Colloidal gold immunochromatography method and kit for detecting malaria protozoa antigen

Abstract

The invention provides a colloidal gold immunochromatography method for detecting malaria protozoa antigen and a kit thereof, the chromatography method is based on a double antibody sandwich method for detection, and in the preparation process of a sample pad used in the method, a sample pad containing poly-(oxo-1, 3, 4-triazole-1, 3, 4-triazole-1, 3-triazole-1, 3-triazole-1, 3-triazole-1, 3-triazole-1, 3-triazole-1 the sample pad is soaked in a sample pad treatment solution containing 2, 2-ethidene dimethylene)-alpha-nonylphenyl-omega-hydroxyl and sulfosuccinic acid dioctyl sodium salt. According to the kit prepared by adopting the reagent 1 and the reagent 2 as a sample pad treatment formula, in the range of 0.1 ng / mL-2 mg / mL, the color development intensity is synchronously and positively improved along with the increase of the malaria recombinant antigen protein concentration; the sample pad prepared by the method disclosed by the invention can be used for counteracting the HOOK effect existing when the test strip is used for positive detection.

Description

Technical Field

[0001] The invention relates to the field of biological detection, in particular to a colloidal gold immunochromatography method for detecting malarial protozoan antigens and a kit thereof. Background Art

[0002] Colloidal gold labeling technology is a labeling method developed in the 1980s, following the three major labeling technologies of fluorescein, radioisotopes and enzymes. In 1971, Faulk and Taylor introduced colloidal gold into immunobiochemistry. Since then, this labeling method has been widely used in the fields of drug testing, biomedical testing, agricultural and veterinary drug testing, clinical disease testing, etc. At present, there are hundreds of commercial colloidal gold immunochromatography kits at home and abroad, such as the common ones: early pregnancy test kits, drug detection kits, fecal occult blood test kits, infectious disease detection kits, etc.

[0003] The application of colloidal gold in immunoassay mainly involves labeling antigens or antibodies with colloidal gold, and then fixing the antibodies or antigens that can react immunologically with the labeled antigens or antibodies on a matrix, so that the binding of the antigens or antibodies is displayed as a color change visible to the naked eye. The degree of binding between colloidal gold and protein, the pH of the reaction system, and the concentration of the protein in the reaction will all affect the binding between protein and colloidal gold.

[0004] Malaria is a life-threatening disease caused by parasites, which is transmitted to humans through the bites of infected female mosquitoes. The pathogen is Plasmodium. Clinical manifestations include intermittent, periodic, and paroxysmal chills, high fever and sweating, as well as anemia and splenomegaly. Plasmodium is mainly transmitted by Anopheles mosquitoes, and its transmission route is affected by many factors, including natural factors (temperature, humidity, rainfall, altitude, etc.) and social factors (institutions, culture, etc.). As a worldwide infectious disease with a high mortality rate, malaria has always been a concern of countries around the world.

[0005] For a long time, the main method for malaria diagnosis was microscopic examination of blood smears. Microscopic blood smear examination is the most widely used diagnostic method for field malaria diagnosis and is recommended by the World Health Organization as the gold standard for clinical diagnosis in endemic areas. This low-cost method can assess parasitemia and determine the type of Plasmodium, but microscopic testing has many limitations, has high requirements for the skills of professional technicians and the quality of smears, and has certain limitations for the detection of low-level parasitemia and mixed infection cases. The detection sensitivity of a professional microscopist can only detect infections with at least 50-100 parasites per microliter of blood. And studies have shown that due to the different levels of microscopists themselves, the microscopic examination results will vary by ±50-100 parasites. At the same time, it is difficult to detect mixed infections of Plasmodium with microscopic smears, and misdiagnosis of mixed infections with Plasmodium vivax or Plasmodium falciparum may lead to relapses, thereby causing serious malaria complications.

[0006] The colloidal gold immunochromatography detection method, which is based on the immunoassay method, does not require sample extraction or extraction. It can directly detect the collected whole blood, serum, and plasma samples for screening and auxiliary diagnosis of malaria infection. The colloidal gold immunochromatography technology based on the double antibody sandwich method can quickly and efficiently detect malarial parasites in whole blood, serum, and plasma. The sampling and detection costs are low, and complex instruments and equipment and very professional personnel are not required during the detection process; personnel with a certain cultural foundation can directly perform relevant operations according to the instructions.

