CN111879924A - Colloidal gold immunochromatography test paper for rapidly diagnosing hemoglobin and combining globin-hemoglobin compound and preparation method thereof - Google Patents
Colloidal gold immunochromatography test paper for rapidly diagnosing hemoglobin and combining globin-hemoglobin compound and preparation method thereof Download PDFInfo
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- 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
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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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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Abstract
The invention relates to the technical field of immunodiagnosis, and discloses a colloidal gold immunochromatographic test paper for quickly diagnosing hemoglobin and a combined globin-hemoglobin compound and a preparation method thereof, aiming at the problem that the detection result is inaccurate because the hemoglobin in the digestive tract is easily damaged by pepsin in the prior art, and specifically comprising the following steps: 1) preparing a first detection line T and a first quality control line C; 2) preparing a second detection line T and a second quality control line C; 3) preparing a gold label pad; 4) preparing detection test paper: and sequentially connecting and assembling the water absorption pad, the sample pad, the microsphere probe treatment pad, the modified nitrocellulose membrane coated with the antibody and the PVC bottom plate to obtain a finished product. The invention adopts the combined detection of the hemoglobin and the hemoglobin-globin complex, improves the sensitivity of occult blood detection including upper gastrointestinal hemorrhage, has simple and quick operation, visual and accurate result display, high sensitivity, good specificity and simple preparation process.
Description
Technical Field
The invention relates to the technical field of immunodiagnosis, in particular to colloidal gold immunochromatography test paper for quickly diagnosing hemoglobin and combining a globin-hemoglobin compound and a preparation method thereof.
Background
Hemoglobin (Hb) in feces, also known as Fecal Occult Blood (FOB), is currently clinically recognized as one of tumor markers for diagnosing colorectal cancer and colon cancer. Hemoglobin readily binds to haptoglobin to form a stable, irreversible Complex called the hemoglobin-haptoglobin Complex (Hb-Hp Complex). All digestive tract diseases cause a small amount of bleeding, and the patients can have occult blood stool, so occult blood examination has important value for diagnosing various digestive tract hemorrhagic diseases, is an effective means for screening the digestive tract diseases by general investigation, and the occult blood test becomes a hotspot of continuous and deep research of people.
The immunochromatography one-step method is currently a method for specifically binding human hemoglobin antigen by using an anti-human hemoglobin antibody, is simple, convenient and quick, basically eliminates the interference of factors such as diet, medicines and the like, and is recommended by the world health organization gastroenteroscopy society as a more confirmed method for fecal occult blood experiments. Hemoglobin is extremely unstable, upper gastrointestinal bleeding occurs, and hemoglobin is easily damaged by pepsin when passing through the stomach, so that the clinical condition of missed examination occurs.
Patent No. CN201410797586.9, patent name "joint detection kit for predicting and diagnosing colon tumor and precancerous lesion and use thereof", the present invention provides a joint detection kit for predicting and/or diagnosing colon tumor and/or precancerous lesion and use thereof, the kit comprising reagents and means for detecting methylated Septin9 gene in blood sample from subject, and reagents and means for detecting heme and/or hemoglobin in stool sample from subject. The invention combines the detection means of two different biological detection mechanisms together, generates synergistic action in the interpretation and diagnosis of the large intestine tumor and the precancerous lesion, and greatly improves the detection rate of the large intestine tumor and the precancerous lesion.
The method has the defects that hemoglobin, hemoglobin and instability thereof, upper gastrointestinal hemorrhage and hemoglobin passing through the stomach are easily damaged by pepsin in the detection method, and the detection result of clinical missed detection is not accurate enough.
Disclosure of Invention
The invention aims to overcome the problem that hemoglobin in the digestive tract is easily damaged by pepsin to cause inaccurate detection results in the prior art, and provides the colloidal gold immunochromatographic test paper for quickly diagnosing the hemoglobin and the haptoglobin-hemoglobin compound and the preparation method thereof. The detection test paper has the advantages of simple and rapid operation, visual and accurate result display, high sensitivity, good specificity, low manufacturing and detection cost, high detection efficiency and simple preparation process, and can be widely applied to basic level sample detection.
In order to achieve the purpose, the invention adopts the following technical scheme:
the colloidal gold immunochromatographic assay test paper for rapidly diagnosing hemoglobin and combining a globin-hemoglobin compound comprises a PVC (polyvinyl chloride) base plate, wherein the PVC base plate is sequentially provided with a sample pad, a gold-labeled probe treatment pad, a nitrocellulose membrane and a water absorption pad, the nitrocellulose membrane is arranged in the middle of the PVC base plate, the two ends of the nitrocellulose membrane are respectively provided with the gold-labeled probe treatment pad and the water absorption pad, the bottom sides of one ends of the gold-labeled probe treatment pad and the water absorption pad are respectively connected with the two ends of the nitrocellulose membrane, and the bottom sides of the other ends of the gold-labeled probe treatment pad and; the upper side of one end of the gold-labeled probe treatment pad connected with the PVC base plate is provided with a sample pad, the bottom side of one end of the sample pad is connected with the upper side of the gold-labeled probe treatment pad, the bottom side of the other end of the sample pad is connected with the PVC base plate, and the non-connecting section of the nitrocellulose membrane is sequentially provided with a detection line T coated with mouse anti-human binding globin and a quality control line C coated with goat anti-mouse IgG antibody from one side of the gold-labeled probe treatment pad.
The principle applied by the product is a colloidal gold immunochromatography and a double-antibody sandwich method, when a sample contains hemoglobin or a combined globin-hemoglobin compound, the hemoglobin or the combined globin-hemoglobin compound is firstly combined with a mouse anti human hemoglobin monoclonal antibody-gold-labeled probe in a labeling pad after sample addition to form an antigen-antibody compound, and the compound is captured by a mouse anti human hemoglobin monoclonal antibody or a mouse anti human combined globin monoclonal antibody coated on a nitrocellulose membrane along with the progress of a chromatography process to form a mouse anti human hemoglobin monoclonal antibody-hemoglobin-mouse anti human hemoglobin monoclonal antibody-gold-labeled probe or a mouse anti human combined globin monoclonal antibody-combined globin-hemoglobin compound-mouse anti human hemoglobin monoclonal antibody-gold-labeled probe, the color is purple red, the color is clear and identifiable, no auxiliary equipment is needed, and the operation is convenient.
