CN114414797A - Haptoglobin, hemoglobin and transferrin collinear detection test strip and preparation method thereof, detection card and kit - Google Patents
Haptoglobin, hemoglobin and transferrin collinear detection test strip and preparation method thereof, detection card and kit Download PDFInfo
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Abstract
The invention discloses a haptoglobin, hemoglobin and transferrin collinear detection test strip, a preparation method thereof, a detection card and a kit. The detection line of the test strip is coated with a mixed antibody; the mixed antibody comprises a first anti-human haptoglobin monoclonal antibody, a first anti-human hemoglobin monoclonal antibody and a first anti-human transferrin monoclonal antibody; the quality control line is coated with a quality control antibody; the gold label pad is coated with a colloidal gold labeled antibody; the colloidal gold labeled antibody comprises a colloidal gold labeled second anti-human haptoglobin monoclonal antibody, a colloidal gold labeled second anti-human hemoglobin monoclonal antibody and a colloidal gold labeled second anti-human transferrin monoclonal antibody; the mixed antibody and the antibody marked by the colloidal gold are respectively combined with different antigen epitopes of corresponding detection proteins; the colloidal gold-labeled antibody can also bind to the quality control antibody. The invention has extremely high sensitivity, can detect 4ng/mL haptoglobin and has specificity.
Description
Technical Field
The invention belongs to the field of immunodetection, and relates to a haptoglobin, hemoglobin and transferrin collinear detection test strip, a preparation method thereof, a detection card and a kit.
Background
Gastrointestinal hemorrhage is one of the common clinical symptoms, and can be divided into upper gastrointestinal hemorrhage and lower gastrointestinal hemorrhage. And partial digestive tract bleeding is trace, the appearance of the excrement is not abnormally changed, and the excrement is difficult to discover by naked eyes, so that most of early digestive tract malignant tumor patients cannot discover and intervene in treatment in time, and the best opportunity of treatment is delayed. The fecal occult blood test is one of important methods for detecting acute and chronic gastrointestinal hemorrhage and gastrointestinal malignant tumors, is the only examination item which is confirmed by random clinical tests at present and can reduce death caused by colon cancer and rectal cancer, and is important for early diagnosis and treatment of patients with colon cancer and rectal cancer.
Fecal occult blood, also known as fecal occult blood, refers to bleeding which is manifested by small amount of bleeding in the digestive tract, damaged red blood cells, no abnormal change in the appearance of feces, and no evidence under naked eyes or microscope. The method has the main significance of detecting the gastrointestinal hemorrhage and the gastrointestinal malignant tumor caused by various reasons and an important screening index for early diagnosis, and is also an effective method for discovering occult blood changes. For patients with digestive tract tumors, 20% of patients in the early stage can have occult blood tests positive, and the occult blood tests of patients in the late stage can be positive even more than 90%, and the patients can show sustainable positive. Therefore, it can be used as an index for screening digestive tract tumor. For digestive tract hemorrhage, fecal occult blood tests of patients with digestive tract ulcer are mostly positive, or are positive in a staged manner.
The occult blood of stool, namely occult blood, has chemical methods and immunological methods in the current clinical detection method. The chemical method has low specificity, poor sensitivity, great influence by diet and drugs, and difficult discrimination of results, so the adoption rate is low. The colloidal gold method in immunology detects hemoglobin or transferrin in feces by taking hemoglobin or transferrin as an antigen and carrying out immunoreaction with an anti-hemoglobin or transferrin antibody preset on test paper. The sensitivity and the specificity are greatly improved, and the method is widely applied clinically. The former can detect the hemorrhage of any part of the lower digestive tract, while the latter can detect the hemorrhage of the upper digestive tract, because the erythrocyte matrix is completely digested after the hemorrhage of the upper digestive tract is acted by digestive enzyme, the immunoreaction is not generated any more. Since transferrin is mainly present in plasma, it is hardly present in feces of healthy persons, but is present in large amounts in feces at the time of bleeding of the digestive tract. Meanwhile, the stability of the transferrin is obviously higher than that of the hemoglobin. The method is used for detecting transferrin while detecting hemoglobin aiming at upper gastrointestinal hemorrhage, wherein one item is positive when being detected, so that the effect of complementary advantages can be achieved, the detection rate is higher than that of a single detection method, and the false negative result can be effectively reduced.
Haptoglobin (Hp), a haptoglobin protein, is an acidic glycoprotein with a molecular weight of 85000, synthesized primarily in the liver. The main function is to combine with free hemoglobin (Hb) to form a stable complex, present new antigenic determinants, can be recognized and combined by a hemoglobin clearance receptor (CD163) on the surface of mononuclear cells and macrophages, and then is degraded by phagocytosis, thereby removing free hemoglobin in the blood circulation. The clinical determination of haptoglobin is mainly used for diagnosing hemolytic anemia. The haptoglobin content of various hemolytic anemias is significantly reduced, even to an undetectable extent. In mild hemolysis, the plasma is cleared of all free hemoglobin bound to haptoglobin, and no free hemoglobin is detected in the plasma, only a reduction in haptoglobin is seen. When the amount of free hemoglobin exceeds the haptoglobin binding capacity, it is detected. Therefore, haptoglobin can be used as a sensitive index for early prompting occult blood.
