makes up the vacancy in the field, is that Site Detection and family's detection provide convenient；The inspection
Survey method is diluted by a step and cracks blood sample method, and good controls in the range of human body can be obtained (50-250g/L)
Content of hemoglobin is in good dose curve rule in HOOK effects area, and it is bent that 50-250g/L is carried out into the parameters of logistic tetra-
Line is fitted, and tries to achieve equation of linear regression, and coefficient correlation | r | values are good.
Brief description of the drawings
Fig. 1 is that content of hemoglobin measures curve map with detected signal value dose relationship；
Fig. 2 is content of hemoglobin and detected signal value dose relationship linear analysis figure (1-250g/L)；
Fig. 3 is content of hemoglobin and detected signal value dose relationship linear analysis figure (50-250g/L) figure；
Fig. 4 is the structural representation of hemoglobin Test paper, in figure, 1- colloidal gold pad 2-T line 3-C line 4- nitric acid
Cellulose membrane 5- sample pad 6- sample suction pads；
Fig. 5 is the external structural representation for getting stuck, and in figure, A- sample well B- viewing windows is covered on S-.
Specific embodiment
Technical scheme of the present invention is further described with reference to specific embodiment.
Embodiment 1
A kind of hemoglobin detection kit for realizing above-mentioned detection method, including by hemoglobin Test paper (such as Fig. 4
It is shown), it is external get stuck (as shown in Figure 5), sample lysate and the interior IC-card for having set respective standard curve and decision method, its
In,
The hemoglobin Test paper, width is 3-5mm, and length is 7-8cm, and the Test paper is by containing collaurum
The colloidal gold pad 1 of the antihuman hemoglobin antibody of mark, it is coated with T lines 2 and C lines 3 (are coated with the hemoglobin antibodies of pairing
With anti-mouse IgG) nitrocellulose filter 4 (NC films, are loose structure film, and aperture is 8-15um), sample pad 5, sample suction pad 6 and modeling
Material base plate (figure is omited) composition, the T lines are the hemoglobin antibodies of pairing, and C lines are anti-mouse IgG, and the antihuman hemoglobin resists
Body is marked with colloid gold particle as first antibody, and sample pad, colloidal gold pad, nitrocellulose filter and sample suction pad are from plastic bottom board
One end is arranged in order to the other end, wherein,
The sample suction pad is absorbent filter,
The nitrocellulose filter is coated with the method with antagonist：With the phosphate buffers of 0.01M pH 7.4
(PBS) hemoglobin antibodies of pairing are configured to final concentration 1-2mg/mL, film instrument will be sprayed on nitrocellulose filter with 1.2-
The parameter of 1.4ul/cm is rule, and is coated with T lines, while being coated with the anti-mouse IgG of 1-2mg/ml on nitrocellulose filter top, is made
It is C lines, dries, it is standby,
The preparation method of the colloidal gold pad is：Take 100ml collaurum liquid to be placed in beaker, use 0.2M K2CO3It is adjusted to
PH7.0-8.0, adds 1-2mg mark hemoglobin antibodies, is stirred at room temperature 1 hour, and closing, 12000rpm is centrifuged 30 points
Clock, abandons supernatant, is redissolved to 60ml with working solution, is then equably sprayed on non-woven fabrics (can also be glass) respectively with metal spraying machine
On, drying for standby；
It is described it is external get stuck, comprising upper lid S and lower cover (figure is omited), two hemoglobin Test papers are placed in parallel in
Inside the lower cover, the upper lid counter sample pad part is provided with sample well A, and for adding sample and dilution, upper lid is right
Nitrocellulose filter medium position is answered to be provided with viewing window B, for observing the C lines on nitrocellulose filter, T lines, result of determination.
The sample lysate, main formula is：0.02M Tris-HCL, 0.15molNaCL, 1%Triton100 tune
Section PH to 7.4.
The IC-card, built-in standard curve and decision method, standard curve is drawn by test of many times result and obtained, should
IC-card supports the use NS3001B type immune chromatography result interpretations recorder (Newscen Coast Bio-Pharmaceutical Co., Ltd.'s product)
Use, for the quantitative judgement to testing result, specifically, the interpretation of hemoglobin test paper is determined and obtains signal value, using IC
Card is changed so as to obtain concentration value signal value and concentration value, is 50-250g/L to hemoglobin detection range.
Above-mentioned paper box assembly method is：It is less than under conditions of 30% in relative humidity, takes plastic bottom board, blood will be coated with
NC film of the Lactoferrin with antagonist is pasted onto the middle part of base plate, and colloidal gold pad is pasted in NC film T lines side, another in colloidal gold pad
Paste sample pad in side；Sample suction pad is pasted in NC film C lines side；Each paste composition interface mutually laminates 1-2mm, the big plate for pasting
It is cut into 3-5mm test strips wide.Then by test strips be respectively placed in plastics get stuck lower cover groove in, then cover lid, press
Tightly, paper box is formed.