With the above test strip, a sample containing hemoglobin and a haptoglobin-hemoglobin complex can be detected. Detecting the sample by using a test strip, wherein when red strips appear on the detection line T and the quality control line C, the sample contains hemoglobin or a haptoglobin-hemoglobin compound; if only the quality control line C has a red strip, the sample does not contain the hemoglobin and the haptoglobin-hemoglobin compound or the content of the hemoglobin and the haptoglobin-hemoglobin compound in the sample is lower than the detection value; if the quality control line C does not have a red strip, the test strip is invalid
Preferably, the gold-labeled probe treatment pad is a gold-labeled pad labeled with a mouse anti-human hemoglobin antibody-colloidal gold conjugate.
The colloidal gold probe is essentially a coating process in which a polymer such as a protein is adsorbed on the surface of a colloidal gold particle. The colloidal gold has negative charge in weak alkali environment, can form firm electrostatic combination with the positive charge group of protein molecules, and does not influence the biological characteristics of the protein. The colloidal gold probe comprises corresponding antibody and color developing particles, so that the colloidal gold probe plays a role in combination and color development in the whole process, and the better gold-labeled probe has the advantages of proper particle size, good uniformity and high color transparency.
Preferably, the concentration of the coated mouse anti-human hemoglobin monoclonal antibody coated by the detection line T is 0.5-2.0 mg/ml; the concentration of the mouse anti-human haptoglobin monoclonal antibody for coating the detection line T is 0.5-2.0 mg/ml.
When the concentration of the coating antibody is lower than 0.5mg/ml, the hemoglobin in the sample is insufficiently captured by the mouse anti-human hemoglobin monoclonal antibody in the reaction process; or the haptoglobin-hemoglobin compound in the sample is insufficiently captured by the mouse anti-human haptoglobin monoclonal antibody, so that the condition of missing detection appears clinically. When the concentration of the coating antibody is higher than 2mg/ml, the heteroprotein in the sample can be captured nonspecifically in the chromatography process, and the clinical false positive risk can occur, so the optimal concentration range of the mouse anti-human hemoglobin monoclonal antibody for coating the test line T and the mouse anti-human haptoglobin monoclonal antibody is 0.5mg/ml-2 mg/ml.
Preferably, the concentration of the goat anti-mouse IgG antibody coated by the quality control line C is 1.0-3.0 mg/ml.
When the concentration of the quality control line coated antibody is lower than 1mg/ml, the combination of the mouse IgG monoclonal antibody-gold-labeled conjugate in the labeling pad and the goat anti-mouse polyclonal antibody is insufficient in the reaction process, and the problem of weak quality control line clinically occurs. When the concentration of the quality control line coated antibody is higher than 3mg/ml, the sheep anti-mouse polyclonal antibody treated on the nitrocellulose membrane can generate a diffusion phenomenon due to overflow, and the integral appearance is influenced. Therefore, the optimal concentration range of goat anti-mouse IgG polyclonal antibody coated with the quality control line C is 1mg/ml to 3 mg/ml.
The preparation method of the colloidal gold immunochromatographic test paper for rapidly detecting hemoglobin and combining a globin-hemoglobin compound specifically comprises the following steps:
1) preparing a first detection line T and a first quality control line C: diluting a mouse anti-human hemoglobin monoclonal antibody, spraying the diluted mouse anti-human hemoglobin monoclonal antibody on a nitrocellulose membrane at a speed of 1.0-1.2ul/cm to serve as a first detection line T, diluting a goat anti-mouse IgG antibody solution, spraying the diluted goat anti-mouse IgG antibody solution on the nitrocellulose membrane to serve as a first quality control line C, and drying at 36-38 ℃ to obtain a detection line and a quality control line;
2) preparing a second detection line T and a second quality control line C: diluting a mouse anti-human haptoglobin-hemoglobin compound monoclonal antibody, spraying the diluted mouse anti-human haptoglobin-hemoglobin compound monoclonal antibody on a nitrocellulose membrane at the speed of 1.0-1.2ul/cm to serve as a second detection line T, diluting a sheep anti-mouse IgG antibody solution, spraying the diluted goat anti-mouse IgG antibody solution on the nitrocellulose membrane to serve as a second quality control line C, and drying at 36-38 ℃ to obtain a detection line and a quality control line;
3) preparing a gold label pad: diluting the purified gold-labeled probe solution into a solution with the concentration of OD30-OD70, spraying the solution on a polyester fiber film, and treating the solution by using blast drying equipment to obtain a gold-labeled pad;
4) preparing detection test paper: and sequentially connecting and assembling the water absorption pad (6), the sample pad (1), the gold-labeled probe treatment pad (2), the nitrocellulose membrane (5) coated with the antibody and the PVC bottom plate (7) to obtain a finished product.
The invention adopts two pieces of test paper for joint detection, so that a first detection line T, a first quality control line C, a second detection line T and a second quality control line C are arranged. In the step 3), the positive reference substance of 50ng/ml hemoglobin, 100ng/ml hemoglobin, 10 mu g/ml hemoglobin, 1mg/ml hemoglobin, 50ng/ml haptoglobin-hemoglobin compound, 200ng/ml haptoglobin-hemoglobin compound, 2 mu g/ml haptoglobin-hemoglobin compound and the negative reference substance are used for testing, and the reading result in 5-10 minutes shows that the effect is best when the using concentration of the mouse anti human hemoglobin monoclonal antibody-gold standard conjugate is OD30-OD 70.
Preferably, the preparation process of the purified gold-labeled antibody comprises the following steps:
A. obtaining colloidal gold: adding 1.8-2.2ml of 0.8-1.2% trisodium citrate aqueous solution into 98-102ml of boiling 0.008-0.012% chloroauric acid aqueous solution to obtain colloidal gold;
B. labeling colloidal gold: with K of 0.18-0.22mol/L2CO3Adjusting the pH of the colloidal gold to 8.4-8.6, adding the mouse anti-human hemoglobin monoclonal antibody for marking into the colloidal gold after the pH is adjusted, and marking for 20-25 min;
C. purifying the gold-labeled antibody: and D, adding 8-12% of BSA aqueous solution by mass fraction into the labeled colloidal gold solution obtained in the step B until the final concentration of BSA is 0.8-1.2%, stopping adding, continuing to stir for 25-30min, continuing to add 8-12% of PEG20000 until the final concentration of PEG20000 is 0.18-0.22%, continuing to stir for 10-15min, centrifuging, removing the supernatant, and adding a re-dissolved casein sodium solution to obtain the purified gold-labeled antibody.
