CN109142723B - Human immunodeficiency virus rapid detection test card and application thereof - Google Patents
Human immunodeficiency virus rapid detection test card and application thereof Download PDFInfo
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- CN109142723B CN109142723B CN201810953554.1A CN201810953554A CN109142723B CN 109142723 B CN109142723 B CN 109142723B CN 201810953554 A CN201810953554 A CN 201810953554A CN 109142723 B CN109142723 B CN 109142723B
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
- G01N33/56988—HIV or HTLV
Abstract
The invention provides a test card for rapidly detecting HIV human antibody and HIV antigen, which comprises a bottom plate, a sample pad, a nitrocellulose membrane, a water absorption pad and a fluorescent microsphere pad, wherein the fluorescent microsphere pad adsorbs fluorescent microspheres marked by type I HIV antigen, fluorescent microspheres marked by type II HIV antigen, fluorescent microspheres marked by p24 antibody and fluorescent microspheres marked by quality control protein, and the nitrocellulose membrane sequentially adsorbs type I HIV antigen, type II HIV antigen, p24 antibody and antibody resisting the quality control protein. In addition, the invention also provides a preparation method of the test card, application for rapidly detecting HIV human antibody and antigen, and the like.
Description
Technical Field
The invention belongs to the technical field of immunodetection, and particularly relates to a test card for rapidly detecting human immunodeficiency virus (including type I and type II) antibodies and human immunodeficiency virus p24 antigens, application thereof and the like.
Background
AIDS, acquired immunodeficiency syndrome, is a disease that is gradually the target of many opportunistic diseases due to the destruction of the immune system after the infection by retrovirus of human immunodeficiency virus (abbreviated as HIV), and contributes to various clinical symptoms, which are collectively called syndromes, rather than simple one, and the syndrome can be transmitted by directly contacting oral cavity, genitals, anus, etc. of mucosal tissue (mucosa) or blood, semen, vaginal secretion, breast milk with virus.
Due to the high infectivity, high transmission range and high mortality of AIDS, a corresponding laboratory diagnosis method is actually established from the discovery of AIDS; different detection methods are respectively established due to different detection purposes. Detection methods are broadly divided into two categories:
1. etiology detection
1) Pathogen isolation: the strict requirements are made in the P3 laboratory, by highly concentrating the patient's serum, observing under an electron microscope, isolating the HIV virus.
2) In situ hybridization: the cloned HIV cDNA fragment is labeled with an isotope, subjected to nucleic acid hybridization with tissue cells of a patient, and subjected to autoradiography to determine virus-infected cells.
3) And (3) PCR: the HIV cDNA fragments were amplified and examined for HIV infection by specific nucleic acid probes that were fluorescently labeled.
4) Determination of reverse transcriptase: the diagnosis of HIV infection is carried out by measuring the activity of reverse transcriptase in the serum of a patient.
5) Lymphocyte function assay: by measuring lymphocyte function, mainly on T4And (5) assisting lymphocytes to diagnose whether HIV infection exists.
6) Detection of p24 antigen p24 antigen in blood was detected by immune reaction as early diagnosis of HIV infection.
HIV antibody detection
There are many methods for detecting HIV antibodies, such as Enzyme linked immunosorbent assay (ELISA), gelatin particle agglutination assay (PA), Latex agglutination assay (LA), and various Rapid detection assays (Rapid tests). The antigens used in these experiments are early lysates of whole viruses, often false positive due to the presence of host cell components, and are difficult and dangerous to prepare and purify. The reagent uses recombinant or synthetic polypeptide HIV antigen, selects antigenic determinant related to immune reaction, and has raised sensitivity and specificity, and the preparation of the antigen is safe, easy and repeatable, but the recombinant antigen sometimes has false positive result due to the component containing carrier, especially HIV virus is easy to mutate, and the polypeptide antigen has reduced sensitivity due to lack of proper stereo conformation, so that it has false negative.
For example, chinese patent application CN105842462A discloses an immunochromatographic test strip for detecting HIV antibodies, which uses fluorescence immunochromatographic technique and double-antigen sandwich method to detect HIV-I and HIV-2 antibodies in human serum; chinese patent application CN1858594A discloses a multi-index rapid detection method for human immunodeficiency virus antibodies, which utilizes the fixation of antigen protein fragments including p24, gp41, gp36 and the like to carry out detection; chinese patent application CN102445538A discloses a reagent for rapidly detecting urine of AIDS (HIV-1/2), which is based on a colloidal gold technology to detect HIV recombinant antigens p41, p42 and p 36.