The operating method of above-mentioned hemoglobin detection kit is：The blood sample of 10ul is added to 1ml samples during detection
In this lysate, mix, wait 2 minutes, the sample-adding during 100 μ L add hemoglobin detection reagent card is taken from sample lysate
End, in 15-20 minutes, is judged testing result with NS3001B type immune chromatography result interpretation recorders.
Embodiment 2
1 material
Hemoglobin detection kit described in 1.1 embodiment of the present invention 1
1.2 Hb enterprises internal control product：Human hemoglobin dry powder, purchased from Sigma, product article No.：H7379, weighs dry powder by pure
After water is diluted to high concentration, then by filtering sheep blood serum press gradient dilution to 0.05g/L, 0.1g/L, 0.5g/L, 1g/L, 5g/L,
20g/L.Enterprise internal control product 50g/L, 100g/L, 150g/L, 200g/L, 250g/L are people's definite value whole blood.
1.3 clinical samples：Obtained in relevant hospital by company, totally 120 parts, wherein being diagnosed as anaemia patient whole blood sample 60
Part, Hb detected values are from 60-90g/L.60 parts of normal human serum sample, Hb detected values are from 110-160g/L.
1.3 laboratory apparatus：The interpretation of Newscen Coast Bio-Pharmaceutical Co., Ltd. NS3001B type immune chromatography results is recorded
Instrument.
2 methods
Hb enterprises internal control product concentration 0.05g/L, 0.1g/L, 0.5g/L, 1g/L, 5g/L, 20g/L, 50g/L, 100g/L,
200g/L, 250g/L, each concentration point draw 10 μ L, are separately added into 1mL sample lysates, mix, and after 2 minutes, draw 100 μ L
The sample-adding end of reagent card is added to, each concentration point is set in three repetitions, 15-20 minutes, uses NS3001B type immunochromatography knots
Fruit interpretation recorder records testing result respectively.
3 results
Testing result see the table below 1.
The testing result of the hemoglobin internal control product of table 1
According to the data obtained of upper table 1, with concentration value as X-axis, the average value of gained signal value is Y-axis, does dose curve, such as
Shown in Fig. 1, in 0.05-0.5g/L concentration ranges, increasing detected signal value with HC strengthens；Near 0.5-1g/L,
Increase with HC, detection signal value changes are not notable；In 1-250g/L concentration ranges, increase with HC
Plus detection signal weakens.Show：In 1-250g/L concentration ranges, in significantly " HOOK " effect negative correlation area, and in certain
Correlation.
In accordance with the above, the dose curve of 1-250g/L is done into the parameters of logistic tetra- song using ELISACalc softwares
Line Fitting Analysis.As shown in Fig. 2 being analyzed according to Fitting curve equation, as shown in table 2, correlation coefficient r ^2 values show for 0.999.
Content of hemoglobin is detected using HOOK effects area 1-250g/L, there is good dose curve rule.
The parameter Logistic curve matchings of table 2 four
Because normal person's content of hemoglobin is in the range of 110-160g/L, for severe anemia patient, hemoglobin contains
Amount is not less than 30g/L, therefore in view of the availability of clinical meaning and sample, is used to examine using the reagent device of present method invention
Content of hemoglobin in the range of 50-250g/L is surveyed, the examination to Anemic patients provides scientific basis.According to the data obtained of table 1, will
The dose curve of 50-250g/L does the parameter curves of logistic tetra- and analyzes using ELISACalc softwares.As shown in figure 3, root
Analyzed according to Fitting curve equation, as shown in table 3, correlation coefficient r ^2 values show to detect blood in the range of 50-250g/L for 0.999.
, there is good dose curve rule in Lactoferrin content.
The parameter Logistic curve matchings of table 3 four
Clinical sample is detected：Sample loading alternative is same as above to be detected to 120 parts of clinical whole bloods, wherein 57 are detected as
Anaemia, 58 are detected as normal value.Compared with clinic, positive, negative desired value is 100%, another 5 is suspect results.It is comprehensive
The concordance rate of result is 95.8%.Quantitative determination effect is preferable.Can be determined that this method is applied to immune layer by result above
Analysis sandwich method detection content of hemoglobin credible result, the advantages of realize scene, family quick, easy detection.
It is above-mentioned with reference to embodiment the detection method and hemoglobin detection kit of a kind of hemoglobin are carried out it is detailed
Thin description, is illustrative rather than limited, can include several embodiments according to limited scope, therefore do not taking off
Changing and modifications under present general inventive concept, should belong within protection scope of the present invention.