In the preparation process of the mouse anti-human hemoglobin monoclonal antibody-gold-labeled conjugate, the coupling process of colloidal gold and the antibody is particularly important, and because the particle size selected by the product is larger, the phenomena of gold bleaching and dead gold are easy to occur in the coupling process. In the reaction process, the phenomenon of gold bleaching and dead gold can be greatly reduced by adding the complex solution.
Preferably, the diameter of the colloidal gold particles is 40-100 nm.
The chloroauric acid reacts with trisodium citrate serving as a reducing agent to form gold particles with a certain particle size, the performance of the product, the stability of the labeled conjugate and the color of the labeled conjugate are affected by the difference of the particle sizes, the color of the particles is reddish when the particle size is less than 40nm, and the sensitivity is weak; the particle size of more than 100nm is black, uneven in shape and easy to precipitate, so that the particle size of the colloidal gold particles is most suitable to be 40-100 nm.
Preferably, the volume ratio of the mouse anti-human hemoglobin monoclonal antibody used for marking in the step B to the colloidal gold after pH adjustment is 1:1-1: 1.2.
The mouse anti-human hemoglobin monoclonal antibody and the chloroauric acid are coupled according to the proportion of 1:0.5, 1:1, 1:1.2, 1:1.4, 1:1.6 and 1:2.0, the final concentration values of the conjugates are all larger than OD100, the conjugates are diluted to OD40 by using gold-labeled diluent, and then treated on a polyester fiber membrane, vacuum-frozen and dried overnight (12-24 hours), and tested with a positive reference substance of 50ng/ml hemoglobin, 100ng/ml hemoglobin, 10. mu.g/ml hemoglobin, 1mg/ml hemoglobin, 50ng/ml haptoglobin-hemoglobin complex, 200ng/ml haptoglobin-hemoglobin complex, 2. mu.g/ml haptoglobin-hemoglobin complex and a negative reference substance, and the results of 5-10 minute readings show that the results are optimal when the volume ratio is 1:1-1: 1.2.
Preferably, the centrifugation conditions in step C are: the centrifugation temperature is 4-6 ℃, the centrifugation rotation speed is 9900 and 10000rpm, and the centrifugation time is 30-40 min.
The protein is stable under the condition of 2-8 ℃, the centrifugation speed of the gold-labeled conjugate is generally set at 12000rpm according to the literature, the centrifugation time is about 0.5 hour, and the centrifugation speed is controlled at 9900-10000rpm and the centrifugation time is 30-40 minutes because the particle size of the colloidal gold particles selected by the product is about 70nm, so the effect is optimal.
Preferably, the adding amount of the casein sodium solution in the double solution in the step C accounts for 34-36% of the total volume of the label.
Therefore, the invention has the following beneficial effects:
(1) the colloidal gold immunochromatographic assay test paper for rapidly detecting hemoglobin and combining with the globin-hemoglobin compound is prepared, a product for detecting the antigen by using the antibody test paper is prepared, the detection time is short, only 5-10min is needed, and the requirement of on-site detection can be met;
(2) hemoglobin and its instability, upper gastrointestinal bleeding, hemoglobin passing through the stomach, is easily destroyed by pepsin, and clinical missed examination occurs. The haptoglobin-hemoglobin compound is relatively stable and is not easy to be damaged by pepsin, so that the developed two-in-one detection reagent for the hemoglobin and the haptoglobin-hemoglobin compound can be used for simultaneously detecting the upper and lower gastrointestinal hemorrhage;
(3) the operation is simple and convenient, and other equipment and instruments are not needed; the detection result is displayed visually, can be judged by naked eyes and is suitable for personal use; the detection efficiency is high, the detection result is more direct, and the detection error caused by the antibody latency is avoided;
(4) the hemoglobin-based protein complex has no cross with various animal hemoglobins such as bovine hemoglobin, porcine hemoglobin, sheep hemoglobin, rabbit hemoglobin and the like, so that clinical false positive can be obviously reduced, and interference of related substances is reduced;
(5) the preparation process is simple, the test strip can be stored at normal temperature, special equipment and instruments are not needed, the test strip only needs to be kept dry, and the storage life can reach 2 years.
Drawings
FIG. 1 is a schematic diagram of the structure of the test strip of the present invention.
FIG. 2 is a schematic view of the structure of the test strip detection window of the present invention.
In the figure: 1. the kit comprises a sample pad, 2, a gold-labeled pad for marking brucella antibody-colloidal gold, 3, a detection line T for coating the brucella antibody, 4, a quality control line C for coating goat anti-mouse IgG antibody, 5, a nitrocellulose membrane, 6, a water absorption pad, 7 and a PVC bottom plate.
Detailed Description
The invention is further described with reference to specific embodiments.
The colloidal gold immunochromatographic assay test paper for rapidly diagnosing brucellosis comprises a PVC (polyvinyl chloride) base plate 7, wherein the PVC base plate 7 is sequentially provided with a sample pad 1, a microsphere probe treatment pad 2, a nitrocellulose membrane 5 and a water absorption pad 6, the nitrocellulose membrane 5 is arranged in the middle of the PVC base plate 7, the two ends of the nitrocellulose membrane 5 are respectively provided with the microsphere probe treatment pad 2 and the water absorption pad 6, the bottom sides of one ends of the microsphere probe treatment pad 2 and the water absorption pad 6 are respectively connected to the two ends of the nitrocellulose membrane 5, and the bottom side of the other end of the microsphere probe treatment pad 2 and the water absorption pad 6 are respectively; the upper side of one end of the microsphere probe treatment pad 2 connected with the PVC base plate 7 is provided with a sample pad 1, the bottom side of one end of the sample pad 1 is connected with the upper side of the microsphere probe treatment pad 2, the bottom side of the other end of the sample pad 1 is connected with the PVC base plate 7, and the non-connecting section of the nitrocellulose membrane 5 is sequentially provided with a detection line T3 coated with a mouse anti-brucella antibody and a quality control line C4 coated with a goat anti-mouse IgG antibody from one side of the microsphere probe treatment pad 2.
