Test strip for testing renal function of nano antibody

Abstract

The invention provides a technical scheme for constructing a nano-antibody renal function test strip by adopting a nano-antibody. The test strip can be used for conveniently collecting a random urine sample and detecting the amount of human serum albumin in the sample by using an immunochromatography method so as to realize rapid detection of the renal function. According to the technical scheme, a double-antibody sandwich method and an antibody competition method are applied at the same time, the albumin recognition capacity can be improved, false positives are reduced, larger relative color intensity change exists when positive and negative are distinguished, and accurate interpretation of results is facilitated.

Description

Technical Field

[0001] The present invention relates to the field of antibody technology and biomedicine, and in particular to the application of nano antibodies in detection, diagnosis and treatment. Background Art

[0002] Antibodies have the ability to specifically bind to antigens and can recognize different proteins, including human serum albumin (HSA). They are widely used in the field of biomedicine, and there is no shortage of antibodies against HSA on the market. In terms of detection, food safety supervision requires timely detection of pesticide residues, illegal additives, etc. Various rapid test strips based on the principle of immunochromatography have played a huge role in food supervision due to their simplicity, convenience, rapidity and low cost. In clinical diagnosis, immunoassay is an important detection technology. A specific example is the widely used new crown antigen test, which uses two antibodies against the nucleoprotein (N protein) of the new crown virus and adopts the double antibody sandwich method to prepare an immunochromatographic rapid test strip to qualitatively or quantitatively detect the presence of the new crown virus. In terms of therapeutic drugs, antibodies have become an important type of drug. Not only can antibodies themselves be used as drugs, but antibody drugs also include antibody-derived antibody-toxin protein conjugates (immunotoxin), antibody-drug conjugates (ADC), bispecific antibodies and multi-specific antibodies, etc.

[0003] Antibodies are divided into polyclonal antibodies and monoclonal antibodies. Polyclonal antibodies are produced by immunized animals and are a mixture of antibodies. The antibody composition and titer of each batch are different. Monoclonal antibodies are produced by a single gene, which overcomes the problem of batch-to-batch differences of polyclonal antibodies. However, monoclonal antibodies have the following characteristics and shortcomings: long R&D cycle, long production cycle, high preparation cost, difficult to be cleared in the body, and weak penetration ability.

[0004] We refer to common polyclonal antibodies and monoclonal antibodies as conventional antibodies. Nanobodies are simplified forms of conventional antibodies. In the early days, a rare antibody with only heavy chains was found in the antibodies of animals such as alpacas. By retaining only a single domain that binds to the antigen, a nanobody with a size of nanometers was constructed, also known as a single domain antibody. Nanobodies retain the high affinity and high specificity of conventional antibodies to antigens, and have the following advantages: high stability of a single domain; ability to be efficiently expressed in prokaryotic or eukaryotic cells; short growth cycle and low preparation cost when expressed in prokaryotic cells; weak immunogenicity to humans; small molecular weight and strong penetration ability, and can even penetrate the blood-brain barrier. Nanobodies have been increasingly widely used in biomedicine, such as the approved bispecific nanoantibody Ozoralizumab, which uses the high affinity of anti-human serum albumin nanoantibodies to albumin to prolong the retention time in the body, and uses another fused anti-tumor necrosis factor TNFα nanoantibody to target drug action molecules for the treatment of rheumatoid arthritis.

[0005] Human serum albumin is abundant in human blood, with a concentration of about 42 g / L, accounting for about 60% of the total blood protein. In the blood, human serum albumin can bind to various molecules and ions (such as fatty acids, bile pigments, amino acids, steroid hormones, metal ions, drug molecules, etc.), while maintaining normal blood osmotic pressure. Clinically, human serum albumin can be used to treat shock and burns, to supplement blood loss caused by massive bleeding due to surgery, accidents, etc., and can also be used as a plasma expander. Human serum albumin is a single-chain polypeptide composed of 585 amino acids and a molecular weight of 66 kDa. Based on the large demand for human serum albumin, people have conducted in-depth research on recombinant human serum albumin. At present, there are many recombinant expression systems reported, including Escherichia coli, Pichia pastoris, rice, tobacco chloroplasts, cattle, pigs, etc. Recombinant human serum albumin not only needs high yield and low production cost, but also needs to be consistent with human serum albumin from blood in terms of physiological and biochemical properties, physical structure, biological function, immunogenicity, etc. There is a great market demand for high-purity recombinant human serum albumin products.

