CN107727855B - Sample pad for detecting HIV antibody in urine, sample pad treatment solution and test strip - Google Patents
Sample pad for detecting HIV antibody in urine, sample pad treatment solution and test strip Download PDFInfo
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Abstract
The invention discloses a sample pad, a sample pad treatment solution and a test strip for detecting HIV antibodies in urine, wherein the sample pad treatment solution comprises a buffer solution with the pH value of 8-11, an aqueous rheological aid and a blocking agent, wherein the addition amount of the aqueous rheological aid is 0.05-10 g and the addition amount of the blocking agent is 0.05-10 g per 100mL of the buffer solution. The sample pad treatment solution of the test strip for detecting the HIV antibody in urine can eliminate the influence of urine on the stability of the immune gold and improve the dissolution release speed and the membrane surface chromatography form of the immune gold by selecting the buffer solution with proper pH value and the aqueous rheological additive and the sealant with proper concentration.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a sample pad, sample pad treatment solution and test paper for detecting HIV antibodies in urine.
Background
Human Immunodeficiency Virus (HIV) is a Virus that infects cells of the Human immune system, causing various diseases by destroying the immune cells of the Human body, resulting in the immune system becoming defenseless. According to statistics of national disease control centers, from 2007, AIDS becomes the infectious disease causing the highest death rate in China and continues to the present. It is noted that at present, 32.1% of infected people in China are not found. The only way to determine whether a person is infected with HIV is to perform HIV testing. The data show that the amount of HIV detected in china rose rapidly during the year 2008-2015. Statistically, over 1.43 million people across the country have received HIV testing, accounting for 10% of the total population, for 2015 alone.
Such large-scale HIV detection is a serious challenge to human, material, financial and time, and especially, with the global spread of aids and the risk cases of medical staff being infected, the development and innovation of HIV detection technology are required to be accurate, rapid and non-invasive.
The AIDS diagnosis program is that the AIDS diagnosis is confirmed by western blot after the AIDS diagnosis program is screened to be positive. The primary screening method comprises two main detection methods:
1. blood detection: the main methods include immunochromatography (colloidal gold method or latex method), chemiluminescence method, and time-resolved fluorescence method, i.e., collecting blood sample of patient, and qualitatively or quantitatively detecting human immunodeficiency virus (HIV-1/HIV-2) antibody in human serum, plasma and whole blood in vitro. These methods are mainly used to determine whether infection is caused by detecting the presence or absence of HIV antibodies in blood of a suspicious individual.
However, the blood test method is a traumatic blood test, and blood transmission is also one of 3 major approaches to AIDS transmission, wherein chemiluminescence and time-resolved fluorescence require large-scale equipment and are only equipped in hospitals of county level and above. Secondly, these 2 methods are time consuming and require specialized operators and training. The common defects of the three are as follows:
A. the risk of medical personnel being punctured and infected during the blood drawing process;
B. the risk of infection exists in the garbage and treatment of various medical instruments (such as needle heads, acupuncture needles, dental instruments, cosmetic instruments and the like) after blood drawing;
C. the blood sample of collection needs the centrifugation, thereby is difficult to carry out patient self-checking and guarantees patient's privacy.
2. And (3) detecting oral mucosa exudate: the main method is immunochromatography (colloidal gold method or latex method). Namely, the human immunodeficiency virus HIV-1/2 antibody in the human oral mucosa exudate is detected by the exudate after the oral swab is continuously wiped on the gum line.
The method is a minimally invasive test, with much less risk than blood transmission, but still presents a risk of transmission, such as a high risk group of AIDS-exposed people.
The presence of HIV-specific antibodies in urine has been extensively studied and confirmed. The CFDA approved urine detection method without/with extremely low infectivity is only an enzyme-linked immunosorbent assay at present, but takes long time (3h) and can only carry out qualitative detection. The colloidal gold method which can also carry out qualitative detection is preferred and considered due to simple operation and short time consumption, but the similar products of the test strips for detecting the HIV antibodies in urine are not found in the existing market because the urine antibody content is low and the sensitivity is high, and in addition, the urine detection has a report that saliva has a higher false positive problem compared with blood.
The sample pad is an important component of a test strip for detecting HIV antibodies in urine, and the sample pad needs to be treated by a sample pad treatment solution before use. Due to the difference of detection samples and items, when the existing sample pad treatment solution is applied to a test strip for detecting HIV antibodies in urine, the problems are all great, for example: the sample pad treatment solution of the HIV blood detection test strip and the sample pad treatment solution of the HCV and HBV test strips can not realize color development on HIV negative and positive samples, and the color development of the sample pad treatment solution of the HIV saliva detection test strip on the negative and positive samples is close, so that the positive and negative samples are very poor in differentiation. Therefore, there is a need to develop a sample pad treatment solution for a test strip for detecting HIV antibodies in urine.