[0007] At present, the malaria antigen detection reagents used in China are mainly colloidal gold test strips, and they are mainly imported brands such as Bio-Rad, InBios, Panbio, Abbott, etc. However, due to the supply of raw materials and product sensitivity, there are relatively few products in China. Imported test strips are expensive and product supply is easily affected by the international environment. Therefore, it is very necessary to develop colloidal gold test strips with high sensitivity.

[0008] At present, the kits for malaria antigen detection on the market have low detection sensitivity and accuracy; and when using the double antibody sandwich method to detect malarial parasite antigens, the hook effect (HOOK) is prone to occur. The hook effect refers to the phenomenon that within a certain concentration range, the concentration of the analyte is proportional to the detection signal value; however, when exceeding a certain concentration limit, the signal value decreases with the increase in concentration. Because the entire curve of concentration and signal value resembles a hook, it is called the hook effect, in which excess antibody is called the proband effect; excess antigen is called the postband effect. When the antigen-antibody HOOK occurs, it often exceeds the detection limit and cannot accurately and quantitatively detect the relevant values. Only when the antigen-antibody ratio is appropriate will the strongest reaction occur and the accurate value can be detected within the detection limit. Summary of the invention

[0009] In order to solve the above problems, the present invention provides a colloidal gold immunochromatography method for detecting malarial protozoan antigens. The chromatography method is based on a double antibody sandwich method for detection. During the preparation of a sample pad used in the method, the sample pad is soaked in a sample pad treatment solution containing poly-(oxy-1,2-ethylenedimethylene)-α-nonylphenyl-ω-hydroxy and dioctyl sodium sulfosuccinate.

[0010] In one embodiment, the sample pad is soaked in a sample pad treatment solution containing 1% to 1.5% by mass volume of poly-(oxy-1,2-ethylenedimethylene)-α-nonylphenyl-ω-hydroxy and 0.08% to 0.1% by mass volume of dioctyl sodium sulfosuccinate.

[0011] In one embodiment, the sample pad is a glass fiber pad.

[0012] In one embodiment, the present invention provides a colloidal gold immunochromatography kit for detecting malarial protozoa antigens, wherein the kit is a detection kit for detecting malarial protozoa antigens based on a double antibody sandwich method, and the kit comprises a sample pad, wherein the sample pad is soaked in a sample pad treatment solution containing poly-(oxy-1,2-ethylenedimethylene)-α-nonylphenyl-ω-hydroxy and dioctyl sodium sulfosuccinate.

[0013] In one embodiment, the sample pad is soaked in a sample pad treatment solution containing 1%-1.5% mass volume concentration of poly-(oxy-1,2-ethylenedimethylene)-α-nonylphenyl-ω-hydroxy and 0.08%-0.1% mass volume concentration of dioctyl sodium sulfosuccinate.

[0014] In one embodiment, a sample pad treatment liquid is provided, wherein the sample pad treatment liquid is used to soak a sample pad used in a colloidal gold immunochromatography method, wherein the chromatography method is based on a double antibody sandwich method for detection, and the sample pad treatment liquid contains poly-(oxy-1,2-ethylenedimethylene)-α-nonylphenyl-ω-hydroxy and dioctyl sodium sulfosuccinate.

[0015] In one embodiment, the sample pad treatment solution contains 1%-1.5% by mass volume concentration of poly-(oxy-1,2-ethylenedimethylene)-α-nonylphenyl-ω-hydroxy and 0.08%-0.1% by mass volume concentration of dioctyl sodium sulfosuccinate.

[0016] In one embodiment, the sample pad treatment solution is used to prepare a sample pad in a colloidal gold immunochromatography kit for detecting malarial protozoa antigens.

[0017] In the method of the present invention, the reagent 1 and reagent 2 shown in the present application are used as the sample pad treatment formula to prepare the kit. In the range of 0.1ng / mL-2mg / mL, as the concentration of the malaria recombinant antigen protein increases, the color intensity is synchronously positively increased. There is no HOOK effect that is prone to occur in the double antibody plus heart method. The sample pad prepared by the method of the present invention can offset the HOOK effect that exists in the test strip during positive detection.