The clinical detection mode is generally adopted, and hemoglobin-transferrin joint detection kits, hemoglobin-haptoglobin joint detection kits and the like are available in the market at present. Such as: the invention patent CN101965515A provides a method and device for detecting occult blood, comprising detecting transferrin (Tf) and hemoglobin (Hb) in a GI sample, and comparing the amounts of transferrin and hemoglobin detected with respective predetermined values of transferrin and hemoglobin, and providing for the detection of transferrin and hemoglobin using a lateral flow sandwich immunoassay device, which solution lacks haptoglobin detection. The invention patent CN111879924A provides a colloidal gold immunochromatographic test paper for quickly diagnosing hemoglobin and combining a globin-hemoglobin compound and a preparation method thereof, and the technical scheme is lack of detection of transferrin and can not detect free haptoglobin. In addition to hemoglobin, transferrin, and haptoglobin can be found in feces when bleeding occurs in the digestive tract. Haptoglobin enters the digestive tract only when the digestive tract bleeds, and forms a haptoglobin-hemoglobin complex when being combined with hemoglobin.
The addition of haptoglobin is a non-negligible boost for both of the first two detection methods: the small amount of bleeding and stool combined with globin formed a complex, while the anti-hemoglobin antibody could not recognize the complex, and the test result was negative. The detection of the haptoglobin can not only identify the free haptoglobin in the digestive tract, but also identify a haptoglobin-hemoglobin compound, effectively avoid the leakage detection of trace occult blood, and greatly improve the sensitivity of occult blood detection.
CN111879924A uses haptoglobin-hemoglobin complex as an index of gastrointestinal bleeding, and the structure of the complex is shown in fig. 1. Since the original epitopes c and d are covered after the formation of the complex, epitope b is also covered in a large part, and only epitope a is completely exposed for recognition by the antibody. Therefore, the epitope recognized by the antibody against the complex is mainly a and secondarily b, and is very different from the epitope of free haptoglobin (haptoglobin), and thus free haptoglobin cannot be recognized. According to the invention, the detection of the index (the conjugated globin-hemoglobin compound) is to adopt an anti-haptoglobin antibody and an anti-compound antibody for sandwich capture, assist in gastrointestinal hemorrhage detection, select an antibody pair for specifically recognizing the conjugated globin and the recognition compound, and cannot recognize free conjugated globin. When hemoglobin is destroyed or the content is extremely low, haptoglobin in excrement cannot be detected even if bleeding occurs, and detection is also missed to a certain extent even if the haptoglobin is jointly detected with hemoglobin and transferrin.
None of the products referred to in the above patents are scored with mixed antibodies non-collinear, but rather individually.
Disclosure of Invention
In order to discover digestive tract diseases as early as possible and detect fecal occult blood with high sensitivity, a detection reagent and a detection method which can detect free haptoglobin, hemoglobin and transferrin in human fecal samples and can detect related compounds are needed, and the invention solves the problems.
On one hand, the invention discloses a haptoglobin, hemoglobin and transferrin collinear detection test strip, which comprises a gold-labeled pad and a detection pad overlapped and connected with the end part of the gold-labeled pad; the detection pad is provided with a detection line and a quality control line; the detection line is coated with the mixed antibody; the mixed antibody comprises a first anti-human haptoglobin monoclonal antibody, a first anti-human hemoglobin monoclonal antibody and a first anti-human transferrin monoclonal antibody; the quality control line is coated with a quality control antibody; the gold label pad is coated with a colloidal gold labeled antibody; the colloidal gold labeled antibody comprises a colloidal gold labeled second anti-human haptoglobin monoclonal antibody, a colloidal gold labeled second anti-human hemoglobin monoclonal antibody and a colloidal gold labeled second anti-human transferrin monoclonal antibody;
the first anti-human haptoglobin monoclonal antibody and the second anti-human haptoglobin monoclonal antibody marked by colloidal gold are respectively combined with different antigen epitopes of haptoglobin; the first antihuman hemoglobin monoclonal antibody and the second antihuman hemoglobin monoclonal antibody marked by colloidal gold are respectively combined with different antigen epitopes of hemoglobin; the first anti-human transferrin monoclonal antibody and the second anti-human transferrin monoclonal antibody marked by colloidal gold are respectively combined with different antigen epitopes of transferrin;
the colloidal gold-labeled antibody can also bind to the quality control antibody.
In some embodiments, the quality control antibody is an anti-mouse IgG; the colloidal gold labeled antibodies all belong to murine anti-human monoclonal antibodies. Further, the mixed antibodies are all murine anti-human monoclonal antibodies. Further, the anti-mouse IgG is selected from one or more of goat anti-mouse IgG, rabbit anti-mouse IgG and horse anti-mouse IgG.