[0006] Renal function refers to the function of the kidney to excrete metabolic products in the body and maintain the stability of electrolytes such as sodium, potassium, and calcium in the body and the acid-base balance. Renal function is mainly achieved through glomerular filtration and tubular reabsorption. The glomerulus is a blood filter, which is a filter membrane composed of the walls of tiny arteries. From the inside to the outside, there are three layers of structure: the inner layer is the endothelial cell layer, which is a flat cell attached to the glomerular basement membrane. There are countless small holes of varying sizes on it, and the small holes have a very thin diaphragm; the middle layer is the glomerular basement membrane, which is divided into three layers from the inside to the outside under an electron microscope, namely the inner loose layer, the dense layer and the outer loose layer, which is the main part for controlling the size of filtered molecules; the outer layer is the epithelial cell layer, which is also called podocytes. Its irregular protrusions are called foot processes, and there are many narrow gaps between them. After the blood is filtered through the filter membrane, the filtrate enters the glomerular capsule. Under normal circumstances, human serum albumin and most other proteins in the blood cannot be filtered and remain in the blood. Only small molecules such as urea, glucose, electrolytes and certain small molecules of proteins can be filtered. Therefore, although albumin exists in large quantities in the blood, only trace amounts exist in normal urine. Clinically, the amount of albumin that can be detected in urine is directly related to renal function and is an important indicator for the diagnosis of chronic kidney disease. In urine collected over 24 hours, 30-300 mg of albumin is called microalbuminuria; more than 300 mg is called macroalbuminuria. Summary of the invention

[0007] The present invention provides a technical solution for constructing an immunochromatographic test strip for renal function detection using nano antibodies. The human serum albumin content in urine is an important indicator of renal function. By adopting the technical solution of the present invention, the albumin in urine can be accurately, conveniently and quickly detected using the nano antibody renal function test strip, providing a basis for judging whether the renal function is complete or not.

[0008] The primary purpose of the present invention is to provide a technical solution for constructing an immunochromatographic test strip. The specific binding of antigen and antibody is the key to the immune response. In the technical solution, the antigen is human serum albumin, and the antibody is two anti-human serum albumin nanoantibodies, named nanoantibody A and nanoantibody B respectively. The immunochromatographic test strip is provided with two detection lines, respectively called T1 line and T2 line. Albumin and nanoantibody B are fixed on the T1 line and T2 line, respectively. Therefore, during the detection, at the T1 line, it is a competitive immune reaction, and at the T2 line, it is a double antibody sandwich immune reaction.

[0009] Nanobodies are the characteristic and key material of this technical solution, and have special requirements. First, the binding site. The double antibody sandwich method requires that two nanobodies bind to two independent epitopes of antigen albumin; that is, they can each bind to the antigen, and their respective binding does not affect the binding of the other. Second, specificity. The nanoantibodies used have the ability to specifically recognize antigen albumin, and can distinguish human serum albumin from (1) serum albumin of other species and other proteins with similar structures (such as bovine serum albumin), and (2) other proteins that may appear in urine (such as β2-microglobulin). This technical solution adopts the double antibody sandwich method at the T2 line. The color development of the T2 band is the superposition of the specific antigen recognition ability of the two nanoantibodies, which further enhances the specificity of antigen recognition. Third but more importantly, it is affinity. The requirements for nanoantibodies A and B are appropriate affinity, which can not only effectively capture antigen albumin, or capture the complex of albumin and nanoantibody A, but also produce large color changes near the critical value of detection, at the same time at the T1 line and T2 line.