[0018] In the sample pad formula, reagent 1 and reagent 2 are present at the same time, and when the concentration of reagent 1 is 1%-1.5% and the concentration of reagent 2 is 0.08%-0.1%, the prepared test strip has high sensitivity and can detect 0.1ng / mL of recombinant antigen protein in a positive solution; the sensitivity of detection can be improved and the accuracy of detection can be increased. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0020] Figure 1 is a schematic diagram of the reagent strip of the present invention;

[0021] Figure 2 It is the picture of the test strip of the present invention and actual cutting. DETAILED DESCRIPTION

[0022] In order to enable those skilled in the art to better understand the technical solutions in this application, the present invention will be further described below in conjunction with embodiments. Obviously, the described embodiments are only part of the embodiments of this application, rather than all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of this application.

[0023] Example 1. Preparation of colloidal gold solution

[0024] 1. Weigh 0.2 g / mL of tetrachloroauric acid into a graduated tube, then add ultrapure water to prepare 5 mL of chloroauric acid aqueous solution;

[0025] 2. Weigh trisodium citrate in a graduated tube at a volume of 0.158 g / mL, and then add ultrapure water to prepare 5 mL of trisodium citrate aqueous solution;

[0026] 3. Place a 2-liter Erlenmeyer flask on a magnetic stirrer with heating function, add 1L of ultrapure water into the Erlenmeyer flask, turn on the magnetic stirrer, and heat;

[0027] 4. When the bottom layer of the conical flask is heated to 95-100℃ and the upper layer is 90-95℃, add 1mL of chloroauric acid aqueous solution into the conical flask. Set the stirrer speed to the maximum speed. At this time, the aqueous solution in the conical flask is yellow;

[0028] 5. Then quickly add 1mL + 0.3mL of trisodium citrate aqueous solution. Continue heating and stirring for 6-8 minutes; at this time, the aqueous solution in the conical flask is purple-black at first, then turns purple, and finally turns dark wine red. Stop heating immediately. Transfer the reaction-completed solution to another magnetic stirrer and cool it to room temperature with a water bath;

[0029] 6. After the colloidal gold solution to be prepared is cooled, 1 mL of the solution is taken, and then the spectrum of 200 nm-800 nm band is scanned with a micro-ultraviolet spectrophotometer (Nanodrop type). The colloidal gold solution prepared by this method has a maximum absorption peak at 552 nm, and an OD value of 1.27, without any impurity peaks; that is, available colloidal gold is obtained.

[0030] Example 2. Labeling of Anti-malarial Protozoa Antibodies

[0031] The present invention adopts a direct labeling method. That is, at room temperature, commercially available anti-malarial protozoan antibodies are directly reacted with colloidal gold solution, and then centrifuged to obtain the desired gold label. The specific method is as follows:

[0032] 1. Take 100 ml of colloidal gold solution and put it into a beaker;

[0033] 2. Adjust the pH of the colloidal gold solution to 7.5 with 0.1 M potassium carbonate solution and stir on a magnetic stirrer;

[0034] 3. Dilute the anti-malarial parasite labeled antibody to a concentration of 0.9 mg / ml with 2 mM Tris buffer at pH 7.4, then add 1 ml of the diluted protein solution to the colloidal gold solution, adding the above solution while stirring;

[0035] 4. Stir on a magnetic stirrer for 25 minutes;

[0036] 5. Add 10% OVA aqueous solution to 100 ml of the above solution at a volume of 24 μl / ml and continue stirring for 30 minutes;

[0037] 6. Add 1.5% PEG20000 to the above solution at a volume of 20 μl / ml and continue stirring for 1 hour;

[0038] 7. Transfer the above reaction solution into a centrifuge tube;

[0039] 8. Centrifuge at 8000-15000 rpm / min at 4°C for 30 minutes, for example, 12000 rpm / min; remove the supernatant;

[0040] 9. Redissolve the precipitate with 1 part of 2 mM Tris buffer at pH 7.4 per 100 parts to obtain the labeled gold-labeled malarial parasite antibody.