In some embodiments, the detection pad is prepared by the following method: immersing the nitrocellulose membrane in 4-6% BSA solution, coating the dried nitrocellulose membrane with a mixed antibody to form a detection line, and coating a quality control antibody on the detection line to form a quality control line; and a gap exists between the detection line and the quality control line.
Further, the detection pad is prepared by the following method: immersing the nitrocellulose membrane in a 5% BSA solution, coating the dried nitrocellulose membrane with a mixed antibody to form a detection line, and coating a quality control antibody on the detection line to form a quality control line; and a gap exists between the detection line and the quality control line.
In some embodiments, the mixed antibody is diluted to a set concentration by phosphate coating diluent No. 4 and then coated on a detection pad to form a detection line;
the formulation for preparing 1L of phosphate coated diluent No. 4 is as follows: na (Na)2HPO4 0.23g、NaH2PO41.15g, NaCl 9g, sucrose 30g, Proclin 3000.4 ml, BSA 1g and S91.041g, adjusting the pH to 7.45, and adding water to 1L.
Further, the pH was adjusted by hydrochloric acid and NaOH.
In some embodiments, the concentration of the first anti-human hemoglobin monoclonal antibody, the first anti-human transferrin monoclonal antibody, and the first anti-human haptoglobin monoclonal antibody is each independently 0.1-0.17 mg/mL.
In some embodiments, the mixed antibody is obtained by:
respectively diluting the concentrations of the first anti-human hemoglobin monoclonal antibody, the first anti-human transferrin monoclonal antibody and the first anti-human haptoglobin monoclonal antibody to 0.5mg/mL, 0.3mg/mL and 0.3 mg/mL; and then mixing the first anti-human hemoglobin monoclonal antibody, the first anti-human transferrin monoclonal antibody and the first anti-human haptoglobin monoclonal antibody in equal volume to obtain a mixed antibody.
In some embodiments, further comprising a sample pad, absorbent pad, and a base plate; the sample pad, the gold mark pad, the detection pad and the water absorption pad are sequentially connected on the bottom plate in an overlapping way at the end parts; the detection line is closer to the gold pad than the quality control line. The detection sample is feces or feces diluent.
In a second aspect, the invention discloses a method for preparing the above haptoglobin, hemoglobin and transferrin collinear detection test strip, which comprises the following steps:
step one, immersing a nitrocellulose membrane into a 5% BSA solution, and drying to obtain a pretreated NC membrane;
and secondly, scribing on the pre-treated NC membrane, namely coating the mixed antibody on the pre-treated NC membrane to form a detection line, and coating the quality control antibody on the pre-treated NC membrane to form a quality control line to obtain the detection pad. And a gap exists between the detection line and the quality control line.
In some embodiments, in the second step, the mixed antibody is diluted to a set concentration by phosphate coating diluent No. 4 and then coated to the detection line;
the formulation for preparing 1L of phosphate coated diluent No. 4 is as follows: na (Na)2HPO4 0.23g、NaH2PO41.15g, NaCl 9g, sucrose 30g, Proclin 3000.4 ml, BSA 1g and S91.041g, adjusting the pH to 7.45, and adding water to 1L.
Further, the pH was adjusted by hydrochloric acid and NaOH.
In a third aspect, the invention discloses a detection card, which comprises the haptoglobin, hemoglobin and transferrin collinear detection test strip and a card box, wherein the card box is used for containing the haptoglobin, hemoglobin and transferrin collinear detection test strip;
the card box is provided with an observation window and a sample adding hole; the position of the observation window corresponds to the positions of the detection line and the quality control line; the position of the sample adding hole corresponds to the position of a sample pad of the haptoglobin, hemoglobin and transferrin collinear detection test strip.
In some embodiments, a two-dimensional code is also provided on the cartridge for tracking, anti-counterfeiting and/or sampling the exemplary video link.
In some embodiments, an information entry area is also provided on the cartridge for entering information, such as a number (ID) and DATE (DATE).
In a fourth aspect, the invention discloses a kit, which comprises the haptoglobin, hemoglobin and transferrin collinear detection test strip.
The test strip, the test card and the kit adopt the immunochromatography technical principle, the first anti-human hemoglobin monoclonal antibody, the first anti-human transferrin monoclonal antibody and the first anti-human haptoglobin monoclonal antibody with different concentrations are mixed and coated at the position of a test line (T line) on an NC membrane according to a certain proportion through repeated tests, a second anti-human hemoglobin monoclonal antibody, a second anti-human transferrin monoclonal antibody and a second anti-human haptoglobin monoclonal antibody are marked on a gold pad by a colloidal gold solution according to a certain proportion through continuous tests, bleeding markers in excrement are detected by adopting a double-antibody sandwich method, whether the hemoglobin, the transferrin or the haptoglobin exist, and when any one marker is positive, the bleeding markers (the hemoglobin, the transferrin or the haptoglobin) are combined with the corresponding monoclonal antibody marked by the colloidal gold, the chromatography moves forward along the paper strip and passes through the mixed antibody pre-fixed on the nitrocellulose membrane, so that a red strip is displayed at the detection line, and the free colloidal gold labeled antibody is combined with goat anti-mouse IgG at the quality control line, so that a red strip is displayed at the quality control line. Samples with all 3 markers negative show only red bands at the control line.