[0010] Preferably, Nanobody A and Nanobody B are Nanobody 4p and Nanobody 2p, respectively, which we obtained previously (see patent application: Ye Qizhuang et al., application number CN2023105650880).

[0011] Specifically, the structure of the immunochromatographic test strip includes a sample pad, a binding pad, a chromatography pad, an absorption pad, and a bottom plate. The sample pad, the binding pad, the chromatography pad, and the absorption pad are overlapped by 2 mm and pasted on the bottom plate. On the binding pad, a color-labeled nanobody A is adsorbed. The color labeling is for the purpose of facilitating naked eye observation, including the use of colloidal gold, colloidal silver, colloidal carbon, and colored microspheres of various colors. Preferably, colloidal gold is used to color-label the nanobody. The labeled nanobody A is red (or wine red). The binding pad of the nanobody labeled with colloidal gold is also called a gold-labeled pad. On the chromatography pad, two detection lines, T1 and T2 lines, are arranged in parallel; the T1 line contains albumin, and the T2 line contains nanobody B. Preferably, the T1 line is at one end close to the absorption pad, and the T2 line is at one end close to the binding pad. When the color label of the nanobody A uses colloidal gold, the T1 and T2 lines appear as red strips during detection.

[0012] The immunochromatographic test strip constructed according to the technical solution determines the amount of albumin in the sample by comparing the relative strength of the chromaticity of the T1 line and the T2 line when interpreting the results. At the T1 line, antigen albumin is fixed, and the color-labeled nanoantibody A binds to the albumin. The chromaticity of the T1 line decreases as the amount of albumin in the sample increases. At the T2 line, nanoantibody B is fixed, and the color-labeled nanoantibody A indirectly binds to nanoantibody B through the albumin in the sample. The chromaticity of the T2 line increases as the amount of albumin in the sample increases. The critical value for interpretation is set to 20 mg / L, and the preferred detection range is set to 0-320 mg / L. In the sample, when the albumin concentration is around 20 mg / L, the chromaticity of the T1 line and the T2 line are similar; when the albumin concentration is less than 20 mg / L, the chromaticity of the T1 line is stronger than that of the T2 line, which is a negative result; when the albumin concentration is greater than 20 mg / L, the chromaticity of the T1 line is weaker than that of the T2 line, which is a positive result. Since the color development principle at the T2 line is the double antibody sandwich method, there will be a hook effect common to the double antibody sandwich method, so the detection range is set below 320 mg / L. This technical solution actually detects in the sample albumin concentration range of 20 mg / L to 320 mg / L. Even at a high concentration close to 320 mg / L, the chromaticity of the T2 line is weakened, but it can be clearly shown that the chromaticity of the T1 line is weaker than that of the T2 line, which does not affect the determination of the test results. In actual detection, when the sample albumin concentration is as high as 1500 mg / L, the T2 line can still be seen, which is judged as a positive result.

[0013] The immunochromatographic test strip constructed according to the present technical solution is commonly known as a "naked strip", or "strip type". It is simple to manufacture, has low production costs, and is very convenient to use. It is a preferred product packaging form. Another preferred packaging form is to put the test strip into a plastic card shell, which is called an immunochromatographic test strip card, or "card type". The card type and the strip type are the same renal function test products, but different packaging methods; the strip type product is a naked test strip. When testing, the test strip is directly inserted vertically into the urine for sampling; the card type product is when testing, urine is added to the sample addition hole of the test strip card. After sampling, the urine and the color-labeled nanoantibodies are chromatographed together on the test strip to form two strips of different chromaticities at T1 and T2.

[0014] The immunochromatographic test strip or immunochromatographic test card constructed according to the technical solution can be conveniently used to collect random urine samples and detect the amount of albumin in the samples to achieve rapid detection of renal function.