[0041] Example 3. Preparation of sample pad

[0042] 1. Preparation of sample pad treatment solution

[0043] (1) Weigh 4 g of borax per 100 ml and put it into a beaker, then add 80 ml of water into the beaker, and place the beaker on a magnetic stirrer and stir. A total of 35 groups of liquids are prepared, numbered 1-35;

[0044] (2) Add 0.3 g of ethylenediaminetetraacetate to each of the beakers No. 1 to No. 35 and continue stirring;

[0045] To the above-mentioned beaker solutions No. 1-35, 0g, 0.5g, 1g, 1.5g, 2g of poly-(oxy-1,2-ethylene dimethylene)-α-nonylphenyl-ω-hydroxy (commercially available, recorded as reagent 1) and 0g, 0.02g, 0.04g, 0.06g, 0.08g, 0.1g, 0.12g of dioctyl sodium sulfosuccinate (commercially available, recorded as reagent 2) were added respectively, and stirring was continued; the corresponding relationship between the amounts of the above-mentioned reagent 1 solution and reagent 2 contained in the No. 1-35 solutions is as shown in Table 1 below:

[0046] Table 1 Comparison table of the number of the prepared solutions (No. 1-35) and the content of reagent 1 and reagent 2 in the prepared solutions

[0047]

[0048] (3) Add 1 g of PVP to each of the above solutions 1-35 and continue stirring until completely dissolved;

[0049] (4) Add 0.02 mL of Niusha PC-950 to the above solution No. 1-35 and continue stirring;

[0050] (5) Add 0.005 mL of Tween 20 to the above solution No. 1-35 and continue stirring;

[0051] (6) After the solution is completely dissolved and clarified, the pH of solution No. 1-35 is adjusted to 8.2 with hydrochloric acid or sodium hydroxide, and then the volume is adjusted to the required volume of 100 mL with purified water to obtain a sample pad treatment solution;

[0052] (7) Keep aside at room temperature.

[0053] 2. Preparation of Sample Pad

[0054] (1) Prepare commercially available glass fiber mat (length * width: 300mm * 254mm);

[0055] (2) Soak the glass fiber mat at a rate of 1 piece per 100 mL of solution; first, soak the glass fiber mats numbered 1-35 in the sample pad treatment solution corresponding to the numbered 1-35 for 2 minutes, then take them out and gently filter out excess water with a glass rod. Turn the glass fiber mat over and soak it in the solution again for 2 minutes, and gently filter out excess water with a glass rod. The sample mat is now obtained.

[0056] (3) Place the obtained sample pad in a 37°C oven and dry overnight (17-20 hours).

[0057] (4) After drying, the sample pad is sealed and stored in a dry environment.

[0058] 3. Preparation of Sheets

[0059] (1) Paste the nitrocellulose membrane onto the PVC base plate;

[0060] (2) Preparation of coating solution:

[0061] Test line: dilute the anti-malarial parasite coated antibody to 1 mg / ml with 0.01 M PB and prepare the test line at 2 μl / cm per piece;

[0062] Quality control line: dilute goat anti-mouse IgG to 0.8 mg / ml with 0.01 M PB and prepare the quality control line at 2 μl / cm per slide;

[0063] Coating: Coat the solution onto nitrocellulose membrane using a stripping agent.

[0064] (3) Place the coated sheet in a 37°C oven and dry overnight.

[0065] (4) The dried sheets should be kept sealed and stored in a dry place.

[0066] 4. Preparation of Gold Label

[0067] (1) Dilute the gold label to the working concentration using 0.01 M PBS solution, for example, 45%, 50%.

[0068] (2) The diluted solution was sprayed with gold labels using a gold labeling machine at a rate of 1.6 μl / cm per strip. The spraying base pad was a glass fiber pad (length * width: 300 mm * 84 mm);

[0069] (3) After spraying, place the gold label in a 37°C oven and dry overnight;

[0070] (4) The dried gold label strip is sealed in a ziplock bag and then stored in a dry environment away from light.