However, none of the above prior arts relates to selection of corresponding antigen or antigen fragment, so that it is difficult to avoid false negative result when detecting HIV virus which is easy to mutate, therefore, the present inventors have studied elaborately and developed a new HIV human antibody and HIV antigen rapid detection test card which can detect human antibodies of type I and type II HIV and p24 antigen of HIV at the same time rapidly, with high sensitivity and low variation coefficient, and has high accuracy, can avoid occurrence of false negative result, and is particularly suitable for primary screening of HIV.
Disclosure of Invention
The invention aims to solve the technical problem of providing a test card for quickly detecting HIV human antibody and HIV antigen, which can carry out quick, high-sensitivity and low-variation coefficient detection, is convenient for field detection, bedside detection and other preliminary screening, has high accuracy, particularly avoids false negative results, and is suitable for popularization and use. In addition, the invention also provides a preparation method and application of the test card.
Specifically, in a first aspect, the present invention provides a test card for rapid detection of HIV human antibodies and HIV antigens, which comprises a bottom plate (1), a sample pad (2), a nitrocellulose membrane (8) and a water absorption pad (9) which are sequentially attached on the bottom plate, wherein the nitrocellulose membrane (8) is connected with the water absorption pad (9), wherein a fluorescent microsphere pad (3) attached on the bottom plate is connected between the sample pad (2) and the nitrocellulose membrane (8), the kit comprises a nitrocellulose membrane (8), a sample pad (2), a water absorption pad (9), a fluorescent microsphere labeled with a type I HIV antigen, a fluorescent microsphere labeled with a type II HIV antigen, a fluorescent microsphere labeled with a p24 antibody and a fluorescent microsphere labeled with a quality control protein, wherein three separated lines which are vertical to the direction from the sample pad (2) to the water absorption pad (9) and sequentially adsorb the type I HIV antigen, the type II HIV antigen, the p24 antibody and the antibody of the quality control protein are arranged on the nitrocellulose membrane (8).
Preferably in the test card of the first aspect of the invention, the amino acid sequence of said type I HIV antigen is as set forth in SEQ ID NO: 1 is shown. This antigen was developed by the present inventors from a number of HIV proteins and is critical to the high accuracy of the test card of the first aspect of the invention.
Also preferred in the test card of the first aspect of the invention is a test card wherein the amino acid sequence of said type II HIV antigen is as set forth in SEQ ID NO: 2, respectively. This antigen was developed by the present inventors from a number of HIV proteins and is critical to the high accuracy of the test card of the first aspect of the invention.
Fluorescent microspheres have been commercialized in large quantities, however, the present inventors found that their effects for use in the present invention are uneven, and found that fluorescent microspheres having a carboxyl group (such as those available from Ocean Nanotech) having an absorption peak at a wavelength of 480nm are particularly suitable for use in the present invention. Therefore, it is preferable that in the test card according to the first aspect of the present invention, the fluorescent microsphere is a carboxyl group-carrying fluorescent microsphere having an absorption peak at a wavelength of 480 nm.
Preferably, in the test card of the first aspect of the present invention, the labeled fluorescent microspheres are prepared by: after shaking and mixing the fluorescent microspheres and EDC, washing with PBS buffer solution, adding the antigen, antibody or quality control protein, shaking and mixing, then adding PBS buffer solution containing glycine and BETA SA, shaking and sealing, and finally washing with PBS buffer solution containing BETA SA and Proclin300 and diluting. The fluorescent microsphere marked by the type I HIV antigen, the fluorescent microsphere marked by the type II HIV antigen, the fluorescent microsphere marked by the p24 antibody and the fluorescent microsphere marked by the quality control protein can be prepared by the method.
The inventor researches to find that the mutation of p24 antigen on HIV is not usually on the epitope, so that the existing p24 antigen can be effectively recognized by preparing the antibody. Preferably, in the test card of the first aspect of the present invention, the amino acid sequence of the antigen of the p24 antibody is as set forth in SEQ ID NO: 3, respectively.