The research and development of three-in-one (hemoglobin, transferrin or haptoglobin) detection test strips, detection cards and kits are finally completed by debugging and improving process parameters in multiple aspects such as the formula of a coating solution, the formula of a marking solution and the like of an NC membrane, the monoclonal antibodies corresponding to the three proteins are coated on a colloidal gold combination pad together, the diagnosis of gastrointestinal hemorrhage is assisted by a method of coating the monoclonal antibodies on the same detection line together, the detection rate of colorectal cancer is effectively improved, and the five-year survival rate of colorectal cancer patients is improved.
The conception, the specific structure and the technical effects of the present invention will be further described with reference to the accompanying drawings to fully understand the objects, the features and the effects of the present invention.
Drawings
FIG. 1 is a schematic representation of the structure of the haptoglobin-hemoglobin complex.
Fig. 2 is a schematic structural diagram of an embodiment of the test strip according to the present invention.
Fig. 3 is a schematic structural diagram of an embodiment of the detection card according to the present invention.
Fig. 4 is a schematic structural diagram of another embodiment of the test strip according to the present invention.
Fig. 5 is a schematic structural diagram of another embodiment of the detection card according to the present invention.
Detailed Description
In order to make the technical means, the characteristics, the purposes and the functions of the invention easy to understand, the invention is further described with reference to the specific drawings. However, the present invention is not limited to the following embodiments.
It should be understood that the structures, ratios, sizes, and the like shown in the drawings and described in the specification are only used for matching with the disclosure of the specification, so as to be understood and read by those skilled in the art, and are not used to limit the conditions under which the present invention can be implemented, so that the present invention has no technical significance, and any structural modification, ratio relationship change, or size adjustment should still fall within the scope of the present invention without affecting the efficacy and the achievable purpose of the present invention.
Example 1
Fig. 2 shows a specific embodiment of the test strip for collinear detection of haptoglobin, hemoglobin and transferrin according to the present invention. In this embodiment, the haptoglobin, hemoglobin and transferrin collinear detection test strip comprises a base plate 7, a sample pad 1, a gold-labeled pad 2, a detection pad 5 and an absorbent pad 6. The sample pad 1, the gold label pad 2, the detection pad 5 and the absorbent pad 6 are sequentially overlapped and connected at the end parts on the bottom plate 7. As shown in fig. 2, one end of the sample pad 1 is pressed against one end of the gold label pad 2, and the other end of the gold label pad 2 is pressed against one end of the detection pad 5; one end of the absorbent pad presses against the other end of the detection pad 5. The detection pad 5 is provided with a detection line (T)3 and a quality control line (C) 4. The detection line (T)3 is closer to the gold pad 2 than the quality control line (C) 4.
The bottom plate 7 is made of PVC or PS material; the material of the detection pad 5 is nitrocellulose membrane.
The detection line (T)3 is coated with a mixed antibody; the quality control line (C)4 is coated with a goat anti-mouse IgG antibody; the gold-labeled pad 2 is coated with a colloidal gold-labeled antibody.
The mixed antibody comprises a first anti-human haptoglobin monoclonal antibody, a first anti-human hemoglobin monoclonal antibody and a first anti-human transferrin monoclonal antibody; the colloidal gold labeled antibody comprises a colloidal gold labeled second anti-human haptoglobin monoclonal antibody, a colloidal gold labeled second anti-human hemoglobin monoclonal antibody and a colloidal gold labeled second anti-human transferrin monoclonal antibody. The first anti-human haptoglobin monoclonal antibody and the second anti-human haptoglobin monoclonal antibody marked by colloidal gold are respectively combined with different antigen epitopes of haptoglobin; the first antihuman hemoglobin monoclonal antibody and the second antihuman hemoglobin monoclonal antibody marked by colloidal gold are respectively combined with different antigen epitopes of hemoglobin; the first anti-human transferrin monoclonal antibody and the second anti-human transferrin monoclonal antibody labeled by colloidal gold are respectively combined with different antigen epitopes of transferrin. The colloidal gold labeled antibodies all belong to mouse anti-human monoclonal antibodies and can be combined with goat anti-mouse IgG.
Example 2
The preparation method of the test strip specifically comprises the following steps:
1) pretreating an NC membrane: immersing a nitrocellulose membrane (NC membrane) into a 5% BSA solution, taking out and drying for later use.