[0071] 5. Assembly of test strips

[0072] (1) Cut the prepared gold label into small strips with a length of 300 mm and a width of 8 mm, and stick them on the lower edge of the nitrocellulose membrane of the sheet, pressing 1-2 mm over the nitrocellulose membrane;

[0073] (2) Cut the sample pads numbered 1-35 into strips 300 mm long and 20 mm wide, and stick them on the lower end of the sheet; the lower edge of the sample pad should extend 3 mm beyond the lower edge of the sheet, and the upper edge of the sample pad should extend beyond the white edge of the gold label strip;

[0074] (3) A small strip of absorbent filter paper 300 mm long and 17 mm wide is glued to the lower edge of the bottom plate;

[0075] (4) The pasted reagent plates No. 1-35 are pressed tightly respectively, and then cut into small strips with a width of 4.0 mm using a cutting machine; the schematic diagram of the reagent strip of the present invention is shown in FIG. Figure 1 As shown, and the actual cut test strips are as follows Figure 2 shown.

[0076] 6. Assembly of the kit

[0077] The cut 4.0 mm test strips are placed into the slots of the lower plate of the plastic card case, and the upper card case is tightly closed; thus, a malarial protozoan antigen detection kit is obtained.

[0078] 7. Preparation of loading diluent

[0079] According to the formula, 0.6 parts of sodium dihydrogen phosphate, 8 parts of sodium chloride, 0.2 parts of potassium dihydrogen phosphate and 2 parts of Niusha PC-950 were added per 100 parts of water to prepare the solution, and the pH of the solution was adjusted to 7.8 to obtain the loading dilution solution.

[0080] Example 4 Effects of different sample pad treatment solutions on detection sensitivity and accuracy

[0081] 1. Test of recombinant antigen protein: The malarial parasite recombinant antigen protein was diluted to 1 ng / mL and 0.1 ng / mL with the loading diluent. The above 35 sets of kits numbered 1-35 were tested for positive results using the recombinant antigen protein at these two concentrations. The test results showed that when the concentration of the recombinant antigen protein was 0.1 ng / mL, the T line position of the kits numbered 1-3, 6-8, 11-13, 16-18, 21, 22, 26, and 31 had no color development; the T line position of the kits numbered 4, 5, 9, 10, 14, 15, 19, 20, 27, and 32 was shaded, and was judged as weakly positive; the kits numbered 23, 24, 25, 28, 29, 30, 33, 34, and 35 were light red, and were judged as positive. When the concentration of recombinant antigen protein is 1 ng / mL, the T lines of kits No. 1, 2, 6, 7, 11, 12, 16, and 17 have unlimited color; the T lines of kits with other numbers are colored, which is positive. The test was performed using the loading buffer as a negative matrix, and the test results were: the T lines of kits No. 1-20, 21-24, 26-29, 31, and 32 had no color and were judged as negative; the T lines of kits No. 25, 30, 33, 34, and 35 had color and were judged as false positive. Therefore, when testing low-concentration malarial antigen recombinant antigen protein, the kits with obvious positive color and all negative negative color results are No. 23, 24, 28, and 29.

[0082] Table 2 Malaria recombinant antigen detection results

[0083]

[0084] Note: In the table, “-” indicates negative, “- / +” indicates weak positive, “+” indicates positive, “++” indicates moderately positive, “+++” indicates strongly positive, and “++++” indicates extremely strongly positive.

[0085] Table 3 Test results for negative matrix

[0086]

[0087] Note: “-” indicates negative and “+” indicates positive.