The present inventors have found that a control protein also has an influence on the effect of the present invention, and it is preferable to use a hapten and an antibody thereof in order to avoid the influence of a substance in a human sample on the control line. Preferably in the test card of the first aspect of the invention, the quality control protein is DNP-BSA (dinitrofluorobenzene-bovine serum albumin), i.e. DNP (hapten) -conjugated BSA; the corresponding antibody is preferably an antibody against said hapten, i.e. an anti-DNP antibody. DNP is used as a hapten of the quality control protein, so that the influence of a human sample on a quality control line can be effectively avoided.
In a second aspect, the present invention provides a method of preparing a test card of the first aspect of the invention, comprising:
(a) preparing the fluorescent microspheres marked by the type I HIV antigens, the fluorescent microspheres marked by the type II HIV antigens, the fluorescent microspheres marked by the p24 antibodies and the fluorescent microspheres marked by the quality control protein, and then mixing and adsorbing the fluorescent microspheres on a fluorescent microsphere pad (3);
(b) streaking the type I HIV antigen, the type II HIV antigen, the p24 antibody and the antibody against the quality control protein on a nitrocellulose membrane (8), respectively; and the combination of (a) and (b),
(c) assembling the fluorescent microsphere pad (3) obtained in the step (a) and the nitrocellulose membrane (8) obtained in the step (b) with the base plate (1), the sample pad (2) and the absorbent pad (9) to form the test card.
The method for preparing the fluorescent microspheres marked by the HIV antigens and the fluorescent microspheres marked by the quality control proteins can be the following method:
after shaking and mixing the fluorescent microspheres and EDC, washing with PBS buffer solution, adding the antigen, antibody or quality control protein, shaking and mixing, then adding PBS buffer solution containing glycine and BETA SA, shaking and sealing, and finally washing with PBS buffer solution containing BETA SA and Proclin300 and diluting. The fluorescent microsphere marked by the type I HIV antigen, the fluorescent microsphere marked by the type II HIV antigen, the fluorescent microsphere marked by the p24 antibody and the fluorescent microsphere marked by the quality control protein can be prepared by the method.
In a third aspect, the present invention provides the use of a test card according to the first aspect of the invention for the manufacture of a test product for detecting human antibodies and HIV antigens with a low coefficient of variation.
The test card of the first aspect of the invention has good precision and low coefficient of variation. Preferably in the use according to the third aspect of the invention, the low coefficient of variation is a coefficient of variation not exceeding 15%.
In a first aspect, the present invention provides a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 1 is shown as SEQ ID NO: 2 in the preparation of HIV antibody detection products.
The application of this aspect may be an amino acid sequence as set forth in SEQ ID NO: 1 or the amino acid sequence of the HIV antigen is shown as SEQ ID NO: 2, or a combination thereof.
In addition, this aspect may also be their use with polypeptides directed against the amino acid sequences as set forth in SEQ ID NO: 3, p24 of p24 antigen. Preferably wherein the detection product of an HIV antibody is a detection product of an HIV human antibody and an HIV antigen, more preferably a test card according to the first aspect of the invention.
The invention has the advantages of rapid, high-sensitivity and low-variation coefficient detection of HIV human antibody and antigen, convenient field detection, bedside detection and other preliminary screening, high accuracy, particularly avoidance of false negative result, and suitability for popularization and use.
The present invention incorporates publications which are intended to describe the invention more clearly and which are incorporated herein by reference in their entirety as if reproduced in their entirety.
For the purpose of facilitating understanding, the present invention will be described in detail below with reference to specific embodiments and the accompanying drawings. It is to be expressly understood that the description is illustrative only and is not intended as a definition of the limits of the invention. Many variations and modifications of the present invention will be apparent to those skilled in the art in light of the teachings of this specification.
Drawings
Fig. 1 shows a schematic structural diagram of the HIV rapid detection test card of the present invention, wherein the reference numerals respectively represent: 1: a PVC base plate; 2: a sample pad; 3: a fluorescent microsphere pad; 4: a type I antigen test line; 5: a type II antigen test line; 6: p24 antibody test line; 7: DNP antibody test line; 8: NC film; 9: a water absorbent pad; 10: and (3) sampling.