2) Preparing a detection line T and a quality control line C: diluting a mouse anti-human hemoglobin monoclonal antibody 1 (namely a first anti-human hemoglobin monoclonal antibody), a mouse anti-human transferrin monoclonal antibody 1 (namely a first anti-human transferrin monoclonal antibody), a mouse anti-human haptoglobin monoclonal antibody 1 (namely a first anti-human haptoglobin monoclonal antibody) (3 antibodies are purchased from NORDIC LIFE, the goods numbers are 10081, 10079 and 10077 respectively) to 0.3-0.5mg/mL by using a phosphate coating diluent No. 4 (the formula is shown in a table 1), spraying the mixture on a cellulose membrane at the speed of 1.5ul/cm to serve as a detection line T; diluting goat anti-mouse IgG antibody (purchased from Hangzhou Bo Ying biology, product number P200201), spraying on a nitrocellulose membrane to serve as a 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 1, the mouse anti-human transferrin monoclonal antibody 1 and the mouse anti-human haptoglobin monoclonal antibody 1 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.
TABLE 1 formulation No. 4 phosphate coated Diluent
3) Preparing a gold label pad: diluting the purified colloidal gold-labeled antibody according to the ratio of 1:5, coating the antibody on a glass fiber pad, and treating the glass fiber pad in a freeze dryer to obtain the gold-labeled pad.
The preparation process of the purified colloidal gold-labeled antibody comprises the following steps:
A. obtaining colloidal gold: a certain amount of ultrapure water is added into a glass container, stirred and heated. After the ultrapure water is boiled, a certain amount of 1% tetrachloroauric acid is added, and after the ultrapure water is boiled again, a certain amount of 2% trisodium citrate is added. After about 3 minutes the heat was adjusted to slightly boil. The time was counted from the addition of the tetrachloroauric acid and boiled for about 15 minutes. And after the time is up, the liquid is cooled at room temperature, the liquid is clear transparent wine red, and the colloidal gold particles have a maximum absorption peak at 522nm and the particle size is 40 nm.
B1, colloidal gold labeled secondary anti-human hemoglobin monoclonal antibody: with 0.1mol/L of K2CO3The pH value of the colloidal gold is adjusted to 8.2 by the solution, a mouse anti-human hemoglobin monoclonal antibody 2 (purchased from NORDIC LIFE, the product number is 10082) with the concentration of 2mg/ml is added into the colloidal gold after the pH value is adjusted, the volume ratio of the mouse anti-human hemoglobin antibody 2 to the colloidal gold after the pH value is adjusted is 1:1, and the labeling is carried out for 30min, so as to obtain the mouse anti-human hemoglobin monoclonal antibody 2-colloidal gold conjugate (namely the second anti-human hemoglobin monoclonal antibody marked by the colloidal gold).
B2, colloidal gold labeled secondary anti-human transferrin monoclonal antibody: with 0.1mol/L of K2CO3The pH value of the colloidal gold is adjusted to 8.2 by the solution, a labeled mouse anti-human transferrin monoclonal antibody 2 (purchased from NORDIC LIFE, the product number is 10080) with the concentration of 4mg/ml is added into the colloidal gold after the pH value is adjusted, the volume ratio of the labeled mouse anti-human transferrin antibody 2 to the colloidal gold after the pH value is adjusted is 1:1, and the labeling is carried out for 30min, so as to obtain a mouse anti-human transferrin monoclonal antibody 2-colloidal gold conjugate (namely a colloidal gold labeled second anti-human transferrin monoclonal antibody).
B3, colloidal gold labeled secondary anti-human haptoglobin monoclonal antibody: the pH of the colloidal gold was adjusted to 8.2 with 0.1mol/L K2 CO3 solution, and 2mg/ml labeled mouse anti-human haptoglobin monoclonal antibody 2 (purchased from NORDIC LIFE, cat # 10078) was added to the pH-adjusted colloidal gold, and the volume ratio of the labeled mouse anti-human haptoglobin monoclonal antibody 2 to the pH-adjusted colloidal gold was 1:1, followed by labeling for 30min, to obtain a mouse anti-human haptoglobin monoclonal antibody 2-colloidal gold conjugate (i.e., a colloidal gold-labeled secondary anti-human haptoglobin monoclonal antibody).
C. Purified colloidal gold labeled 3 antibodies: and D, respectively adding 10 mass percent of Bovine Serum Albumin (BSA) aqueous solution into the solutions of the colloidal gold-labeled second anti-human haptoglobin monoclonal antibody, the colloidal gold-labeled second anti-human hemoglobin monoclonal antibody and the colloidal gold-labeled second anti-human transferrin monoclonal antibody obtained in the steps B1-B3 until the final concentration of BSA is 0.5%, stopping adding, continuously stirring for 25min, centrifuging at 14000rpm for 30min, discarding the supernatant, and adding a casein sodium solution accounting for 25 percent of the total volume of the markers (precipitates after centrifugation), thereby obtaining the purified 3 antibodies labeled with the colloidal gold.