[0088] According to the above experimental results, it can be known that: 1. When the content of reagent 1 poly-(oxy-1,2-ethylenedimethoxy)-α-nonylphenyl-ω-hydroxy in the sample pad treatment solution is 1%-1.5% (W / V) and reagent 2 dioctyl sodium salt of sulfosuccinate is 0.08%-0.1% (W / V), the prepared malaria antigen detection kit has a good positive color development result for the detection of a lower concentration of 0.1ng / mL of malaria recombinant antigen; 2. When the sample pad formula contains surfactant reagents other than the buffer system and there is no reagent 1 or reagent 2, the detection of a low concentration of 0.1ng / mL of recombinant antigen protein is negative; even if surfactant 1 or surfactant 2 is tried alone in the sample pad formula, the test result is still negative at a low concentration of recombinant antigen protein; when reagent 1 or surfactant 2 is tried alone, the test result is still negative. When the concentration of reagent 2 is relatively high, although the test result of the low-concentration recombinant antigen protein of 0.1ng / mL is positive, the test result of the negative matrix is ​​also positive, that is, the test result shown by the test strip is incorrect, and a false positive occurs; 3. When reagent 1 and reagent 2 are present in the sample pad formula at the same time, and the concentration of reagent 1 is 1%-1.5% and the concentration of reagent 2 is 0.08%-0.1%, the ratio of reagent 1 and reagent 2 within this range, the prepared sample pad, the test results for the negative reagent are all negative, and the test results for the recombinant antigen protein of 0.1ng / mL are all positive; 4. When both are present in the sample pad formula, the concentration of reagent 1 or reagent 2 is lower than the above range, the test result for the recombinant antigen protein of 0.1ng / mL is negative or weakly positive. When both are present in the sample pad formula, the concentration of reagent 1 or reagent 2 is higher than the above range, the test result for the recombinant antigen protein of 0.1ng / mL is weakly positive or positive, while the test result for the negative matrix is ​​positive, that is, the test strip cannot detect the negative normally, and a false positive occurs. Therefore, when reagent 1 and reagent 2 are present in the sample pad formula, and the concentration of reagent 1 is 1%-1.5% and the concentration of reagent 2 is 0.08%-0.1%, the prepared test strip has high sensitivity and can detect 0.1ng / mL of recombinant antigen protein in the positive solution; the sensitivity of the detection can be improved and the accuracy of the detection can be increased.

[0089] Example 5 Effect of the Sample Pad Treatment Solution of the Present Invention on Detection of the Hook Effect

[0090] 1. Preparation of test strips: According to the method of Example 4, test strips with sample pad formulas that do not contain reagent 1 poly-(oxy-1,2-ethylenedimethoxy)-α-nonylphenyl-ω-hydroxy and reagent 2 dioctyl sodium sulfosuccinate are prepared, and test strips containing the sample pads of reagent 1 poly-(oxy-1,2-ethylenedimethoxy)-α-nonylphenyl-ω-hydroxy with a content of 1% (W / V) and reagent 2 dioctyl sodium sulfosuccinate with a content of 0.1% (W / V) in Example 4 are prepared. Then, a kit is formed for use. The kit prepared without the above reagents 1 and 2 is recorded as Kit A; the kit prepared with reagents 1 and 2 is recorded as Kit B.

[0091] 2. Prepare recombinant malaria antigen protein solutions with concentrations of 1 ng / mL, 10 ng / ml, 1 μg / / mL, 10 μg / / mL, 100 μg / mL, 1 mg / mL, 1.5 mg / mL, and 2 mg / mL as positive solutions.

[0092] 3. Negative solution was prepared as shown in Example 4.

[0093] 4. Testing: Testing the kit prepared in 1 above with a negative solution and a positive solution respectively.

[0094] 5. The test results are as follows:

[0095] Table 4 Test results of different concentrations of malaria recombinant antigens

[0096]

[0097]

[0098] Note: In the table, “-” indicates negative, “- / +” indicates weak positive, “+” indicates positive, “++” indicates moderately positive, “+++” indicates strongly positive, and “++++” indicates extremely strongly positive.

[0099] From the above test results, it can be seen that when the prepared kit A is tested, as the concentration of the malarial recombinant antigen protein in the test solution increases, its color development result gradually increases from negative to strongly positive, but when the concentration of the malarial recombinant antigen protein exceeds 100μg / mL and reaches 1mg / mL, the color development result becomes weaker and positive; when the concentration of the malarial recombinant antigen protein reaches 2mg / mL, it is negative; this result shows that the kit A has a HOOK effect. As for the kit B for synchronous detection, as the concentration of the malarial recombinant antigen protein increases, the color intensity of its detection result continues to increase, from negative to extremely strong positive. Combined with the test results of Example 4, this result shows that the kit prepared by using reagents 1 and reagents 2 shown in this application as the sample pad treatment formula, within the range of 0.1ng / mL-2mg / mL, as the concentration of the malarial recombinant antigen protein increases, the color development intensity is synchronously positively increased. There is no HOOK effect that is easy to occur in the double-antibody plus heart method. That is, the sample pad prepared by the above-mentioned method can offset the HOOK effect that exists when the test strip is positively detected.