Disclosure of Invention
In one aspect, in order to overcome the above-mentioned drawbacks of the prior art, the present invention provides a sample pad treatment solution for a test strip for detecting HIV antibodies in urine.
In order to realize the purpose of the invention, the invention adopts the following technical scheme:
the sample pad treatment solution comprises a buffer solution with the pH value of 8-11, an aqueous rheological aid and a blocking agent, wherein the addition amount of the aqueous rheological aid is 0.05-10 g and the addition amount of the blocking agent is 0.05-10 g per 100mL of the buffer solution.
A large number of experiments show that when the pH value of the buffer solution is 8-11, the false positive can be eliminated, the strong and medium positive samples can be detected, and when the pH value of the buffer solution is acidic or neutral, the problems that the gold particles are discolored, cannot flow or the false positive elimination effect is poor can occur. The addition of the aqueous rheological additive can slow down the flow of water molecules in urine, provide a liquid environment for the reaction of gold particles, and provide a bracket in the process of spraying the gold particles, so that biological raw materials are stably fixed without drifting and uniform distribution, and the uniformity of the distribution of the biological raw materials is ensured.
In some of these embodiments, the buffer is 10-200 mM Tris-HCl buffer.
In some of the embodiments, the sample pad treatment solution further comprises 0.01-1M Na2CO3Or K2CO3。
In some of these embodiments, the sample pad treatment solution further comprises S9.
In some embodiments, the S9 is added in an amount of 0.01-1 g per 100mL of the buffer.
In some of these embodiments, the aqueous rheology aid is one or more of hydroxypropyl methylcellulose, PAA, PEO.
In some of these embodiments, the aqueous rheology aid is a mixture of hydroxypropyl methylcellulose and PAA, the hydroxypropyl methylcellulose comprising 45-70% of the total weight of the mixture.
In some of these embodiments, the blocking agent is rabbit serum.
In another aspect, the invention further provides a sample pad treated with the sample pad treatment solution of the test strip for detecting HIV antibodies in urine.
In another aspect, the invention further provides a test strip for detecting HIV antibodies in urine, the test strip includes a base plate, and the sample pad, a nitrocellulose membrane and absorbent paper disposed on the base plate, the two ends of the nitrocellulose membrane are respectively connected with the sample pad and the absorbent paper, the end of the nitrocellulose membrane near the nitrocellulose membrane of the sample pad is sprayed with a colloidal gold-labeled HIV recombinant antigen and a colloidal gold-labeled mouse anti-human IgG antibody, and the nitrocellulose membrane is provided with a detection line coated with the HIV recombinant antigen and a control line coated with a goat anti-mouse IgG antibody.
In some of these embodiments, the colloidal gold labeled HIV recombinant antigen comprises recombinant gp 160; the HIV recombinant antigen coated on the detection line comprises recombinant gp41 and gp 160.
In some embodiments, the colloidal gold labeled HIV recombinant antigen further comprises recombinant gp36, and the HIV recombinant antigen coated on the detection line further comprises recombinant gp 36.
In some embodiments, the concentration of the colloidal gold labeled recombinant gp160 antigen is 1.5-3.5 ug/ml; the concentration of the colloidal gold labeled mouse anti-human IgG antibody is 4.5-6.5 ug/ml; the concentration of the colloidal gold labeled recombinant gp36 antigen is 4.5-6.5 ug/ml; the dosage of the colloidal gold labeling raw material is 2-3.5 ul/cm; the coating concentration of the HIV recombinant antigen gp41 is 0.5-2.0mg/ml, the coating concentration of the HIV recombinant antigen gp160 is 0.2-0.8mg/ml, and the coating concentration of the HIV recombinant antigen gp36 is 0.5-1.0 mg/ml; the dosage of the HIV recombinant antigen is 0.12-0.15ul/mm, the concentration of the goat anti-mouse IgG antibody is 0.8-1.0mg/ml, and the dosage of the goat anti-mouse IgG antibody is 0.09-0.22 ul/mm.
The invention also provides a preparation method of the test strip for detecting the HIV antibody in urine, which comprises the following steps:
1) preparing sample pad treating solution, and coating on glass cellulose membrane with coating concentration of 40ul/cm2Drying to obtain a sample pad;
2) combining the antibody or antigen for detecting HIV with the colloidal gold particles to form a colloidal gold labeled antibody or antigen for detecting HIV, and uniformly spraying the colloidal gold labeled antibody or antigen on a sample pad by using a gold spraying instrument; airing;
3) diluting an HIV recombinant antigen to a working concentration by using an HIV coating diluent, scribing on a nitrocellulose membrane to form a detection line, diluting a goat anti-mouse antibody to the working concentration, scribing on the nitrocellulose membrane to form a quality control line, and drying;
4) and overlapping the sample pad, the nitrocellulose membrane and the absorbent paper on a bottom plate to obtain the sample pad.