D. The purified 3 kinds of colloidal gold-labeled antibodies were mixed at an equivalent ratio of 1:1:1 and used.
4) Preparing detection test paper: and (3) connecting and assembling the water absorption pad, the sample pad, the gold label pad, the nitrocellulose membrane (namely the detection pad) coated with the antibody and the bottom plate in sequence to obtain the test strip.
Example 3
In order to realize that 3 detection lines can be scribed on one line, the process needs to be searched, the sensitivity is ensured, and the specificity cannot be influenced, and the detection method is mainly realized by researching the formula of the coated diluent, the concentration and mutual proportion of 3 antibodies, membrane treatment and the like.
1. By adjusting different coating conditions (1-4) of 3 monoclonal antibodies on the detection line, the detection effect of different samples (1# -8#) is observed, the absorbance reflects the content of the marker, and the higher the value is, the more targets are combined with the marker. The detailed procedure is shown in test example 1.
2. The effect of the detection was observed for different samples (1# -8#) by pre-treating the NC films using different blocking conditions. The detailed procedure is shown in test example 2.
3. The detection effect on different samples (1# -8#) is observed by studying the formulation of the coated diluent. The detailed procedure is shown in test example 3.
The serial numbers 1-8 (i.e. 1# -8#) respectively represent samples with different contents of hemoglobin (Hb), transferrin (Tf) and haptoglobin (Hp) in the feces, and the positivity of the 3 markers represents that the contents in the samples are respectively (200ng/mL, 40ng/mL and 4 ng/mL); negative indicates the content is 0, if the 1# sample indicates that the 3 markers are positive, the content in the sample is (200ng/mL, 40ng/mL and 4ng/mL), and the 8# sample indicates that the sample does not contain any marker; a positive detection of any of the 3 markers is positive for the sample.
Test example 1
Different coating conditions of 3 monoclonal antibodies on the detection line are Hb: tf: hp ═ 0.3:0.3:0.3mg/mL, Hb: tf: hp ═ 0.5:0.3:0.3mg/mL, Hb: tf: hp ═ 0.5:0.5:0.3mg/mL, Hb: tf: hp is 0.5:0.5:0.5mg/mL, it is observed that for different samples (1# -8#) the test results are shown in table 2, the absorbance reflects the content of the marker, the higher the value, the more the target substance is bound to, the test result of the 1# sample should be as high as possible, the test result of the 8# sample should be as low as possible, the ratio of the test results of the 1# sample to the 8# sample is taken as the evaluation criterion, the 4 method ratios are (7.8, 7.5, 3.7 and 3.1), the test values of the 2# and 5# samples in the first method are obviously reduced, and Hb omission may exist, and the 2 nd coating condition is selected as Hb: tf: hp ═ 0.5:0.3:0.3mg/mL was used as the kit coating conditions. In test example 1, the ratio of 3 monoclonal antibodies (Hb monoclonal antibody: Tf monoclonal antibody: Hp monoclonal antibody) was expressed directly by the corresponding antigen protein, that is, Hb: tf: hp is measured.
Table 2, results of detection of different coating conditions of 3 monoclonal antibodies on detection line
Test example 2
The pretreatment of the NC membrane can realize the effects of improving the sensitivity and reducing the non-specificity. And observing the detection results of the detection cards made of the NC films processed under different closed conditions for different samples (1# -8#), and finding out a proper film processing method. As a result, as shown in Table 3, the absorbance reflects the content of the marker, the higher the value, the more the target substance is bound to, the test result of the sample No. 1 should be as high as possible, the test result of the sample No. 8 should be as low as possible, and the test value of the sample No. 2-7 should not be too low, so that the test effect can be effectively improved by selecting 5% BSA as the pretreatment condition, immersing the NC membrane in 5% BSA solution, taking out and drying the NC membrane.
TABLE 3 comparison of pretreatment results of NC membranes
Test example 3
The optimal formula is determined by adding different surfactants or high molecular polymers into the basic phosphate coating diluent, and the detection effect of the prepared detection card on different samples (1# -8#) is observed. Tween, triton, S9 and sucrose are selected as additives, the detection results are shown in Table 4, the absorbance reflects the content of the marker, the higher the value is, the more the target substance is combined with the marker, the detection result of the sample No. 1 is as high as possible, the detection result of the sample No. 8 is as low as possible, meanwhile, the detection value of the sample No. 2-7 is not too low, and the detection effect can be effectively improved by preferably adding 0.1% of S9 into phosphate buffer.
TABLE 4 Effect of different surfactants or high molecular weight polymers in phosphate-coated dilutions on assay results
Example 4
The test strip described in example 1 or the test strip obtained in example 2 is put into a cartridge to obtain a test card, as shown in fig. 3. The test card includes a cartridge 20 and a test strip. The cartridge 20 is provided with a viewing window 21 and a loading hole 22. The position of the observation window 21 corresponds to the positions of the detection line (T) and the quality control line (C). The position of the sample adding hole 22 corresponds to the position of the sample pad of the test strip for collinear detection of haptoglobin, hemoglobin and transferrin. The card box is also provided with a two-dimensional code and an information input area. And the information entry area is used for entering information and contains a number (ID) and a DATE (DATE).