[0015] The present invention uses a novel biomaterial nano-antibody to construct a device that can accurately, conveniently and quickly detect the albumin content in urine and judge whether the kidney function is sound or not. The significant benefits of the present invention include: (1) Stability. Stability is a major advantage of nano-antibodies over conventional antibodies. The immunochromatographic test strip constructed based on the nano-antibody of the present invention has been proven to be able to withstand high temperatures for a long time. The benefit of high stability is that the product is easier to store and transport; (2) Specificity. The present invention uses two nano-antibodies that recognize different epitopes of antigens. The immunochromatographic test strip constructed in this way has been proven to be able to distinguish between human and bovine serum albumin, showing high specificity. The benefit of high specificity is that the product can effectively distinguish albumin from other proteins and reduce false positives; (3) Accuracy. The immunochromatographic test strip constructed based on the nano-antibody of the present invention adopts a method in which the color changes of the two color-developing strips are opposite to each other, which improves the accuracy of the result judgment and makes the result judgment easier. (4) Economic efficiency. Similar immunochromatographic test strip products on the market commonly use monoclonal and polyclonal conventional antibodies produced by animals. This technical solution uses genetically recombinant nanobodies produced by E. coli, which not only reduces the time required for production, but also greatly reduces production costs. (5) Consistency. Nanobodies are genetically engineered products, produced by the expression of the same gene, and are monoclonal with good consistency, which reduces batch-to-batch differences compared to polyclonal antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is the detection specificity of the immunochromatographic test strip.

[0017] Figure 2 It is the color change and hook effect of the immunochromatographic test strip in the positive detection range.

[0018] Figure 3 This is the verification result of the repeatability and detection limit of the immunochromatographic test strip.

[0019] Figure 4 Thermal stability of immunochromatographic test strips. DETAILED DESCRIPTION

[0020] Example 1, solution preparation: 10 mM PBS buffer: weigh 1.44 g Na2HPO4, 0.24 g KH2PO4, 1.8 g K2HPO4, 0.2 g KCl, 7.9 g NaCl, add water to 1 L, stir to dissolve, and measure with a pH meter (pH 7.2-7.4). 50mM Tris buffer: weigh 6.06 g Tris, add water to 1 L, stir to dissolve, measure with a pH meter, add hydrochloric acid to adjust the pH (pH8.0±0.1, about 2.1 mL concentrated hydrochloric acid). Blocking solution (10% BSA): weigh 5 g bovine serum albumin, add 50 mL water, stir to dissolve. Diluent: Add 200 μL isopropanol to 10 mL 10 mM PBS buffer and mix well. Gold-labeled antibody reconstitution solution: weigh 1 g bovine serum albumin and 8 g trehalose, add 100 mL 50 mM Tris buffer, stir to dissolve, add 100 μL Tween-20, and stir to mix.

[0021] Example 2, colloidal gold labeling of nanoantibody 4p: adjust the pH value of colloidal gold with K2CO3 (add 9 μL 0.2 M K2CO3 to every 1 mL of colloidal gold), stir for 5-10 minutes, slowly add nanoantibody 4p to the colloidal gold solution (add 10 μg nanoantibody 2p to every 1 mL of colloidal gold), and stir at low speed for 30 minutes; add blocking solution to a final concentration of 10% (mass percentage), stir for 20 minutes; let stand for 30 minutes and then centrifuge at 12000 rpm for 30 minutes; remove the supernatant and dissolve the precipitate with 70 μL gold-labeled antibody re-solution to obtain colloidal gold-labeled nanoantibody 4p.

[0022] Example 3, preparation of conjugate pad and chromatography pad: Spray the reconstituted colloidal gold-labeled nanobody 4p on the gold label pad (conjugate pad) and dry for use. Dilute human serum albumin to 3.5 mg / mL with diluent, spray at 0.8 µL / cm, and coat at the T1 line position of the nitrocellulose membrane (chromatographic pad). Dilute nanobody 2p to 0.7 mg / mL with diluent, spray at 1.8 µL / cm, coat at the T2 line position of the nitrocellulose membrane, and dry for use.