Detailed Description
The invention will be described herein below by means of specific examples. Unless otherwise specified, the method can be performed according to the methods listed in the experimental manuals such as "molecular cloning laboratory Manual" (third edition) (Cold Spring Harbor laboratory Press), "cellular laboratory Manual" (science publishers, Beijing, China, 2001), "RNA experimental technical Manual" (science publishers, Beijing, China, 2004), "immunoassay technology" (science publishers, Beijing, China, 1991), and the references cited herein, which are familiar to those skilled in the art; the reagents are commercially available.
EXAMPLE 1 determination of HIV type I, type II recombinant antigens, p24 antibody
According to the design of the inventor, the antigen of the HIV virus is designed into a sequence shown as SEQ ID NO: 1 as a type I HIV virus detection antigen (I antigen for short), and the amino acid sequence shown as SEQ ID NO: 2 as a type II HIV virus detection antigen (II antigen for short). In addition, the inventors have adopted the nucleotide sequence shown as SEQ ID NO: 3, and preparing corresponding p24 monoclonal antibody and polyclonal antibody (rabbit anti-p 24 polyclonal antibody) according to a conventional method by entrusting Beijing China fir Jinqiao biotechnology limited to the HIV p24 antigen of the amino acid sequence shown in the specification.
Example 2HIV antigen, antibody on nitrocellulose membrane
The nitrocellulose membrane was scratched with the recombinant antigen and antibody of example 1 on a nitrocellulose membrane (NC membrane), specifically with a Sartori μ S CN104NC membrane with a pore size of 15 μm and a crawling speed of 150S/4 cm. The specific scribing process is as follows:
1) preparing a membrane scratching buffer solution: 0.01MPBS (phosphate buffer), pH7.0, containing 1% sucrose was prepared and filtered at 0.22. mu.m.
2) The membrane-streaking buffer was used to dilute the antigen and antibody at a given concentration, wherein the type I HIV antigen and type II HIV antigen of example 1 were diluted to 1mg/ml, the type II antigen of example 1 was diluted to 1mg/ml, the p24 mab of example 1 was diluted to 1.8mg/ml, and the DNP antibody (available from DAKO) as a quality control was diluted to 1.5 mg/ml.
3) Respectively scratching films on NC films by using a scratching instrument (available from Hangzhou Peak aviation technologies, Ltd.) at a speed of 1 mu L/cm, scratching films at 0.5cm for the type I antigen of example 1, scratching films at 0.9cm for the type II antigen of example 1, scratching films at 1.3cm for the p24 monoclonal antibody of example 1, scratching films at 1.8cm for the DNP antibody, drying at 37 ℃ for 3-4 hours, and sealing and storing at 1-30 ℃.
EXAMPLE 3 identification of fluorescent microspheres and conjugation with antigens, antibodies
Taking carboxyl group fluorescent microspheres from different sources, and diluting the microspheres with 0.01M PBS (phosphate buffer solution) in a ratio of 1: 10; zeroing with 0.01M PBS, scanning at wavelength of 400-600 nm to see if there is narrow absorption peak at 480nm (indicating uniform particle size of fluorescent microsphere); diluting the fluorescent microsphere to 2mg/ml with 0.01M PBS, spotting 10 μ l on NC membrane, and detecting whether the fluorescence value is greater than or equal to 104. The fluorescent microspheres with carboxyl groups, available from Ocean Nanotech, were tested to be the best and were therefore selected. The specific process of antigen and antibody coupling is as follows:
1) type I antigen-labeled fluorescent microspheres: the fluorescent microspheres were diluted to 1mg/ml with activation buffer (0.05M MES (2- (N-morpholino) ethanesulfonic acid)) during which time they were washed twice with activation buffer and after each washing centrifuged (10000 rpm, 30 min). Mixing 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and fluorescent microspheres according to a mass ratio of 1: 20, shaking at 25 deg.C for 30min, washing with coupling solution (0.01M P BETA S, pH7.4) for 2 times, adding 10 μ g protein per mg microsphere, adding type I antigen of example 1, and shaking at 2-8 deg.C overnight. Blocking with a coupling solution additionally containing 40mM glycine and 1% (w/v) BETA SA as a blocking solution, and shaking at 25 deg.C for 60 min. Washing with a preservative solution (0.01M P BETA S containing 0.5% (w/v) BETA SA (bovine serum albumin), 0.5% (v/v) Proclin300 (available from Shanghai flash crystal molecular Biotech Co., Ltd.)) for 2 times. Adding the preservation solution to the concentration of 1mg/ml, and preserving at the temperature of 2-8 ℃.