Example 5
FIG. 4 shows the working principle of the test strip according to the present invention; fig. 5 shows a test card according to the invention.
As shown in fig. 4, the test strip according to the present invention comprises a base plate 7, here a PVC or PS base plate, for supporting the assembly of 4 kinds of functional pads. The 4 kinds of functional pads are a sample pad 1, a gold label pad 2, a detection pad 5 and a water absorption pad 6, respectively. The sample pad 1 is used to receive a sample. The gold pad 2 serves to adsorb the marker and uniformly release the marker. The detection pad 5 is marked with a detection line (T)3 formed by coating 3 monoclonal antibodies and a quality control line (C)4 formed by coating quality control monoclonal antibodies. After the sample 30 is added to the sample pad 1, it passes through the gold label pad 2 and the detection pad 5 in sequence along the sample pad 1, and finally reaches the absorbent pad 6, completing the whole chromatography process. If the sample 30 contains haptoglobin, hemoglobin and/or transferrin, the colloidal gold aggregation coloration occurs at the detection line 3, and the colloidal gold aggregation coloration also occurs at the quality control line 4. If the sample 30 does not contain haptoglobin, hemoglobin and transferrin, the colloidal gold aggregation does not occur at the detection line 3, the detection line 3 does not develop color, but the colloidal gold aggregation and color development occurs at the quality control line 4. If no color is developed at the position of the quality control line 4 after the detection chromatography of the sample 30 is finished, the detection result is not reliable.
A nitrocellulose membrane is taken as a material, and a first anti-human haptoglobin monoclonal antibody, a first anti-human hemoglobin monoclonal antibody and a first anti-human transferrin monoclonal antibody are coated on the nitrocellulose membrane to form a detection line; meanwhile, a quality control antibody goat anti-mouse IgG is coated on the detection pad to form a quality control line, and the detection pad is obtained. The first anti-human haptoglobin monoclonal antibody, the first anti-human hemoglobin monoclonal antibody and the first anti-human transferrin monoclonal antibody belong to mouse anti-human monoclonal antibodies.
The glass fiber membrane is used as a material, and a second anti-human haptoglobin monoclonal antibody marked by colloidal gold, a second anti-human hemoglobin monoclonal antibody marked by colloidal gold and a second anti-human transferrin monoclonal antibody marked by colloidal gold are uniformly coated on the glass fiber membrane to obtain the gold-labeled pad. The second anti-human haptoglobin monoclonal antibody, the second anti-human hemoglobin monoclonal antibody and the second anti-human transferrin monoclonal antibody belong to mouse anti-human monoclonal antibodies. The second anti-human haptoglobin monoclonal antibody and the first anti-human haptoglobin monoclonal antibody can be respectively combined on different antigen epitopes of haptoglobin; the second anti-human hemoglobin monoclonal antibody and the first anti-human hemoglobin monoclonal antibody can be respectively combined on different antigen epitopes of hemoglobin; the second anti-human transferrin monoclonal antibody and the first anti-human transferrin monoclonal antibody can be respectively combined on different antigen epitopes of transferrin. The second anti-human haptoglobin monoclonal antibody, the second anti-human hemoglobin monoclonal antibody and the second anti-human transferrin monoclonal antibody can be combined with a quality control antibody, namely goat anti-mouse IgG.
The test card shown in fig. 5 includes a cartridge and a test strip. The cartridge includes a loading hole (S)22 and a viewing window 21. The test line (T line) and the quality control line (C line) of the test strip are visible through the observation window 21. Wherein the detection line is coated with mouse anti-Hb (first mouse anti-human hemoglobin monoclonal antibody), mouse anti-Tf (first mouse anti-human transferrin monoclonal antibody) and mouse anti-Hp (first mouse anti-human haptoglobin monoclonal antibody); the quality control line is coated with goat anti-mouse (goat anti-mouse IgG).
The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.
Claims (11)
1. A haptoglobin, hemoglobin and transferrin collinear detection test strip is characterized by comprising a gold-labeled pad and a detection pad overlapped and connected with the end part of the gold-labeled pad; the detection pad is provided with a detection line and a quality control line; the detection line is coated with a mixed antibody; the mixed antibody comprises a first anti-human haptoglobin monoclonal antibody, a first anti-human hemoglobin monoclonal antibody and a first anti-human transferrin monoclonal antibody; the quality control line is coated with a quality control antibody; the gold label pad is coated with a colloidal gold labeled antibody; the colloidal gold labeled antibody comprises a colloidal gold labeled second anti-human haptoglobin monoclonal antibody, a colloidal gold labeled second anti-human hemoglobin monoclonal antibody and a colloidal gold labeled second anti-human transferrin monoclonal antibody;
the first anti-human haptoglobin monoclonal antibody and the second anti-human haptoglobin monoclonal antibody marked by the colloidal gold are respectively combined with different antigen epitopes of haptoglobin; the first anti-human hemoglobin monoclonal antibody and the second anti-human hemoglobin monoclonal antibody marked by the colloidal gold are respectively combined with different antigen epitopes of hemoglobin; the first anti-human transferrin monoclonal antibody and the second anti-human transferrin monoclonal antibody marked by the colloidal gold are respectively combined with different antigen epitopes of transferrin;
the colloidal gold-labeled antibody is also capable of binding to the quality control antibody.