[0023] Example 4, film pasting, film cutting, and assembly: On a PVC base plate of 80 mm X 300 mm X 0.42 mm with a single-sided adhesive, place the sample pad, gold label pad, nitrocellulose membrane coated with human serum albumin and nanoantibody 4p, and absorption pad in sequence, and there should be a little contact (about 2 mm) between the two, and the end of the nitrocellulose membrane coated with human serum albumin is close to the absorption pad. The assembled large plate is cut into 80 mm X 4 mm immunochromatographic test strips. If you need to prepare an immunochromatographic test paper card, take a 100 mm X 16 mm X 5 mm plastic card shell, put the above 80 mm X 4 mm immunochromatographic test strip into the card shell, and press it to seal it, which is an immunochromatographic test paper card.

[0024] Example 5, assembly of the kit: put the assembled immunochromatographic test strips and desiccant into an aluminum foil bag; or put the immunochromatographic test strip card, straw and desiccant into an aluminum foil bag; seal with a heat sealer and label. According to the finished product specifications, put a certain number of aluminum foil bags, urine cups and instructions into a packaging box, and label the outside of the packaging box.

[0025] Example 6, Method of using the immunochromatographic test strip: Open the outer packaging and take out the immunochromatographic test strip from the sealed aluminum foil bag. Use a urine cup to obtain fresh urine, or add artificial urine to the test sample. Insert the sampling end (sample pad) of the immunochromatographic test strip vertically into the urine, and after 10 seconds, take it out and lay it flat. After 10 minutes, observe the relative color depth of the T1 line and the T2 line. When negative, T1>T2; when near the critical value, T1=T2; when positive, T1 <T2。

[0026] Example 7, verification of detection specificity of immunochromatographic test strips: artificial urine was used to prepare bovine serum albumin (BSA) into samples with concentrations of 100, 200 and 500 mg / L, and these samples were used to test the test strips. The test results showed that ( Figure 1 ), at the above three concentrations of bovine serum albumin samples, the T1 line of the test strip shows an obvious band, and the T2 line does not show a band. Therefore, the immunochromatographic test strip is specific to human serum albumin and can clearly distinguish between human serum albumin and bovine serum albumin.

[0027] Example 8, detection range and hook effect of immunochromatographic test strips: Human serum albumin (HSA) was prepared into samples with concentrations of 0, 20, 40, 80, 160 and 320 mg / L using artificial urine, and the test strips were tested using the above samples. The experimental results showed that the chromaticity of the T1 line and the T2 line changed significantly ( Figure 2), when the concentration of human serum albumin is 80 mg / L, the hook effect begins to appear, causing the T2 line band to weaken. When the concentration is 320 mg / L, the hook effect causes the T2 line band to weaken significantly, but it still shows that the T2 line is stronger than the T1 line, and the result is still positive, which does not affect the judgment of the positive result. The results show that within the detection range of 0-320 mg / L, the test results can be correctly judged.

[0028] Example 9, verification of repeatability and detection limit of immunochromatographic test strips: Use artificial urine to prepare human serum albumin into samples with concentrations of 10, 12, 15, 24, 28 and 32 mg / L, take out 30 test strips, divide them into 6 groups, 5 strips in each group, mark the concentrations on the back, use the human serum albumin samples of the above concentrations for testing, let them stand for 10 minutes, shuffle the order, and let another person interpret and record the results. Take another 30 test strips, change people and repeat the above operation once, and now all 60 test strips have been tested. The experimental results show ( Figure 3 ), at a concentration of 12 mg / L, the negative compliance rate was 100%, and C5 could be determined to be 12 mg / L. At a concentration of 28 mg / L, the positive compliance rate was 100%, and C95 could be determined to be 28 mg / mL. We used 10 test cards to test the 28 mg / L sample, and a total of 20 test cards were positive, confirming that its detection limit was 28 mg / L. The T1 and T2 lines showed relative color changes with concentration changes, with good repeatability.