Preparation of a mouse anti-human hemoglobin monoclonal antibody: (1) immunizing BALB/C mice with eight weeks old by using antigen, performing subcutaneous injection, performing injection once every three weeks for five times in total, measuring the antibody titer by ELISA (enzyme-linked immuno sorbent assay) for blood collection, and performing intensive injection three days before fusion; (2) cell fusion and culture: carrying out superstrong immunization on a 3d mouse, removing a neck to kill the mouse, taking out a spleen by aseptic surgery to prepare splenocytes, carrying out cell fusion with SP2/0 cells under the action of PEG, adding the fused cell suspension to a cell culture plate, and selectively culturing the cell suspension in a 5% CO 2 incubator at 37 ℃ by using HAT and HT culture media; (3) and (3) cell screening: screening positive holes by using indirect competition E LISA, and carrying out three times of limiting dilution cloning on strong positive holes with vigorous cell growth; (4) production of ascites monoclonal antibody: six mice are taken, paraffin is injected into the abdominal cavity, each mouse is injected with 2.5ml, cloned hybridoma cell strains are injected after 10 days, ascites is extracted after 12 days, antibodies are purified by an ammonium caprylate-sulfate salting-out method, and the concentration of the antibodies is measured by an ultraviolet absorption method.
Preparation of mouse anti-human haptoglobin monoclonal antibody: (1) immunizing BALB/C mice with eight weeks old by using antigen, performing subcutaneous injection, performing injection once every three weeks for five times in total, measuring the antibody titer by ELISA (enzyme-linked immuno sorbent assay) for blood collection, and performing intensive injection three days before fusion; (2) cell fusion and culture: immunizing 3d mouse with superstrong immunity, removing neck to kill mouse, aseptically operating to take out spleen to prepare splenocyte, performing cell fusion with SP2/0 cell under the action of PEG, adding the fused cell suspension onto cell culture plate, standing at 37 deg.C and 5% CO2Selectively culturing in HAT and HT culture medium in an incubator; (3) and (3) cell screening: screening positive holes by using indirect competition E LISA, and carrying out three times of limiting dilution cloning on strong positive holes with vigorous cell growth; (4) production of ascites monoclonal antibody: six mice are taken, paraffin is injected into the abdominal cavity, each mouse is injected with 2.5ml, cloned hybridoma cell strains are injected after 10 days, ascites is extracted after 12 days, antibodies are purified by an ammonium caprylate-sulfate salting-out method, and the concentration of the antibodies is measured by an ultraviolet absorption method.
Preparing a feces collecting liquid: mixing 25-45g/L chelating agent, 0.2-1g/L preservative, 1-10g/L blocking agent, 6-12g/L buffer, and 25-30g/L polyethylene glycol with water, and adjusting final pH to 9.4.
Is used for collecting and preserving feces discharged by human body and preventing hemoglobin from deteriorating. The preparation of excrement and urine collecting fluid is key technology, and the hemoglobin in the excrement and urine is very unstable, and low concentration hemoglobin is placed at room temperature for more than 8 hours, can appear clinically the risk of lou examining, even place 3 days at high concentration hemoglobin room temperature, sensitivity decline also can be very obvious. Based on the technical difficulty, the inventor develops the excrement collecting fluid through continuous research, can stably store the hemoglobin for one week, greatly improves the clinical detectable rate, places the omission and is convenient for patients and doctors to transport excrement samples.
Example 1
The preparation method of the colloidal gold immunochromatographic test paper for rapidly detecting human hemoglobin and combined globin-hemoglobin compound specifically comprises the following steps:
1) preparing a first detection line T and a first quality control line C: diluting a mouse anti-human hemoglobin monoclonal antibody, spraying the diluted mouse anti-human hemoglobin monoclonal antibody on a nitrocellulose membrane at a speed of 1.0-1.2ul/cm to serve as a first detection line T, diluting a goat anti-mouse IgG antibody solution, spraying the diluted goat anti-mouse IgG antibody solution on the nitrocellulose membrane to serve as a first quality control line C, and drying at 36 ℃ to obtain a detection line and a quality control line; the concentration of the mouse anti-human hemoglobin monoclonal antibody coated by the detection line T is 0.5mg/ml, and the concentration of the goat anti-mouse IgG antibody is 1.0 mg/ml;
2) preparing a second detection line T and a second quality control line C: diluting a mouse anti-human haptoglobin-hemoglobin compound monoclonal antibody, spraying the diluted mouse anti-human haptoglobin-hemoglobin compound monoclonal antibody on a nitrocellulose membrane at the speed of 1.0ul/cm to serve as a second detection line T, diluting a goat anti-mouse IgG antibody solution, spraying the diluted mouse anti-human haptoglobin-hemoglobin compound monoclonal antibody on the nitrocellulose membrane to serve as a second quality control line C, and drying at 36 ℃ to obtain a detection line and a quality control line; the concentration of the mouse anti-human haptoglobin monoclonal antibody is 0.5mg/ml, and the concentration of the goat anti-mouse IgG antibody is 1.0 mg/ml;
3) preparing a gold label pad: coating the purified gold-labeled antibody with the concentration of OD30 after dilution on a glass fiber pad, and treating the glass fiber pad by using a freeze dryer to obtain a gold-labeled pad; the preparation process of the purified gold-labeled antibody comprises the following steps: A. obtaining colloidal gold: adding 1.8ml of 0.8% trisodium citrate aqueous solution into 98ml of boiling 0.008% chloroauric acid aqueous solution to obtain colloidal gold with the diameter of 70 nm; B. labeling colloidal gold: with 0.18mol/L of K2CO3Adjusting the pH value of the colloidal gold to 8.4 by using the solution, adding a labeled mouse anti-human hemoglobin monoclonal antibody with the concentration of 5mg/ml into the colloidal gold after the pH value is adjusted, wherein the volume ratio of the labeled mouse anti-human hemoglobin antibody to the colloidal gold after the pH value is adjusted is 1:1, and labeling for 20min to obtain a mouse anti-human hemoglobin monoclonal antibody-colloidal gold conjugate; C. purifying the gold-labeled antibody: adding 8 percent BSA (bovine serum albumin) aqueous solution to the labeled colloidal gold solution in the step B until the final concentration of BSA is 0.8 percent, stopping adding, continuing to stir for 25min, continuing to add 8 percent PEG20000 to PEG20000 until the final concentration is 0.18 percent, continuing to stir for 10min, centrifuging at 4 ℃ and 9900rpm for 30min, discarding the supernatant, and adding a casein sodium solution accounting for 34 percent of the total volume of the label, namelyObtaining a purified gold-labeled antibody;
4) preparing detection test paper: and sequentially connecting and assembling the water absorption pad, the sample pad, the gold-labeled probe treatment pad, the nitrocellulose membrane coated with the antibody and the PVC bottom plate to obtain the combined test paper for joint detection of the two pieces of test paper.