In order to solve the problems of sensitivity and specificity of urine detection of HIV, the inventor of the invention carries out various researches, and confirms that the sensitivity and the specificity can be improved by applying the solid-phase immunochromatography principle, adopting the sensitivity treatment of an early-stage sample pad and the technology of combining a specific antigen gp160 and a double-antibody sandwich indirect method to detect the HIV antibody in the urine. If the urine sample contains HIV antibody, the HIV antibody is combined with the colloidal gold particle marker to form a complex, the complex is diffused to the nitrocellulose membrane for further chromatography, when the conjugate antigen coated at a detection area (T line) on the nitrocellulose membrane is encountered, the complex is combined with the coating antigen again and is captured at the coating area, when the captured complex reaches a certain amount, a macroscopic T line is formed, the HIV antibody is contained in the sample, and if the conjugate antigen does not appear, the sample is negative or the content of the HIV antibody is lower than the lowest detection limit of the test strip. The control area (line C) is used as the quality control standard of the test strip, and can be used for detecting positive and negative samples.
Compared with the prior art, the invention has the following beneficial effects:
1. the sample pad treatment solution of the test strip for detecting the HIV antibody in urine can eliminate the influence of urine on the stability of the immune gold and improve the dissolution release speed and the membrane surface chromatography form of the immune gold by selecting the buffer solution with proper pH value and the aqueous rheological additive and the sealant with proper concentration;
2. the sample pad treating solution of the test strip for detecting the HIV antibody in urine of the present invention is further added with K2CO3/Na2CO3The reagent can provide a more stable and good alkaline environment for reaction, maintains the alkaline environment after adding the urine sample together with a Tris-HCL buffer solution, thereby eliminating the influence of false positive, improving the dissolution and release speed of the immune gold and the chromatographic morphology of the membrane surface, effectively releasing gold particles even if the pH of urine is acidic (the pH is less than 7.0-7.5), and expanding the applicability of the detection pH;
3. the antibody in the urine is mainly of IgG type, various antibodies can be rapidly enriched by adding the anti-human IgG antibody into the test strip, a concentration effect is achieved, and then the HIV antibody is specifically combined from the concentrated enriched antibody through the specificity of the HIV antigen, so that the sensitivity of the test strip is improved;
4. the test strip for detecting the HIV antibody in the urine reduces the risk that HIV contacters (such as medical staff, prenuptial or pregnant woman examination and the like) are infected due to puncture of collected samples, the urine samples do not need to be concentrated and pretreated, the storage and the transportation are convenient, the leakage risk is low, more processing equipment is not needed, and professional skill training is not needed for medical staff; meanwhile, with the development of medical treatment, the OTC detection is more and more oriented, and the pressure of hospitals and medical care personnel is greatly released. In addition, the method can also carry out personal detection, is simple to operate, protects personal privacy and achieves real POCT detection;
5. the high-sensitivity urine test of the test strip for detecting the HIV antibody in urine ensures the authenticity and reliability of data, and can be used for large-scale primary screening of AIDS.
Detailed Description
The invention will be further described with reference to specific examples, which are not described herein as being applicable to the prior art. Specific examples of the present invention are given below, but the examples are only for the purpose of further elaborating the present invention and do not limit the claims of the present invention. The reagents and starting materials used in the following examples were all commercially available unless otherwise specified.
Example 1 sample pad treatment solution for test strip for detecting HIV in urine
The sample pad treatment solution of this example included Tris-HCl buffer solution at pH9, 100mM, HPMC, PAA, rabbit serum, S9, and 0.09MNa2CO3；
Wherein, the addition amount of HPMC is 0.1g, the addition amount of PAA is 0.1g, the addition amount of rabbit serum is 0.1g, and the addition amount of S9 is 0.1g per 100mL of Tris-HCl buffer solution.
The preparation method of the sample pad treatment solution comprises the following steps:
preparing Tris-HCl with pH of 9 and 100mM as base solution, adding HPMC, PAA, rabbit serum, S9, and adding Na2CO3And obtaining the sample pad treatment solution.
Example 2 sample pad treatment solution for test strip for detecting HIV in urine
The sample pad treatment solution of this example included Tris-HCl buffer solution at pH 10, 10mM, HPMC, PAA, rabbit serum, S9 and 0.5M Na2CO3；
Wherein, the addition amount of HPMC is 0.7g, the addition amount of PAA is 0.3g, the addition amount of rabbit serum is 1g, and the addition amount of S9 is 1g, relative to 100mL of Tris-HCl buffer solution.