[0029] Example 10, thermal stability verification of immunochromatographic test strips: 210 aluminum foil bags containing immunochromatographic test strips were taken and divided into 3 groups, each with 70 bags, and incubated in a 98°C water bath for 0 minutes, 30 minutes and 60 minutes respectively. Human serum albumin was prepared into samples with concentrations of 8, 12, 16, 20, 24, 28 and 32 mg / L using artificial urine. Test strips (70 strips) were taken out of the 70 aluminum foil bags that were not incubated in a 98°C water bath, divided into 7 groups, each with 10 strips, and concentration marks were made on the back. The human serum albumin samples of the above concentrations were used for testing respectively, and the order was disrupted after standing for 10 minutes, and another person was asked to interpret and record the results. In the same way, test strips were taken out from 70 bags of aluminum foil bags that had been incubated in a 98°C water bath for 30 minutes or 60 minutes, divided into 7 groups, 10 strips in each group, and concentration marks were made on the back. The above-mentioned human serum albumin samples of each concentration were used for testing. After standing for 10 minutes, the order was shuffled and another person was asked to read and record the results. The judgment result of each strip was converted into a number. The conversion method was: 2 points for T1 stronger than T2 (negative), 1 point for T1 and T2 with similar chromaticity (near the critical value), and 0 points for T1 weaker than T2 (positive). The results of the 10 strips in each concentration group were added to obtain the score of the group. The scores of the 7 concentration groups that were incubated in a 98°C water bath for 0 minutes, 30 minutes and 60 minutes were plotted. The results show ( Figure 4 ), the scores of each concentration group have a good correlation with its concentration, and the scores of the same concentration group have a good data consistency with its incubation time. The results show that the immunochromatographic test strip based on nanoantibodies disclosed in the present invention shows excellent thermal stability.

[0030] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed in the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be based on the protection scope of the claims.

Claims

1. An immunochromatographic test strip for detecting human serum albumin based on nanoantibodies, characterized in that The test strip contains an antigen and an antibody that bind to each other through an immune reaction, wherein the antigen is albumin; the antibodies are two nanobodies that bind to different epitopes of the antigen, nanobody A and nanobody B; the nanobody A is nanobody 4p, and the nanobody B is nanobody 2p; The detection method is based on competition method and double antibody sandwich method.

2. The immunochromatographic test strip according to claim 1, characterized in that The test strip includes a sample pad, a conjugation pad, a chromatography pad, and an absorption pad, and each component is overlapped by 2 mm in sequence and pasted on a bottom plate; the conjugation pad is adsorbed with a color-marked nano antibody A, and the color mark includes colloidal gold and colored microspheres; two detection lines, T1 and T2 lines, are arranged in parallel on the chromatography pad; the T1 line contains albumin, and the T2 line contains nano antibody B.

3. The immunochromatographic test strip according to claim 2, characterized in that The amount of albumin in the sample is detected by determining the relative chromaticity of the T1 line and the T2 line, and the detection range is 0-320 mg / L; the chromaticity of the T1 line decreases as the amount of albumin in the sample increases; The chromaticity of the T2 line increases with the increase of the amount of albumin in the sample; when the critical value set for the detection is around 20 mg / L, the chromaticity of the T1 line and the T2 line are similar.

4. An immunochromatographic test paper card for detecting human serum albumin based on nanoantibodies, characterized in that The structure is that the immunochromatographic test strip according to any one of claims 1 to 3 is loaded into a card shell.

5. Use of the immunochromatographic test strip according to any one of claims 1 to 3, or the immunochromatographic test card according to claim 4, characterized in that Used to test renal function by detecting the amount of albumin in urine samples.

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