[0100] Example 6 Comparative test of the kit of the present invention with similar kits in whole blood / serum / plasma samples

[0101] 1. Comparative test of similar whole blood / serum / plasma kits

[0102] The kit prepared by the present invention was obtained by purchasing two similar malaria antigen detection kits on the market and the sample pad set prepared by the treatment liquid No. 23, 24, 28, 29 described in Example 4, and tested on 50 positive inactivated whole blood / serum / plasma samples and 50 negative samples. It was found that the kit prepared by the sample pad formula No. 23, 24, 28, 29 described in this patent had a higher detection rate for positive than the commercially available product.

[0103] 2. Test result statistics

[0104] Table 5 Whole blood negative sample test results

[0105]

[0106]

[0107]

[0108]

[0109]

[0110] According to the "WS / T 505-2017 Guidelines for the Evaluation of Qualitative Assay Performance", the calculation method of the positive compliance rate is: PPA = a / (a+c) × 100%, negative compliance rate NPA = d / (b+d) × 100%; for 50 positive whole blood samples and serum samples, the positive compliance rate of the homemade kit is 100%, compared with the positive compliance rate of whole blood samples of Kit 1 of 96%, and the compliance rate of serum samples of 98%; compared with the positive compliance rate of whole blood samples and serum samples of Kit 2 of 98%. That is, for the same batch of positive whole blood / serum samples, the homemade kit in this case has a higher detection sensitivity.

[0111] It should be understood that the disclosed invention is not limited only to the specific method, scheme and material of description, because these all can change.It should also be understood that the terminology used herein is only for the purpose of describing specific embodiment scheme, rather than being intended to limit the scope of the present invention, and the scope of the present invention is only limited to the appended claims.

[0112] Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are also intended to be encompassed by the appended claims.

Claims

1. A colloidal gold immunochromatographic method for detecting malarial parasite antigens, characterized in that: The chromatography method is based on double antibody sandwich method for detection. During the preparation of the sample pad used in the method, the sample pad is soaked in a sample pad treatment solution containing poly-(oxy-1,2-ethylenedimethylene)-α-nonylphenyl-ω-hydroxyl and dioctyl sodium sulfosuccinate.

2. The colloidal gold immunochromatography method according to claim 1, characterized in that: The sample pad is soaked in a sample pad treatment solution containing 1%-1.5% by mass volume of poly-(oxy-1,2-ethylenedimethylene)-α-nonylphenyl-ω-hydroxy and 0.08%-0.1% by mass volume of dioctyl sodium sulfosuccinate.

3. The colloidal gold immunochromatography method according to claim 2, characterized in that: The sample pad is a glass fiber pad.

4. A colloidal gold immunochromatography kit for detecting malarial parasite antigens, characterized in that: The kit is a detection kit for detecting malarial protozoan antigens based on a double antibody sandwich method. The kit comprises a sample pad, which is obtained by soaking in a sample pad treatment solution containing poly-(oxy-1,2-ethylenedimethylene)-α-nonylphenyl-ω-hydroxyl and dioctyl sodium sulfosuccinate.

5. The colloidal gold immunochromatography kit according to claim 4, characterized in that: The sample pad is obtained by soaking in a sample pad treatment solution containing 1%-1.5% mass volume concentration of poly-(oxy-1,2-ethylene dimethylene)-α-nonylphenyl-ω-hydroxyl and 0.08%-0.1% mass volume concentration of dioctyl sodium sulfosuccinate.

6. The colloidal gold immunochromatography method according to claim 5, characterized in that: The sample pad is a glass fiber pad.

7. A sample pad treatment solution, characterized in that: The sample pad treatment liquid is used for soaking the sample pad used in the colloidal gold immunochromatography method. The chromatography method is based on the double antibody sandwich method for detection. The sample pad treatment liquid contains poly-(oxy-1,2-ethylenedimethylene)-α-nonylphenyl-ω-hydroxyl and dioctyl sodium sulfosuccinate.

8. The sample pad treatment solution according to claim 7, characterized in that: The sample pad treatment solution contains 1%-1.5% mass volume concentration of poly-(oxy-1,2-ethylene dimethylene)-α-nonylphenyl-ω-hydroxyl and 0.08%-0.1% mass volume concentration of dioctyl sodium sulfosuccinate.

9. The sample pad treatment solution according to claim 8, characterized in that: The sample pad treatment solution is used for preparing a sample pad in a colloidal gold immunochromatography kit for detecting malarial protozoan antigens.

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