2) Type II antigen labeled fluorescent microspheres: the fluorescent microspheres were diluted to 1mg/ml with activation buffer (0.05M MES) during which they were washed twice with activation buffer and centrifuged after each wash (10000 rpm, 30 min). Mixing 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and fluorescent microspheres according to a mass ratio of 1: 20, shaking at 25 deg.C for 30min, washing with coupling solution (0.01M P BETA S, pH7.4) for 2 times, adding 10 μ g protein per mg microsphere, adding type II antigen of example 1, and shaking at 2-8 deg.C overnight. Blocking with a coupling solution additionally containing 40mM glycine and 1% (w/v) BETA SA as a blocking solution, and shaking at 25 deg.C for 60 min. Washing with a preservative solution (0.01M P BETA S containing 0.5% (w/v) BETA SA (bovine serum albumin), 0.5% (v/v) Proclin300) for 2 times. Adding the preservation solution to the concentration of 1mg/ml, and preserving at the temperature of 2-8 ℃.
3) p24 multi-antibody labeled fluorescent microspheres: the fluorescent microspheres were diluted to 1mg/ml with activation buffer (0.05M MES) during which they were washed twice with activation buffer and centrifuged after each wash (10000 rpm, 30 min). Mixing 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and fluorescent microspheres according to a mass ratio of 1: 20, shaking at 25 deg.C for 30min, washing with coupling solution (0.01M P BETA S, pH7.4) for 2 times, adding p24 polyclonal antibody of example 1 at a concentration of 5 μ g protein per mg microsphere, and shaking at 2-8 deg.C overnight. Blocking with a coupling solution additionally containing 40mM glycine and 1% (w/v) BETA SA as a blocking solution, and shaking at 25 deg.C for 60 min. Washing with a preservative solution (0.01M P BETA S containing 0.5% (w/v) BETA SA (bovine serum albumin), 0.5% (v/v) Proclin300) for 2 times. Adding the preservation solution to the concentration of 1mg/ml, and preserving at the temperature of 2-8 ℃.
4) DNP-BSA labeled fluorescent microspheres: the fluorescent microspheres were diluted to 1mg/ml with activation buffer (0.05M MES) during which they were washed twice with activation buffer and centrifuged after each wash (10000 rpm, 30 min). Mixing 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and fluorescent microspheres according to a mass ratio of 1: 20, shaking at 25 deg.C for 30min, washing with coupling solution (0.01M P BETA S, pH7.4) for 2 times, adding DNP-BSA (available from DAKO) at a concentration of 15 μ g protein per mg microsphere, and shaking at 2-8 deg.C overnight. Blocking with a coupling solution additionally containing 40mM glycine and 1% (w/v) BETA SA as a blocking solution, and shaking at 25 deg.C for 60 min. Washing with a preservative solution (0.01M P BETA S containing 0.5% (w/v) BETA SA (bovine serum albumin), 0.5% (v/v) Proclin300) for 2 times. Adding the preservation solution to the concentration of 1mg/ml, and preserving at the temperature of 2-8 ℃.
5) Mixing the type I antigen, the type II antigen, the p24 antibody and the fluorescent microsphere marked by DNP-BSA in equal volume, adding the mixed fluorescent microsphere to glass fiber, paving a glass fiber pad with the thickness of about 8 square centimeters per milliliter of fluorescent microsphere, and drying at 37 ℃ for 4 hours.
EXAMPLE 4 Assembly of HIV Rapid test card
The NC film, the fluorescent microsphere glass fiber pad (fluorescent microsphere pad) and the sample pad, the absorbent pad, etc. prepared in examples 2 and 3 were attached to a backing, slit, and assembled. The method comprises the following specific steps:
cutting the sample pad into 30cm and 28mm in length and width, cutting the fluorescent glass fiber pad into 30cm and 10mm in width, cutting the reaction membrane into 30cm and 25mm in length and 25mm in width, cutting the water absorption layer into 30cm and 27mm in length and width, sequentially adhering the reaction membrane to a lining card to form a large plate, cutting the reaction membrane into individual test strips with 8cm in length and 4mm in width, putting the test strips into a test card shell, and storing the test strips in a sealed bag filled with a drying agent for each individual person at room temperature. The prepared test card structure is shown in figure 1.