Example 2
The preparation method of the colloidal gold immunochromatographic test paper for rapidly detecting human hemoglobin and combined globin-hemoglobin compound specifically comprises the following steps:
1) preparing a first detection line T and a first quality control line C: diluting a mouse anti-human hemoglobin monoclonal antibody, spraying the diluted mouse anti-human hemoglobin monoclonal antibody on a nitrocellulose membrane at a speed of 1.15ul/cm to serve as a first detection line T, diluting a goat anti-mouse IgG antibody solution, spraying the diluted goat anti-mouse IgG antibody solution on the nitrocellulose membrane to serve as a first quality control line C, and drying at 36.5 ℃ to obtain a detection line and a quality control line; the concentration of the mouse anti-human hemoglobin monoclonal antibody coated by the detection line T is 0.8mg/ml, and the concentration of the goat anti-mouse IgG antibody is 1.5 mg/ml;
2) preparing a second detection line T and a second quality control line C: diluting a mouse anti-human haptoglobin-hemoglobin compound monoclonal antibody, spraying the diluted mouse anti-human haptoglobin-hemoglobin compound monoclonal antibody on a nitrocellulose membrane at the speed of 1.0-1.2ul/cm to serve as a second detection line T, diluting a sheep anti-mouse IgG antibody solution, spraying the diluted goat anti-mouse IgG antibody solution on the nitrocellulose membrane to serve as a second quality control line C, and drying at 36.5 ℃ to obtain a detection line and a quality control line; the concentration of the mouse anti-human haptoglobin monoclonal antibody is 0.8mg/ml, and the concentration of the goat anti-mouse IgG antibody is 1.5 mg/ml;
3) preparing a gold label pad: coating the purified gold-labeled antibody with the concentration of OD40 after dilution on a glass fiber pad, and treating the glass fiber pad by using a freeze dryer to obtain a gold-labeled pad; the preparation process of the purified gold-labeled antibody comprises the following steps: A. obtaining colloidal gold: adding 1.9ml of 0.9% trisodium citrate aqueous solution into 99ml of boiling 0.009% chloroauric acid aqueous solution to obtain colloidal gold with diameter of 60 nm; B. labeling colloidal gold: with 0.19mol/L of K2CO3Adjusting the pH of the colloidal gold to 8.5, adding a monoclonal antibody of a mouse anti-human hemoglobin with the concentration of 5mg/ml into the colloidal gold after the pH adjustment, and marking the mouse anti-human hemoglobinThe volume ratio of the white antibody to the colloidal gold after pH adjustment is 1:1.1, and the labeling is carried out for 22min to obtain a mouse anti-human hemoglobin monoclonal antibody-colloidal gold conjugate; C. purifying the gold-labeled antibody: adding a BSA (bovine serum albumin) aqueous solution with the mass fraction of 9% into the labeled colloidal gold solution in the step B until the final concentration of the BSA is 0.9%, stopping adding, continuing to stir for 26min, continuing to add 9% PEG20000 to PEG20000 until the final concentration is 0.19%, continuing to stir for 11min, centrifuging at 4.5 ℃ and 9920rpm for 32min, discarding the supernatant, and adding a casein sodium solution accounting for 34.5% of the total volume of the label to obtain the purified gold-labeled antibody;
4) preparing detection test paper: and sequentially connecting and assembling the water absorption pad, the sample pad, the gold-labeled probe treatment pad, the nitrocellulose membrane coated with the antibody and the PVC bottom plate to obtain the combined test paper for joint detection of the two pieces of test paper.
Example 3
The preparation method of the colloidal gold immunochromatographic test paper for rapidly detecting human hemoglobin and combined globin-hemoglobin compound specifically comprises the following steps:
1) preparing a first detection line T and a first quality control line C: diluting a mouse anti-human hemoglobin monoclonal antibody, spraying the diluted mouse anti-human hemoglobin monoclonal antibody on a nitrocellulose membrane at a speed of 1.1ul/cm to serve as a first detection line T, diluting a goat anti-mouse IgG antibody solution, spraying the diluted goat anti-mouse IgG antibody solution on the nitrocellulose membrane to serve as a first quality control line C, and drying at 37 ℃ to obtain a detection line and a quality control line; the concentration of the mouse anti-human hemoglobin monoclonal antibody coated by the detection line T is 1.2mg/ml, and the concentration of the goat anti-mouse IgG antibody is 2 mg/ml;
2) preparing a second detection line T and a second quality control line C: diluting a mouse anti-human haptoglobin-hemoglobin compound monoclonal antibody, spraying the diluted mouse anti-human haptoglobin-hemoglobin compound monoclonal antibody on a nitrocellulose membrane at the speed of 1.1ul/cm to serve as a second detection line T, diluting a goat anti-mouse IgG antibody solution, spraying the diluted mouse anti-human haptoglobin antibody solution on the nitrocellulose membrane to serve as a second quality control line C, and drying at 37 ℃ to obtain a detection line and a quality control line; the concentration of the mouse anti-human haptoglobin monoclonal antibody is 1.5mg/ml, and the concentration of the goat anti-mouse IgG antibody is 2 mg/ml;
3) preparing a gold label pad: the purified gold-labeled antibody was diluted to OD50 and then coatedPlacing the glass fiber mat on a freeze dryer for treatment to prepare a gold label mat; the preparation process of the purified gold-labeled antibody comprises the following steps: A. obtaining colloidal gold: adding 2.0ml of 1.0% trisodium citrate aqueous solution into 100ml of boiling 0.010% chloroauric acid aqueous solution to obtain colloidal gold with the diameter of 60 nm; B. labeling colloidal gold: with 0.2mol/L of K2CO3Adjusting the pH value of the colloidal gold to 8.5 by using the solution, adding a labeled mouse anti-human hemoglobin monoclonal antibody with the concentration of 5mg/ml into the colloidal gold after the pH value is adjusted, wherein the volume ratio of the labeled mouse anti-human hemoglobin antibody to the colloidal gold after the pH value is adjusted is 1:1.1, and labeling for 22min to obtain a mouse anti-human hemoglobin monoclonal antibody-colloidal gold conjugate; C. purifying the gold-labeled antibody: adding a BSA (bovine serum albumin) aqueous solution with the mass fraction of 10% into the labeled colloidal gold solution in the step B until the final concentration of the BSA is 1%, stopping adding, continuing to stir for 28min, continuing to add 10% PEG20000 to the final concentration of PEG20000 to be 0.2%, continuing to stir for 12min, centrifuging at 9950rpm at 5 ℃ for 35min, discarding the supernatant, and adding a casein sodium solution accounting for 35% of the total volume of the label to obtain the purified gold-labeled antibody;
4) preparing detection test paper: and sequentially connecting and assembling the water absorption pad, the sample pad, the gold-labeled probe treatment pad, the nitrocellulose membrane coated with the antibody and the PVC bottom plate to obtain the combined test paper for joint detection of the two pieces of test paper.