The preparation method of the sample pad treatment solution comprises the following steps:
preparing Tris-HCl with pH of 10 and 10mM as base solution, adding HPMC, PAA, rabbit serum, S9, and adding Na2CO3And obtaining the sample pad treatment solution.
Example 3 sample pad treatment solution for test strip for detecting HIV in urine
The sample pad treatment solution of this example included Tris-HCl buffer solution at pH 11, 200mM, HPMC, rabbit serum, S9 and 1MNa2CO3；
Wherein, the addition amount of HPMC is 5g, the addition amount of rabbit serum is 1g, and the addition amount of S9 is 0.5g per 100mL of Tris-HCl buffer solution.
The preparation method of the sample pad treatment solution comprises the following steps:
preparing Tris-HCl with pH of 11 and 200mM as base solution, adding HPMC, rabbit serum, S9, and adding 1M Na2CO3And obtaining the sample pad treatment solution.
Example 4 sample pad treatment solution for test strip for detecting HIV in urine
The sample pad treatment solution of this example included Tris-HCl buffer solution at pH9, 100mM, HPMC, rabbit serum and 0.01M Na2CO3；
Wherein the addition amount of HPMC is 0.05g and the addition amount of rabbit serum is 10g per 100mL of Tris-HCl buffer solution.
The preparation method of the sample pad treatment solution comprises the following steps:
preparing Tris-HCl with pH of 9 and 100mM as base solution, adding HPMC and rabbit serum, and adding 0.01M Na2CO3And obtaining the sample pad treatment solution.
Example 5 sample pad treatment solution for test strip for detecting HIV in urine
The sample pad treatment solution of this example included Tris-HCl buffer solution at pH9, 100mM, HPMC, rabbit serum and S9;
wherein, the addition amount of HPMC is 10g, the addition amount of rabbit serum is 1g, and the addition amount of S9 is 0.01g per 100mL of Tris-HCl buffer solution.
The preparation method of the sample pad treatment solution comprises the following steps:
preparing Tris-HCl with the pH value of 9 and 100mM as a base solution, and adding HPMC, rabbit serum and S9 to obtain the sample pad treatment solution.
Example 6 test strip for detecting HIV antibody in urine
The test strip for detecting the HIV antibody in urine comprises a bottom plate, a sample pad and a nitrocellulose membrane, wherein the sample pad and the nitrocellulose membrane are arranged on the bottom plate, the sample pad is treated by the sample pad treatment solution in the embodiment 1, the two ends of the nitrocellulose membrane are respectively lapped with the sample pad and water absorbent paper, a colloidal gold labeled mouse anti-human IgG antibody, a colloidal gold labeled gp160 antigen and a colloidal gold labeled gp36 antigen are sprayed on the sample pad, and the nitrocellulose membrane is provided with a detection line for coating the HIV recombinant antigen gp41 and the HIV recombinant antigen gp160, a detection line for coating the HIV recombinant antigen gp36 and a control line for coating a goat anti-mouse antibody.
The test strip can be used for detecting HIV-1 type and HIV-2 type infection.
In this example, the concentration of the colloidal gold labeled gp160 antigen was 2.5 ug/ml; the concentration of the colloidal gold labeled mouse anti-human IgG antibody is 5.6 ug/ml; the concentration of the colloidal gold labeled gp36 antigen was 5.6 ug/ml. The dosage of the colloidal gold labeling raw material is 2.75 ul/cm.
In the embodiment, the coating concentration of the HIV recombinant antigen gp41 is 1.0mg/ml, the coating concentration of the HIV recombinant antigen gp160 is 0.6mg/ml, the coating concentration of the HIV recombinant antigen gp36 is 0.6mg/ml, and the dosage of the HIV recombinant antigen is 0.12 ul/mm.
In this example, the concentration of the goat anti-mouse antibody was 1.0mg/ml and the amount of the goat anti-mouse antibody was 0.1 ul/mm.