EXAMPLE 5 use of HIV detection test cards
The detection method for detecting HIV antibody and antigen in a sample comprises the following specific processes:
1) the test card prepared in example 4 was taken.
2) Serum and plasma samples were pipetted at 75 μ L and applied to the test card sample pad.
3) After 5 minutes of loading the test card, the test card was immediately inserted into a jack of a fluorometer (available from Hangzhou and Micheli technologies, Inc.) to detect the fluorescence value of each strip, wherein:
the fluorescence value of the I-type antigen strip is more than 500, the fluorescence value of the quality control strip is more than or equal to 1000, and the type I HIV virus antibody can be judged to be positive;
the fluorescence value of the II type antigen strip is more than 500, the fluorescence value of the quality control strip is more than or equal to 1000, and the II type HIV antibody can be judged to be positive;
the fluorescence value of the P24 antibody strip is more than 500, the fluorescence value of the quality control strip is more than or equal to 1000, and the HIV P24 antigen is judged to be positive;
when the fluorescence value of the quality control strip is less than 1000, repeating the step 1) and measuring again.
The results of the detection of HIV national standards provided by the national food and drug testing institute using the above method are shown in Table 1 below.
TABLE 1 HIV national Standard test results
In addition, the standard sample is respectively repeated for 10 times, the Coefficient of Variation (CV) is respectively 8.32%, 9.87% and 10.25%, and the Coefficient of Variation (CV) is less than or equal to 15%, and the method is proved to have good precision. In addition, the sensitivity of the test card can reach 0.05pg, and the linear range can reach 200-1000 times.
In addition, the test card of the invention is subjected to accelerated destruction, is placed at 37 ℃ for 30 days for measurement, the detection result is still as shown in the table above, the CV is less than or equal to 15 percent, and the precision is still good, which shows that the test card of the invention has good stability and can be used for rapid detection of HIV samples in places with poor storage conditions.
In addition, the method is used for detecting 107 common samples, the results are consistent with the confirmed detection results of clinical multiple detection (including virus culture), and no false negative result is found; and 1-3% of false negative results can be generated when the sample is detected by using the colloidal gold of the conventional antigen or the immunofluorescence technology.
<110> Huayimei Biotechnology Ltd, Suzhou
<120> human immunodeficiency virus rapid detection test card and application thereof
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Gln Ala Ile Glu Ala Gln Gln His Leu Leu Gln Leu Thr Val Trp Gly
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Lys Asp Gln Gln Leu Ala Gly Ile Trp Gly Cys Ser Gly Lys His Ile
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Ile Thr Val Leu Ser Glu Gln Ala Gln Ile Gln Gln Glu Lys Asn Met
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Tyr Glu Leu Gln Lys Leu Ser His Trp Asp Ile Phe Ser Asn Trp Phe
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Asp Phe Thr Ser Trp Ile Ala Tyr Ile Arg Ile Gly Leu Tyr Ile Val
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Ala Met Leu Met Leu Lys Asp Thr Ile Asn Glu Glu Ala Ala Glu Trp
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Claims (9)
- The HIV human antibody and HIV antigen rapid detection test card comprises a base plate (1), a sample pad (2), a nitrocellulose membrane (8) and a water absorption pad (9) which are sequentially attached to the base plate, wherein the nitrocellulose membrane (8) is connected with the water absorption pad (9), and is characterized in that a fluorescent microsphere pad (3) attached to the base plate is connected between the sample pad (2) and the nitrocellulose membrane (8), wherein the fluorescent microsphere pad (3) adsorbs fluorescent microspheres marked by I-type HIV antigens, fluorescent microspheres marked by II-type HIV antigens, fluorescent microspheres marked by p24 antibodies and fluorescent microspheres marked by quality control proteins, four separated lines vertical to the direction from the sample pad (2) to the water absorption pad (9) and used for sequentially adsorbing I-type HIV antigens, II-type HIV antigens, p24 antibodies and antibodies against the quality control proteins are arranged on the cellulose membrane (8) in the direction from the sample pad (2) to the water absorption pad (9), wherein the amino acid sequence of the type I HIV antigen is shown as SEQ ID NO: 1 is shown in the specification; or, the amino acid sequence of the type II HIV antigen is shown as SEQ ID NO: 2, respectively.