Example 4
The preparation method of the colloidal gold immunochromatographic test paper for rapidly detecting human hemoglobin and combined globin-hemoglobin compound specifically comprises the following steps:
1) preparing a first detection line T and a first quality control line C: diluting a mouse anti-human hemoglobin monoclonal antibody, spraying the diluted mouse anti-human hemoglobin monoclonal antibody on a nitrocellulose membrane at a speed of 1.15ul/cm to serve as a first detection line T, diluting a goat anti-mouse IgG antibody solution, spraying the diluted goat anti-mouse IgG antibody solution on the nitrocellulose membrane to serve as a first quality control line C, and drying at 37.5 ℃ to obtain a detection line and a quality control line; the concentration of the mouse anti-human hemoglobin monoclonal antibody coated by the detection line T is 1.8mg/ml, and the concentration of the goat anti-mouse IgG antibody is 2.5 mg/ml;
2) preparing a second detection line T and a second quality control line C: diluting a mouse anti-human haptoglobin-hemoglobin compound monoclonal antibody, spraying the diluted mouse anti-human haptoglobin-hemoglobin compound monoclonal antibody on a nitrocellulose membrane at the speed of 1.15ul/cm to serve as a second detection line T, diluting a goat anti-mouse IgG antibody solution, spraying the diluted mouse anti-human haptoglobin antibody solution on the nitrocellulose membrane to serve as a second quality control line C, and drying at 37 ℃ to obtain a detection line and a quality control line; the concentration of the mouse anti-human haptoglobin monoclonal antibody is 1.5mg/ml, and the concentration of the goat anti-mouse IgG antibody is 2.5 mg/ml;
3) preparing a gold label pad: coating the purified gold-labeled antibody with the concentration of OD60 after dilution on a glass fiber pad, and treating the glass fiber pad by using a freeze dryer to obtain a gold-labeled pad; the preparation process of the purified gold-labeled antibody comprises the following steps: A. obtaining colloidal gold: adding 2.1ml of 1.0% trisodium citrate aqueous solution into 101ml of boiling 0.01% chloroauric acid aqueous solution to obtain colloidal gold with the diameter of 80 nm; B. labeling colloidal gold: with 0.21mol/L of K2CO3Adjusting the pH value of the colloidal gold to 8.5 by using the solution, adding a labeled mouse anti-human hemoglobin monoclonal antibody with the concentration of 5mg/ml into the colloidal gold after the pH value is adjusted, marking for 24min according to the volume ratio of the labeled mouse anti-human hemoglobin antibody to the colloidal gold after the pH value is adjusted being 1:1.1, and obtaining a mouse anti-human hemoglobin monoclonal antibody-colloidal gold conjugate; C. purifying the gold-labeled antibody: adding 11 mass percent BSA (bovine serum albumin) aqueous solution into the labeled colloidal gold solution obtained in the step B until the final concentration of BSA is 1.1%, stopping adding, continuing stirring for 28min, continuing adding 11 percent PEG20000 to PEG20000 until the final concentration is 0.21%, continuing stirring for 14min, centrifuging at 9980rpm for 38min at 5 ℃, discarding the supernatant, and adding 35.5 percent casein sodium solution accounting for the total volume of the label to obtain the purified gold-labeled antibody;
4) preparing detection test paper: and sequentially connecting and assembling the water absorption pad, the sample pad, the gold-labeled probe treatment pad, the nitrocellulose membrane coated with the antibody and the PVC bottom plate to obtain the combined test paper for joint detection of the two pieces of test paper.
Example 5
The preparation method of the colloidal gold immunochromatographic test paper for rapidly detecting human hemoglobin and combined globin-hemoglobin compound specifically comprises the following steps:
1) preparing a first detection line T and a first quality control line C: diluting a mouse anti-human hemoglobin monoclonal antibody, spraying the diluted mouse anti-human hemoglobin monoclonal antibody on a nitrocellulose membrane at a speed of 1.2ul/cm to serve as a first detection line T, diluting a goat anti-mouse IgG antibody solution, spraying the diluted goat anti-mouse IgG antibody solution on the nitrocellulose membrane to serve as a first quality control line C, and drying at 38 ℃ to obtain a detection line and a quality control line; the concentration of the mouse anti-human hemoglobin monoclonal antibody coated by the detection line T is 2.0mg/ml, and the concentration of the goat anti-mouse IgG antibody is 3.0 mg/ml;
2) preparing a second detection line T and a second quality control line C: diluting a mouse anti-human haptoglobin-hemoglobin compound monoclonal antibody, spraying the diluted mouse anti-human haptoglobin-hemoglobin compound monoclonal antibody on a nitrocellulose membrane at the speed of 1.2ul/cm to serve as a second detection line T, diluting a goat anti-mouse IgG antibody solution, spraying the diluted mouse anti-human haptoglobin-hemoglobin compound monoclonal antibody on the nitrocellulose membrane to serve as a second quality control line C, and drying at 38 ℃ to obtain a detection line and a quality control line; the concentration of the mouse anti-human haptoglobin monoclonal antibody is 2.0mg/ml, and the concentration of the goat anti-mouse IgG antibody is 3.0 mg/ml;
3) preparing a gold label pad: coating the purified gold-labeled antibody with the concentration of OD70 after dilution on a glass fiber pad, and treating the glass fiber pad by using a freeze dryer to obtain a gold-labeled pad; the preparation process of the purified gold-labeled antibody comprises the following steps: A. obtaining colloidal gold: adding 2.2ml of 1.2% trisodium citrate aqueous solution into 102ml of boiling 0.012% chloroauric acid aqueous solution to obtain colloidal gold with the diameter of 40-100 nm; B. labeling colloidal gold: with 0.22mol/L of K2CO3Adjusting the pH value of the colloidal gold to 8.6 by using the solution, adding a labeled mouse anti-human hemoglobin monoclonal antibody with the concentration of 5mg/ml into the colloidal gold after the pH value is adjusted, labeling for 25min, and thus obtaining a mouse anti-human hemoglobin monoclonal antibody-colloidal gold conjugate, wherein the volume ratio of the labeled mouse anti-human hemoglobin antibody to the colloidal gold after the pH value is adjusted is 1: 1.2; C. purifying the gold-labeled antibody: adding a BSA (bovine serum albumin) aqueous solution with the mass fraction of 12% into the labeled colloidal gold solution in the step B until the final concentration of the BSA is 1.2%, stopping adding, continuing to stir for 30min, continuing to add 12% PEG20000 to PEG20000 until the final concentration is 0.22%, continuing to stir for 15min, centrifuging at 6 ℃ and 10000rpm for 40min, discarding the supernatant, and adding a casein sodium solution accounting for 36% of the total volume of the label to obtain the purified gold-labeled antibody;
4) preparing detection test paper: and sequentially connecting and assembling the water absorption pad, the sample pad, the gold-labeled probe treatment pad, the nitrocellulose membrane coated with the antibody and the PVC bottom plate to obtain the combined test paper for joint detection of the two pieces of test paper.