The preparation method of the test strip for detecting the HIV antibody in urine in the embodiment is as follows:
1) preparing sample pad treating solution, and coating on glass cellulose membrane with coating concentration of 40ul/cm2Placing at 25 ℃ and humidity of 10-30%, and drying for 18-22h to obtain a sample pad;
2) combining the mouse IgG and the colloidal gold particles to form a colloidal gold labeled mouse IgG antibody probe; the gp160 antigen and the colloidal gold particles are labeled to form a colloidal gold labeled HIV specific antigen probe (before the gp160 antigen is combined with the colloidal gold particles, the gp160 antigen needs to be dissolved in an HIV coating diluent which comprises a Tris-HCl buffer solution with pH of 7.4 and 20mM, S9 and urea, wherein the addition amount of S9 is 0.1g and the addition amount of urea is 5g per 100mL of the Tris-HCl buffer solution); the gp36 antigen is labeled with the colloidal gold particles to form a colloidal gold labeled HIV specific antigen probe. Uniformly mixing the 3 kinds of gold particles, uniformly spraying the mixture on a sample pad by using a gold spraying instrument, wherein the width of gold spraying is 6mm, the length of the gold spraying is 2.8cm, the mixture is dried for 18-22h at 25 ℃, and the humidity is 10% -30% for later use;
3) coating dilutions of HIV-specific antigens gp41 and gp160 with HIV (said coated dilutions comprising Tris-HCl buffer at pH 7.4, 20mM, S9 and urea; the method comprises the following steps of (1) diluting S9 with the addition of 0.1g and urea with the addition of 5g) to a working concentration per 100mL of Tris-HCl buffer solution, scribing with a film spraying machine to form a T1 detection line on a nitrocellulose membrane, diluting HIV specific antigen gp36 with Tris-HCl buffer solution with the pH of 7.4 and 20mM to the working concentration, scribing with the film spraying machine to form a T2 detection line on the nitrocellulose membrane, diluting goat anti-mouse antibody to the working concentration, scribing with the film spraying machine to form a C quality control line on the nitrocellulose membrane, and drying at the temperature of 25 ℃ and the humidity of 10-30% for 18-22 h;
3) and sequentially overlapping the sample pad, the nitrocellulose membrane and the absorbent paper on the bottom plate to obtain the composite material.
Test example 1 selection test of auxiliary agent for sample pad treatment liquid
Adding one or more rheological additives to adjust the rheological property of liquid and prevent drying of gold particles, selecting aqueous rheological additives according to urine characteristics, and respectively selecting ①, hydroxypropyl methyl cellulose 0.1%, 1%, 10%, ②, PEO 0.1%, 1%, 10%, ③, PAA 0.1%, 1%, 10%, ④, sodium alginate 0.1%, 1%, 10% (wherein hydroxypropyl methyl cellulose 0.1% means 0.1g of hydroxypropyl methyl cellulose dissolved in 100mL of Tris-9.00.1M-HCl buffer solution with pH of 9.00 under the same solvent condition, and the rest are in turn analogized), respectively coating on glass cellulose membrane with coating concentration of 40ul/cm2At 25 deg.C and humidity10 to 30 percent, drying for 18 to 22 hours to obtain the sample pad.
The spraying of gold particles and the preparation of nitrocellulose membrane were the same as in example 6.
The test paper strip was prepared in the same manner as in example 6.
The test paper prepared in this test example was tested as follows:
selecting healthy people as a control, detecting HIV positive patients with confirmed diagnosis, and preparing internal reference products of strong yang, middle yang and weak yang from the negative urine of the healthy people and the urine of the HIV positive patients. Respectively dripping 2 drops (60-80ul) of samples to a sample pad of the test strip prepared in the test example by using a rubber head dropper, wherein the samples move to the gold particles and the cellulose nitrate membrane along the test strip due to the capillary action, and when the samples completely dissolve the gold particles and flow to the cellulose nitrate membrane, the result begins to be displayed; observation after 15 minutes showed the result (Note: color development was ineffective after 30 minutes). The test results are shown in table 1.
TABLE 1 Effect of different aqueous rheological Agents on samples
Note: a total of 10 color development gradients: C1-C9 and B. Wherein B represents "blank", with no bands; C1-C9 represent the color intensity, wherein a larger number indicates a lighter color. C8+ indicates that the color development is between C8 and C7, and so on.
From the results in Table 1, it can be seen that the effect of the different types of rheology auxiliaries is, at low concentrations, in turn hydroxypropyl methylcellulose > PEO ≈ PAA > sodium alginate. Compared with other aqueous rheological additives, the hydroxypropyl methyl cellulose effectively weakens the flow of water, and simultaneously provides sufficient liquid phase reaction environment for gold particles, so that the reaction is fully carried out, and the sensitivity is improved. PEO and PAA lock the flow of water to some extent, but are used at higher concentrations than hydroxypropyl methylcellulose. Sodium alginate is the weakest. Therefore, hydroxypropyl methylcellulose is preferred, and polyoxyethylenes and polyphenyleneic acids are selected as candidates. In addition, the inventor also unexpectedly finds that when the hydroxypropyl methyl cellulose and the PAA are used as the aqueous rheological aid at the same time and in a proper concentration range (namely, the hydroxypropyl methyl cellulose accounts for 45-70% of the total weight of the hydroxypropyl methyl cellulose and the PAA), the hydroxypropyl methyl cellulose and the PAA play a synergistic role, the sensitivity can be further improved, and the gold particles are not separated from water.