Claims (5)
1. a kind of detection method of hemoglobin, it is characterised in that：Detected using immuno-sandwich method, comprised the following steps that：
After being marked with the hemoglobin discharged in the first antibody combination blood of colloid gold particle, chromatographed forward by capillary force,
When chromatographing SA coated to nitrocellulose filter, the compound system of " colloid gold particle-antibody-antigen-antibody " is formed,
And it is gathered on nitrocellulose filter, the signal of aubergine can be formed when colloid gold particle is largely assembled, can by instrument
The intensity of accurate quantitative analysis interpretation detection signal, so as to realize quantitative determination, the first antibody is antihuman hemoglobin antibody, the
Two antibody are the hemoglobin antibodies of pairing, and the first antibody and SA can be in combination with the different tables of target antigen
Position, so as to form compound system.
2. a kind of hemoglobin detection kit for realizing detection method described in claim 1, it is characterised in that：Including by blood red
Protein Detection test paper, it is external get stuck, sample lysate and the interior IC-card for having set respective standard curve and decision method, wherein,
The hemoglobin Test paper, width is 3-5mm, and length is 7-8cm, and the Test paper is by containing colloid gold label
Antihuman hemoglobin antibody colloidal gold pad, be coated with nitrocellulose filter, sample pad, sample suction pad and the plastics of T lines and C lines
Base plate is constituted, and the T lines are the hemoglobin antibodies of pairing, and C lines are anti-mouse IgG, and the antihuman hemoglobin antibody is used as the
One antibody labeling has a colloid gold particle, sample pad, colloidal gold pad, nitrocellulose filter and sample suction pad from plastic bottom board one end successively
Arrange to the other end；
It is described it is external get stuck, comprising upper lid and lower cover, two hemoglobin Test papers are placed in parallel in the lower cover
Portion, the upper lid counter sample pad part is provided with sample well, for adding sample and dilution, upper lid correspondence nitrocellulose
Film medium position is provided with viewing window, for observing the C lines on nitrocellulose filter, T lines, result of determination；
The sample lysate, main formula is：0.02M Tris-HCL, 0.15molNaCL, 1%Triton100 adjust PH to
7.4；
The IC-card of the IC-card has been built-in standard curve and decision method, the IC-card supports the use NS3001B type immunochromatographies
As a result interpretation recorder is used, and is 50-250g/L to hemoglobin detection range for the quantitative judgement to testing result.
3. hemoglobin detection kit according to claim 2, it is characterised in that：The sample suction pad is absorbent filter.
4. hemoglobin detection kit according to claim 2, it is characterised in that：It is described to be coated with the nitre with antagonist
The preparation method of acid cellulose film is：The hemoglobin antibodies of pairing are configured to by end with the phosphate buffers of 0.01M pH 7.4
Concentration 1-2mg/mL, spray film instrument is rule on nitrocellulose filter with the parameter of 1.2-1.4ul/cm, is coated with T lines, together
When be coated with the anti-mouse IgG of 1-2mg/ml on nitrocellulose filter top, as C lines, dry, it is standby.
5. hemoglobin detection kit according to claim 2, it is characterised in that：The preparation method of the colloidal gold pad
For：Take 100ml collaurum liquid to be placed in beaker, use 0.2M K2CO3PH7.0-8.0 is adjusted to, 1-2mg marks is added and is used blood red egg
Bai Kangti, is stirred at room temperature 1 hour, and closing, 12000rpm is centrifuged 30 minutes, abandons supernatant, is redissolved to 60ml, Ran Houyong with working solution
Metal spraying machine is equably sprayed on non-woven fabrics or glass respectively, drying for standby.
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| CN201710082997.3A CN106872694A (en) | 2017-02-16 | 2017-02-16 | The detection method and hemoglobin detection kit of a kind of hemoglobin |
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| CN201710082997.3A CN106872694A (en) | 2017-02-16 | 2017-02-16 | The detection method and hemoglobin detection kit of a kind of hemoglobin |
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Cited By (3)
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| WO2019029083A1 (en) * | 2017-08-10 | 2019-02-14 | 深圳先进技术研究院 | Test strip for detecting chemerin and derived polypeptide thereof, and preparation method therefor |
| CN110346578A (en) * | 2019-06-06 | 2019-10-18 | 安徽惠邦生物工程有限公司 | Joint quantitative detection system for insulin-like growth factor binding protein-1 and fetal fibronectin content |
| CN113113079A (en) * | 2021-02-25 | 2021-07-13 | 安徽桐康医疗科技股份有限公司 | Method for identifying hook effect in quantitative immunochromatography test |
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| CN110346578B (en) * | 2019-06-06 | 2022-02-11 | 安徽惠邦生物工程有限公司 | Combined quantitative detection system for content of insulin-like growth factor binding protein-1 and fetal fibronectin |
| CN113113079A (en) * | 2021-02-25 | 2021-07-13 | 安徽桐康医疗科技股份有限公司 | Method for identifying hook effect in quantitative immunochromatography test |
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