Comparative example 1
The difference from example 3 is that the diameter of the murine anti-human hemoglobin antibody-colloidal gold conjugate is as small as 20 nm.
Comparative example 2
The difference from example 3 is that the step of purifying the gold-labeled antibody in step 2) is omitted.
Comparative example 3
The difference from example 3 is that the reconstituted solution Casein-Na (sodium caseinate solution) was replaced by Casein (Casein solution).
Comparative example 4
The difference from example 3 is that the final centrifugation speed was reduced to 5500r/min and the centrifugation was carried out for 0.4 hours.
Comparative example 5
The difference from example 3 is that the fecal collection was changed to a common buffer.
And (3) performing sensitivity tests on the positive reference substance 50ng/ml hemoglobin, 100ng/ml hemoglobin, 10 mu g/ml hemoglobin, 1mg/ml hemoglobin, 50ng/ml haptoglobin-hemoglobin complex, 200ng/ml haptoglobin-hemoglobin complex, 2 mu g/ml haptoglobin-hemoglobin complex and the negative reference substance respectively, repeating the setting of each concentration for three times, and ensuring that the detection results are completely consistent, thereby proving that the detection result of the test strip is stable and reliable.
The stability test adopts an accelerated stability test, the test strips of the same batch are respectively placed in ovens at 45 ℃ and 55 ℃, and the standard substances are respectively detected in the time periods shown in the following table.
The results of the tests on the finished products obtained in examples 1 to 5 and comparative examples 1 to 5 are shown in Table 1.
Table 1 shows the performance evaluation indexes of the items related to the colloidal gold immunochromatographic test paper
Conclusion analysis: the colloidal gold immunochromatographic test paper with high detection sensitivity, accurate detection result and long service cycle can be prepared in the embodiments 1 to 5.
Comparative examples 1-5 compared to example 3, the diameter of the murine anti-human hemoglobin antibody-colloidal gold conjugate in comparative example 1 was 20nm too small. The coupling mode of the antibody and the colloidal gold is physical adsorption, and although the colloidal gold with undersize grain size is stable, the quantity of the mouse anti-human hemoglobin monoclonal antibody adsorbed on the surface of the colloidal gold is limited, thereby influencing the sensitivity of the product.
The step of purifying the gold-labeled antibody in step 2) was omitted in comparative example 2. During the labeling process, the risk of clinical false positives may arise due to interference of the hetero-protein.
In comparative example 3, in step C, Casein-Na (Casein sodium solution) was replaced with Casein (Casein solution), and the pH was increased from 7.4 to 8. In the reaction process, the phenomenon of gold bleaching and dead gold can be greatly reduced by adding the complex solution; if the re-solution casein sodium solution is changed into casein solution, the colloidal gold particle size colloid used by the product is easy to generate floating gold and dead gold in the marking process, and the marked gold-labeled conjugate is black in color and easy to generate false positive clinically.
In comparative example 4, the final centrifugation speed was reduced to 5500r/min and the centrifugation was carried out for 0.4 hours. The centrifugation speed is slow, the time is short, the centrifugation is insufficient, the concentration value of the gold-labeled conjugate is low, part of effective components are not collected, and the sensitivity is weak.
In comparative example 5, the fecal collection was replaced with a normal buffer. Hemoglobin is extremely unstable, and placing at normal temperature easily leads to the detectivity to descend, and excrement and urine collecting fluid is exclusive prescription, can obviously stabilize hemoglobin, can preserve 7 days at normal temperature, if change excrement and urine collecting fluid into ordinary buffer solution, then lead to the detectivity to descend.
As shown in fig. 2, the test line of the test strip was colored after the dropping of the positive standard, and the test line of the test strip was not colored after the dropping of the negative standard. The hemoglobin and haptoglobin-hemoglobin compound two-in-one detection kit adopts a double-window mode, two different sample adding holes are required to be added to the same sample, and two windows are required to be observed in a reading result. Because the hemoglobin detection reagent and the detection reagent of the haptoglobin-hemoglobin compound are the same in the used marking raw materials, the same test strip cannot be realized.
Clinical sample validation
The inventor collects 100 fecal samples of gastrointestinal hemorrhage, and utilizes the colloidal gold test strip prepared by the invention to carry out detection, and the detection result shows that the samples show that 95 cases of hemoglobin are positive, 2 cases of combined globin-hemoglobin compound are positive, and 40 cases of both cases are positive. The test strip is completely consistent with the results of Alere contrast reagents which are sold in the market abroad, and the test strip is proved to have reliable results, strong specificity, simple and quick operation, can detect clinical samples without any equipment, and has intuitive and accurate detection result display. As can be seen from the data of examples 1-5 and comparative examples 1-5, only the protocol within the scope of the claims of the present invention can satisfy the above requirements in all aspects, and an optimized protocol can be obtained, and a colloidal gold immunochromatographic test strip with optimal performance can be obtained. The change of the mixture ratio, the replacement/addition/subtraction of raw materials or the change of the feeding sequence can bring corresponding negative effects.
The raw materials and equipment used in the invention are common raw materials and equipment in the field if not specified; the methods used in the present invention are conventional in the art unless otherwise specified.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and all simple modifications, alterations and equivalents of the above embodiments according to the technical spirit of the present invention are still within the protection scope of the technical solution of the present invention.