2. The haptoglobin, hemoglobin and transferrin collinear detection test strip of claim 1, wherein the quality control antibody is anti-mouse IgG; the colloidal gold labeled antibodies all belong to mouse anti-human monoclonal antibodies.
3. The haptoglobin, hemoglobin and transferrin collinear detection test strip of claim 1, wherein the detection pad is prepared by the method comprising the following steps of: immersing a nitrocellulose membrane into 4% -6% BSA solution, coating the mixed antibody on the nitrocellulose membrane after drying to form the detection line, and coating the quality control antibody on the detection line to form the quality control line; and a gap exists between the detection line and the quality control line.
4. The haptoglobin, hemoglobin and transferrin collinear detection test strip of claim 1, wherein the mixed antibody is diluted to a set concentration by phosphate coating diluent No. 4 and then coated on the detection pad to form the detection line;
the formula for preparing 1L of the phosphate coated diluent No. 4 is as follows: na (Na)2HPO4 0.23g、NaH2PO41.15g、NaCl 9g30g of sucrose, 3000.4 ml of Proclin, 1g of BSA and 1g of S91.041g, adjusting the pH to 7.45, and adding water to 1L.
5. The haptoglobin, hemoglobin and transferrin collinear detection test strip of claim 1, wherein the concentrations of said first anti-human hemoglobin monoclonal antibody, said first anti-human transferrin monoclonal antibody and said first anti-human haptoglobin monoclonal antibody are each independently 0.1-0.17 mg/mL.
6. The haptoglobin, hemoglobin and transferrin collinear detection test strip of claim 1, wherein said mixed antibody is obtained by:
respectively diluting the concentrations of the first anti-human hemoglobin monoclonal antibody, the first anti-human transferrin monoclonal antibody and the first anti-human haptoglobin monoclonal antibody to 0.5mg/mL, 0.3mg/mL and 0.3 mg/mL; and then mixing the first anti-human hemoglobin monoclonal antibody, the first anti-human transferrin monoclonal antibody and the first anti-human haptoglobin monoclonal antibody in an equal volume to obtain the mixed antibody.
7. The haptoglobin, hemoglobin and transferrin collinear detection test strip of claim 1, further comprising a sample pad, a water absorbent pad and a bottom plate; the sample pad, the gold mark pad, the detection pad and the water absorption pad are sequentially connected and arranged on the bottom plate in an overlapping way at the end parts; the detection line is closer to the gold pad than the quality control line.
8. The method for preparing the haptoglobin, hemoglobin and transferrin collinear detection test strip according to claim 1, which comprises the following steps:
step one, immersing a nitrocellulose membrane into a 5% BSA solution, and drying to obtain a pretreated NC membrane;
and secondly, scribing on the pre-treated NC membrane, namely coating the mixed antibody on the pre-treated NC membrane to form the detection line, and coating the quality control antibody on the pre-treated NC membrane to form the quality control line to obtain the detection pad.
9. The method for preparing a haptoglobin, hemoglobin and transferrin collinear detection test strip according to claim 8, wherein in the second step, the mixed antibody is diluted to a set concentration by phosphate coated diluent No. 4 and then coated at the detection line;
the formula for preparing 1L of the phosphate coated diluent No. 4 is as follows: na (Na)2HPO4 0.23g、NaH2PO41.15g, NaCl 9g, sucrose 30g, Proclin 3000.4 ml, BSA 1g and S91.041g, adjusting the pH to 7.45, and adding water to 1L.
10. A test card comprising the haptoglobin, hemoglobin and transferrin collinear test strip and cartridge of claim 1 for containing said haptoglobin, hemoglobin and transferrin collinear test strip;
the card box is provided with an observation window and a sample adding hole; the position of the observation window corresponds to the positions of the detection line and the quality control line; the position of the sample adding hole corresponds to the position of a sample pad of the haptoglobin, the hemoglobin and the transferrin collinear detection test strip.
11. A kit comprising the haptoglobin, hemoglobin and transferrin collinear detection test strip of claim 1.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN117517680A (en) * | 2024-01-05 | 2024-02-06 | 济南玖方生物科技有限公司 | Method for improving detection accuracy of hemoglobin and transferrin duplex detection colloidal gold immunochromatography kit in feces |
| CN117741165A (en) * | 2023-11-29 | 2024-03-22 | 浙江鼎创医疗科技有限公司 | Colloidal gold immunochromatographic test strip for high-specificity and high-sensitivity HNL detection and preparation method and use method thereof |
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