- 2. The test card of claim 1, wherein the antigen of the quality control protein is a hapten.
- 3. The test card of claim 1, wherein the quality control protein is DNP-BSA and the antibody to the quality control protein is an anti-DNP antibody.
- 4. The test card of claim 1, wherein the amino acid sequence of the antigen of the p24 antibody is set forth in SEQ ID NO: 3, respectively.
- 5. The test card of claim 1, wherein the fluorescent microspheres are carboxyl group-bearing fluorescent microspheres having an absorption peak at a wavelength of 480 nm.
- 6. The test card of claim 1, wherein the labeled fluorescent microspheres are prepared by the method of: after shaking and mixing the fluorescent microspheres and EDC, washing with PBS buffer solution, adding the antigen, antibody or quality control protein, shaking and mixing, then adding PBS buffer solution containing glycine and BETA SA, shaking and sealing, and finally washing with PBS buffer solution containing BETA SA and Proclin300 and diluting.
- 7. A method of preparing a test card according to any one of claims 1 to 6, comprising:(a) preparing the fluorescent microspheres marked by the type I HIV antigens, the fluorescent microspheres marked by the type II HIV antigens, the fluorescent microspheres marked by the p24 antibodies and the fluorescent microspheres marked by the quality control protein, and then mixing and adsorbing the fluorescent microspheres on a fluorescent microsphere pad (3);(b) streaking the type I HIV antigen, the type II HIV antigen, the p24 antibody and the antibody against the quality control protein on a nitrocellulose membrane (8), respectively; and the combination of (a) and (b),(c) assembling the fluorescent microsphere pad (3) obtained in the step (a) and the nitrocellulose membrane (8) obtained in the step (b) with the base plate (1), the sample pad (2) and the absorbent pad (9) to form the test card.
- 8. Use of the test card according to any one of claims 1 to 6 for the preparation of a test product for the detection of human antibodies and HIV antigens of HIV with a low coefficient of variation; the low coefficient of variation is a coefficient of variation not exceeding 15%.
- 9. The amino acid sequence is shown as SEQ ID NO: 1 is shown as SEQ ID NO: use of a type II HIV antigen as set forth in claim 2 in the manufacture of a test product for HIV antibodies, wherein the test product is a test card according to any one of claims 1-6.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810953554.1A CN109142723B (en) | 2018-08-21 | 2018-08-21 | Human immunodeficiency virus rapid detection test card and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810953554.1A CN109142723B (en) | 2018-08-21 | 2018-08-21 | Human immunodeficiency virus rapid detection test card and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109142723A CN109142723A (en) | 2019-01-04 |
| CN109142723B true CN109142723B (en) | 2021-07-13 |
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| CN101576562A (en) * | 2009-06-01 | 2009-11-11 | 无锡中德伯尔生物技术有限公司 | Fluorescence microballoon immunochromatography testing card for testing HIV and preparation method |
| WO2010011775A2 (en) * | 2008-07-23 | 2010-01-28 | The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services | Detection and quantification of antigens |
| CN202975019U (en) * | 2012-07-20 | 2013-06-05 | 天津中新科炬生物制药有限公司 | Human immunodeficiency virus antibody and antigen detection kit |
| CN105132439A (en) * | 2006-07-28 | 2015-12-09 | 宾夕法尼亚大学托管会 | Improved vaccines and methods for using the same |
| CN108025057A (en) * | 2015-09-02 | 2018-05-11 | 扬森疫苗与预防公司 | Stabilized virus I class fusion proteins |
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| CN105132439A (en) * | 2006-07-28 | 2015-12-09 | 宾夕法尼亚大学托管会 | Improved vaccines and methods for using the same |
| WO2010011775A2 (en) * | 2008-07-23 | 2010-01-28 | The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services | Detection and quantification of antigens |
| CN101576562A (en) * | 2009-06-01 | 2009-11-11 | 无锡中德伯尔生物技术有限公司 | Fluorescence microballoon immunochromatography testing card for testing HIV and preparation method |
| CN202975019U (en) * | 2012-07-20 | 2013-06-05 | 天津中新科炬生物制药有限公司 | Human immunodeficiency virus antibody and antigen detection kit |
| CN108025057A (en) * | 2015-09-02 | 2018-05-11 | 扬森疫苗与预防公司 | Stabilized virus I class fusion proteins |
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