Test example 2 elimination test of false positives
Most of urine pH is acidic, the pH is very sensitive to urine detection, gold particles are easy to deposit under acidic conditions, and sodium carbonate can finely adjust acidic substances (uric acid, creatine and the like) in the urine. The sodium ions can also maintain the required ionic state of the antigen-antibody reaction, thereby ensuring the antigen-antibody specific reaction.
Gradient Na with different concentrations2CO3(as shown in Table 2) was added to 0.1M Tris-HCl buffer solution at pH9.0, and then coated on a glass cellulose membrane at a concentration of 40ul/cm2And (5) placing at 25 ℃ and with the humidity of 10-30%, and drying for 18-22h to obtain the sample pad.
The spraying of gold particles and the preparation of nitrocellulose membrane were the same as in example 6.
The test paper strip was prepared in the same manner as in example 6.
The test paper prepared in this test example was tested in the same manner as in test example 1, and the test results are shown in table 2.
TABLE 2Na2CO3Influence of the amount of (2) on the sample
Note: a total of 10 color development gradients: C1-C9 and B. Wherein B represents "blank", with no bands; C1-C9 represent the color intensity, wherein a larger number indicates a lighter color. C8+ indicates that the color development is between C8 and C7, and so on.
As can be seen from Table 2, some false positives in the alkaline buffer system of Tris-HCl pH9.00.1M can be ensured by adding sodium carbonate, while introducing a certain sodium ion concentration to maintain the ions required for antigen-antibody reaction during immunochromatography.
Test example 3 selection test of protein in sample pad treatment solution
The urine hardly contains protein, and the addition of a few blocking proteins can not only avoid nonspecific binding, but also increase the total amount of urine molecules and stabilize the environment in the urine reaction. The blocking protein rabbit serum can be combined with a protein non-specific site in a sample like conventional BSA, and has the greatest advantages of blocking an endogenous Fc fragment in the sample, blocking the combination of an antibody and an Fc receptor in the sample, reducing background and reducing false positive.
Preparing Tris-HCl buffer solution (pH9.0, 0.1M) as base solution, adding hydroxypropyl methylcellulose (0.1%), PAA (0.1%), and 0.09MNa (sodium alginate)2CO3Then coating on a glass cellulose membrane with the coating concentration of 40ul/cm2And (5) placing at 25 ℃ and with the humidity of 10-30%, and drying for 18-22h to obtain the sample pad.
The spraying of gold particles and the preparation of nitrocellulose membrane were the same as in example 6.
The test paper strip was prepared in the same manner as in example 6.
The test paper prepared in this test example was tested in the same manner as in test example 1, and the test results are shown in table 3.
TABLE 3 comparison of the Effect of Rabbit serum, BSA on samples
Note: a total of 10 color development gradients: C1-C9 and B. Wherein B represents "blank", with no bands; C1-C9 represent the color intensity, wherein a larger number indicates a lighter color. C8+ indicates that the color development is between C8 and C7, and so on. Furthermore, the numerical values in the table above before the% characterizing the amount of rabbit serum represent the amount of rabbit serum used by mass (g) per 100mL of buffer; the values in the table above before the% characterizing the amount of BSA represent the amount of BSA used by mass (g) per 100mL of buffer.
As can be seen from Table 3, rabbit serum is superior to BSA. BSA is widely used as a blocking agent in blood and plasma as a blocking protein and is also commonly used in antibody preparation, and antibodies in gold particles are not removed due to the fact that a small amount of anti-BSA antibodies may be contained in the expression process, so that the anti-BSA antibodies react with the BSA to cause the false positive. And the gold particles do not have anti-rabbit antibodies, so that rabbit serum can be well non-specifically combined with other epitopes.
Test example 4 selection test of reinforcing agent in sample pad treatment liquid
The color of different urine is different, some transparent, some yellow, some honey, and the true color is white nitrocellulose membrane in the colloidal gold chromatography process, and the color of the background is darker when a darker sample flows through, so the color of the background is eliminated by adding a toner, the background of the T/C line is clearer, the T/C line is clear at a glance, and the sensitivity is indirectly increased.
Preparing Tris-HCl buffer solution (pH9.0 and 0.1M) as base solution, adding hydroxypropyl methylcellulose (0.1%), PAA (0.1%), and Na (0.09M)2CO31% of rabbit serum and S9 with different concentrations are mixed evenly and then coated on a glass cellulose membrane with the coating concentration of 40ul/cm2And (5) placing at 25 ℃ and with the humidity of 10-30%, and drying for 18-22h to obtain the sample pad.
Wherein S9 is a surfactant, which is commercially known under the name Tetronic 1307. The negative effect of false positive is higher than that of other surfactants (such as S17 and the like), while S9 is relatively mild, and the detection result is not influenced by a small amount of addition.