Claims (10)
1. The colloidal gold immunochromatographic assay test paper for rapidly diagnosing hemoglobin and combining a globin-hemoglobin compound is characterized by comprising a PVC (polyvinyl chloride) base plate (7), wherein the PVC base plate (7) is sequentially provided with a sample pad (1), a gold-labeled probe treatment pad (2), a nitrocellulose membrane (5) and a water absorption pad (6), the nitrocellulose membrane (5) is arranged in the middle of the PVC base plate (7), the gold-labeled probe treatment pad (2) and the water absorption pad (6) are respectively arranged at two ends of the nitrocellulose membrane (5), the bottom sides of one ends of the gold-labeled probe treatment pad (2) and the water absorption pad (6) are respectively connected to two ends of the nitrocellulose membrane (5), and the bottom side of the other end of the gold-labeled probe treatment pad and the water absorption pad (; the upper side of one end of the gold-labeled probe treatment pad (2) connected with the PVC base plate (7) is provided with a sample pad (1), the bottom side of one end of the sample pad (1) is connected with the upper side of the gold-labeled probe treatment pad (2), the bottom side of the other end of the sample pad is connected with the PVC base plate (7), and the non-connecting section of the nitrocellulose membrane (5) is sequentially provided with a detection line T (3) coated with mouse anti-human binding globin and a quality control line C (4) coated with goat anti-mouse IgG antibody from one side of the gold-labeled probe treatment pad (2).
2. The test strip for rapid diagnosis of hemoglobin-globin-hemoglobin complex according to claim 1, wherein said gold-labeled probe-treated pad (2) is a gold-labeled pad labeled with a mouse anti-human hemoglobin antibody-gold colloidal conjugate.
3. The test strip for rapid diagnosis of hemoglobin and combined with globin-hemoglobin complex according to claim 1, wherein the concentration of coated mouse-anti-human hemoglobin monoclonal antibody coated on the detection line T (3) is 0.5-2.0 mg/ml; the concentration of the mouse anti-human haptoglobin monoclonal antibody for coating the detection line T is 0.5-2.0 mg/ml.
4. The test strip for rapid diagnosis of hemoglobin and combined with globin-hemoglobin complex of claim 1, wherein the concentration of goat anti-mouse IgG antibody coated with the control line C (4) is 1.0-3.0 mg/ml.
5. The method for preparing the test paper for the immunochromatography of colloidal gold for the rapid diagnosis of hemoglobin and haptoglobin-hemoglobin complex according to any one of claims 1 to 4, which comprises the following steps:
1) preparing a first detection line T and a first quality control line C: diluting a mouse anti-human hemoglobin monoclonal antibody, spraying the diluted mouse anti-human hemoglobin monoclonal antibody on a nitrocellulose membrane at a speed of 1.0-1.2ul/cm to serve as a first detection line T, diluting a goat anti-mouse IgG antibody solution, spraying the diluted goat anti-mouse IgG antibody solution on the nitrocellulose membrane to serve as a first quality control line C, and drying at 36-38 ℃ to obtain a detection line and a quality control line;
2) preparing a second detection line T and a second quality control line C: diluting a mouse anti-human haptoglobin-hemoglobin compound monoclonal antibody, spraying the diluted mouse anti-human haptoglobin-hemoglobin compound monoclonal antibody on a nitrocellulose membrane at the speed of 1.0-1.2ul/cm to serve as a second detection line T, diluting a sheep anti-mouse IgG antibody solution, spraying the diluted goat anti-mouse IgG antibody solution on the nitrocellulose membrane to serve as a second quality control line C, and drying at 36-38 ℃ to obtain a detection line and a quality control line;
3) preparing a gold label pad: diluting the purified gold-labeled antibody solution into a solution with the concentration of OD30-OD70, spraying the solution on a polyester fiber membrane, and treating the solution by using blast drying equipment to obtain a gold-labeled pad;
4) preparing detection test paper: and sequentially connecting and assembling the water absorption pad (6), the sample pad (1), the gold-labeled probe treatment pad (2), the nitrocellulose membrane (5) coated with the antibody and the PVC bottom plate (7) to obtain a finished product.
6. The method for preparing a test strip for rapid diagnosis of hemoglobin and combined globin-hemoglobin complex according to claim 5, wherein said purified gold-labeled antibody is prepared by the steps of:
A. obtaining colloidal gold: adding 1.8-2.2ml of 0.8-1.2% trisodium citrate aqueous solution into 98-102ml of boiling 0.008-0.012% chloroauric acid aqueous solution to obtain colloidal gold;
B. labeling colloidal gold: with K of 0.18-0.22mol/L2CO3Adjusting the pH of the colloidal gold to 8.4-8.6, adding the mouse anti-human hemoglobin monoclonal antibody for marking into the colloidal gold after the pH is adjusted, and marking for 20-25 min;
C. purifying the gold-labeled antibody: and D, adding 8-12% of BSA aqueous solution by mass fraction into the labeled colloidal gold solution obtained in the step B until the final concentration of BSA is 0.8-1.2%, stopping adding, continuing to stir for 25-30min, continuing to add 8-12% of PEG20000 until the final concentration of PEG20000 is 0.18-0.22%, continuing to stir for 10-15min, centrifuging, removing the supernatant, and adding a re-dissolved casein sodium solution to obtain the purified gold-labeled antibody.
7. The method for preparing a test strip for immunochromatography using colloidal gold for rapid diagnosis of hemoglobin and haptoglobin-hemoglobin complex according to claim 6, wherein said colloidal gold particles have a diameter of 40 to 100 nm.
8. The method for preparing the test paper for immunochromatography of colloidal gold for rapid diagnosis of hemoglobin and haptoglobin-hemoglobin complex according to claim 7, wherein the volume ratio of the mouse anti-human hemoglobin monoclonal antibody for labeling in the step B to the colloidal gold after pH adjustment is 1:1 to 1: 1.2.
9. The method for preparing the test strip for rapid diagnosis of hemoglobin and combined globin-hemoglobin complex according to claim 7, wherein the centrifugation conditions in step C are as follows: the centrifugation temperature is 4-6 ℃, the centrifugation rotation speed is 9900 and 10000rpm, and the centrifugation time is 30-40 min.
10. The method for preparing a test strip for rapid diagnosis of hemoglobin and combined globin-hemoglobin complex according to claim 7, wherein the amount of sodium caseinate in said complex solution added in step C is 34-36% of the total volume of the label.
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