The spraying of gold particles and the preparation of nitrocellulose membrane were the same as in example 6.
The test paper strip was prepared in the same manner as in example 6.
The test paper prepared in this test example was tested in the same manner as in test example 1, and the test results are shown in table 4.
Table 4 comparison of the effect of enhancers on samples
Note: a total of 10 color development gradients: C1-C9 and B. Wherein B represents "blank", with no bands; C1-C9 represent the color intensity, wherein a larger number indicates a lighter color. C8+ indicates that the color development is between C8 and C7, and so on. In addition, the numerical values in the table above before the% characterizing the amount of S9 represent the mass amount (g) of S9 per 100mL of buffer.
As can be seen from Table 4, the toner is similar to "bleaching", and eliminates the color of urine to background color, thereby improving the color definition and indirectly improving the color gradient. And the low dose is as effective as the high dose, so the low dose is preferred.
In summary, cellulose is an effective class of the many auxiliary agents, and hydroxypropyl methyl cellulose contains many side chains, which can absorb moisture to the maximum extent to 'swell', slow down the flow of water, and provide a liquid environment for the reaction of gold particles. The application range of the biological material spray coating is 0.05% -10%, a support is provided in the spray coating process, the biological material is stably fixed and does not drift and uniformly distribute, and the uniformity of the biological material is ensured. K2CO3/Na2CO3Provides a good alkaline environment, maintains the alkaline environment after adding the urine sample together with Tris, thereby eliminating the influence of false positive, improving the dissolution and release speed of the immunogold and the chromatographic morphology of the membrane surface, effectively releasing gold particles even if the urine with acidic pH (pH less than 7.0-7.5) and expanding the detection pH applicability.
Test example 5 sensitivity test of the test strip of the present invention
Healthy people are selected as a control, and HIV positive patients with confirmed diagnosis are subjected to a blood detection test strip (a comparison test strip 1) and a saliva detection test strip (a comparison test strip 2) which are registered by FDA on the market for comparison test, so that the sensitivity and the specificity of the test strip are compared. Respectively dripping 2 drops (60-80ul) of samples on a sample pad of the test strip by using a rubber head dropper, wherein the samples move to the marker pad and the nitrocellulose membrane along the test strip due to the capillary action, and when the samples completely dissolve gold particles and move to the nitrocellulose membrane, the result begins to be displayed; observation after 15 minutes showed the result (Note: color development was ineffective after 30 minutes).
The results are shown in Table 5.
Table 5 test strip sensitivity testing
Note: a total of 10 color development gradients: C1-C9 and B. Wherein B represents "blank", with no bands; C1-C9 represent the color intensity, wherein a larger number indicates a lighter color.
As can be seen from Table 5, the test range of the test strip (reference substance diluted to 10000 times) of the invention is wider than that of saliva and blood test strips (comparison test strips 1 and 2, reference substance diluted to 100 times), and the sensitivity is higher than 100 times. Secondly, the saliva and blood test strip is sensitive to urine samples, all positive urine cannot be detected, the HIV urine test strip can detect urine and blood simultaneously, and the urine test strip can replace the blood test strip to a certain extent.
Test example 6 national reference disk test of the test strip of the present invention
The test paper of the embodiment 6 of the invention is used for testing HIV urine antibody reference substances (urine rapid reagents) of China pharmaceutical and biological product institute, and the results are shown in Table 6.
Table 6 national reference disc test results
The results in Table 6 show that the sensitivity of the reagent strip of example 6 for detecting urine HIV antibody is 100%, the specificity reaches more than 99.9%, and the reagent strip meets the HIV antibody detection standard of China pharmaceutical biological product institute.
Test example 7 clinical sample test of the test strip of the present invention
101 cases of HIV patients diagnosed with western blot (standard for the positive western blot and sample numbers P1-P101) were selected from the disease prevention and control center of a certain province, and 100 healthy human urine samples (standard for the no clinical symptoms, no history of contact with infectious diseases and infection, and sample numbers WF-N1-WF-N100) from Wanfu Biotechnology, Inc. of Guangzhou were used as controls, and the test paper of example 6 of the present invention was used to perform the tests, and the results are shown in tables 7 and 8.
TABLE 7 clinical specimen test results
Note: a total of 10 color development gradients: C1-C9 and B. Wherein B represents "blank", with no bands; C1-C9 represent the color intensity, wherein a larger number indicates a lighter color. "-" indicates negative; "+" indicates positive diagnosis.
TABLE 8 summary of clinical sample test results
As seen from the results in tables 7 and 8, the small batch clinical specimen tests showed: the sensitivity is more than or equal to 99 percent, and the specificity is more than or equal to 99 percent, thereby meeting the expected requirements.
Test example 8 clinical sample tracking test of the test strip of the present invention
The test strip (test strip 1 for short) and the comparative test strip (test strip 2 for short) in the embodiment 6 of the invention are adopted to track the high risk group of 5 HIV positive patients for 2 months, and the window time is compared, and the results are shown in Table 9. The reagent formula and the preparation method of the comparative test strip are the same as those of the test strip in the embodiment 6, and the only difference is that: the detection zone is only coated with HIV specific recombinant antigens gp41 and gp36, and is not coated with HIV specific recombinant antigen gp 160.
TABLE 9 follow-up testing of suspicious patients
Note: a total of 10 color development gradients: C1-C9 and B. Wherein B represents "blank", with no bands; C1-C9 represent the color intensity, wherein a larger number indicates a lighter color. "-" indicates negative; "+" indicates positive diagnosis.
As can be seen from the results in Table 9, the addition of gp160 shortens the window time of some patients by about one week (suspicious individuals 2,5), and when these samples are tested negative to the test strip, the test strip of the present invention shows a stealth band, which can give some prompt or preventive measures to the suspicious individuals to reduce the risk of transmission; or to have a part of the sample developed from a normal occult band into a darker band (suspect 1,3,4) so that the medical staff can diagnose or take treatment early. The later diagnosis by the 'gold standard' western blot shows that 5 samples have bands of gp160 and gp41, which are consistent with the test strip of the invention.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (8)
1. The sample pad treatment solution for the test strip for detecting the HIV antibody in urine is characterized by comprising a buffer solution with the pH value of 8-11, an aqueous rheological aid and a blocking agent, wherein the addition amount of the aqueous rheological aid is 0.05-10 g and the addition amount of the blocking agent is 0.05-10 g per 100mL of the buffer solution; the aqueous rheological aid is a mixture of hydroxypropyl methyl cellulose and PAA, and the hydroxypropyl methyl cellulose accounts for 45-70% of the total weight of the mixture.
2. The sample pad treatment solution for a test strip for detecting HIV antibodies in urine according to claim 1, wherein the buffer solution is 10-200 mM Tris-HCl buffer solution.
3. The sample pad treatment solution for a test strip for detecting HIV antibodies in urine according to claim 1, further comprising 0.01-1M Na2CO3Or K2CO3。
4. The sample pad treatment solution for a test strip for detecting HIV antibodies in urine according to any one of claims 1 to 3, further comprising S9.
5. The sample pad treatment solution for a test strip for detecting HIV antibodies in urine according to claim 4, wherein the S9 is added in an amount of 0.01 to 1g per 100mL of the buffer solution.
6. The sample pad treatment solution for a test strip for detecting HIV antibodies in urine according to claim 1 or 2, wherein the blocking agent is rabbit serum.
7. A sample pad, characterized in that the sample pad is treated with the sample pad treatment solution of the test strip for detecting HIV antibody in urine according to any one of claims 1 to 6.
8. A test strip for detecting HIV antibodies in urine, which is characterized by comprising a bottom plate, and a sample pad, a nitrocellulose membrane and absorbent paper which are arranged on the bottom plate and are disclosed in claim 7, wherein the two ends of the nitrocellulose membrane are respectively lapped with the sample pad and the absorbent paper, the end, close to the nitrocellulose membrane, of the sample pad is sprayed with a colloidal gold-labeled HIV recombinant antigen and a colloidal gold-labeled mouse anti-human IgG antibody, and the nitrocellulose membrane is provided with a detection line coated with the HIV recombinant antigen and a control line coated with a goat anti-mouse IgG antibody.
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| CN106970219A (en) * | 2017-04-28 | 2017-07-21 | 北京金豪制药股份有限公司 | One kind is based on HIV in colloidal gold method detection urine（1+2）Antibody reagent |
| CN107209175A (en) * | 2014-12-11 | 2017-09-26 | 重症监护诊断股份有限公司 | Test device and method for ST2 cardiac biomarkers |
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| CN105738622A (en) * | 2015-10-23 | 2016-07-06 | 北京玛斯玛克生物科技有限公司 | Human urine HIV1/2 antibody detection test paper through colloidal gold chromatographic method and preparation method thereof |
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| CN102841208A (en) * | 2011-06-24 | 2012-12-26 | 北京乐普医疗科技有限责任公司 | Colloidal gold test paper for quickly detecting troponin I and preparation method for colloidal gold test paper |
| CN107209175A (en) * | 2014-12-11 | 2017-09-26 | 重症监护诊断股份有限公司 | Test device and method for ST2 cardiac biomarkers |
| CN106970219A (en) * | 2017-04-28 | 2017-07-21 | 北京金豪制药股份有限公司 | One kind is based on HIV in colloidal gold method detection urine（1+2）Antibody reagent |
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