Colloidal gold test strip for detecting renal injury molecule 1 (kim-1) and preparation method thereof

Abstract

The invention provides a colloidal gold test strip for detecting renal injury molecule 1 (kim-1) and a preparation method thereof, and aims to provide a bedside rapid diagnosis tool which is high in accuracy, strong in specificity, simple to operate and capable of rapidly reporting results. The test strip is formed by sequentially connecting a plastic bottom lining, a nitrocellulose membrane, a gold label pad, a sample pad and a water absorption pad, wherein a detection line and a quality control line are arranged on the nitrocellulose membrane. The detection line contains a KIM-1 monoclonal antibody, the quality control line contains a mouse anti-human IgG antibody, and the colloidal gold pad is coated with a colloidal gold labeled KIM-1 monoclonal antibody, so that a double-antibody sandwich structure is formed to specifically recognize human KIM-1. The preparation method comprises the following steps: spraying an antibody on the nitrocellulose membrane to form the detection line and the quality control line, preparing a colloidal gold antibody compound solution, and assembling each component. By optimizing the antibody concentration and a colloidal gold preparation process, the sensitivity and the stability of the test strip are ensured. The test strip is suitable for rapidly detecting kidney injury, and has important value for early diagnosis and treatment monitoring of acute and chronic kidney injury.

Description

Technical Field

[0001] The invention relates to the field of clinical medical diagnosis, and in particular to a colloidal gold test strip for detecting kidney injury molecule 1 (kim-1) and a preparation method thereof. Background Art

[0002] The results of Northern blot hybridization analysis showed that Kim-1 was not expressed in fetal liver and fetal kidney, but was weakly expressed in normal adult liver, kidney, and spleen, and its expression was significantly enhanced in renal tissue after ischemic injury. Since Kim-1 is only expressed in damaged renal tubular epithelial cells, many research results believe that it can be a sensitive marker for renal injury. In mouse experiments, the results showed that the level of Kim-1 in urine was highly correlated with the severity of renal injury, whether it was ischemic renal injury or drug toxic renal injury. When the degree of renal injury was alleviated or after intervention treatment, the level of Kim-1 in urine would decrease. At the same time, Kim-1 can also provide predictive value for the prognosis of renal injury. Liangos et al. found in the results of a study of 201 hospitalized patients that the level of Kim-1 was closely related to the severity of the patient's prognosis; in a single-center study, the level of Kim-1 in the urine of patients with acute tubular necrosis was 12 times higher than the normal level. In the chronic kidney injury mouse model, mice with severe proteinuria had higher levels of Kim-1, indicating an increased risk of developing end-stage renal disease. Kim-1 can also assess the prognosis of transplanted kidney function. Research results have found that the more Kim-1 is expressed in proximal tubular epithelial cells, the better the recovery of transplanted kidney function; and the follow-up study of transplanted kidney function and kidney injury molecule-1 levels in urine in 145 renal transplant patients found that the higher the level of Kim-1, the poorer the transplanted kidney function is or even completely loses function.

[0003] In summary, as a non-invasive and sensitive renal injury marker, kidney injury molecule 1 (Kim-1) can not only be used for the early diagnosis and treatment monitoring of acute and chronic renal injury, but also for the renal toxicity monitoring of compounds during drug development or the renal toxicity monitoring of clinical drugs.

[0004] At present, the main methods for detecting kidney injury molecule 1 (Kim-1) include enzyme-linked immunosorbent assay (ELISA), immunoturbidimetry, sandwich ELISA, and competitive ELISA to determine the level of Kim-1. However, these methods require matching instruments, are cumbersome to operate, take a long time to test, have large inter-laboratory differences, cannot make rapid early predictions of acute renal failure, and cannot be used for rapid bedside detection and diagnosis. Summary of the invention

[0005] In view of this, in order to overcome the shortcomings of the prior art, the present invention provides a colloidal gold test strip for detecting kidney injury molecule 1 (KIM-1) and a preparation method thereof, which has high accuracy, strong specificity, simple and convenient operation, rapid result reporting and is suitable for bedside rapid diagnosis.

[0006] The technical scheme of the present invention is implemented as follows: a colloidal gold test strip for detecting kidney injury molecule 1 (Kim-1), comprising a plastic backing, a nitrocellulose membrane, a gold label pad, a sample pad and a water absorbent pad connected in sequence, wherein the nitrocellulose membrane is provided with a detection line and a quality control line parallel to each other, the detection line contains a KIM-1 monoclonal antibody liquid, the quality control line contains a mouse anti-human IgG antibody, the gold label pad is coated with a colloidal gold antibody complex solution, the solution contains a monoclonal antibody liquid that specifically recognizes human KIM-1, the KIM-1 monoclonal antibody liquid on the detection line and the colloidal gold-labeled KIM-1 monoclonal antibody liquid on the gold label pad can be paired with each other to form a double antibody sandwich structure.

[0007] Furthermore, the acid cellulose membrane, the gold label pad, the sample pad and the water-absorbing pad are pasted on a plastic backing.

[0008] Furthermore, a method for preparing a colloidal gold test strip for detecting kidney injury molecule 1 (kim-1) comprises the following steps:

[0009] S1, nitrocellulose membrane coating:

[0010] ① Detection line coating: Kim-1 monoclonal antibody solution is sprayed on the nitrocellulose membrane to form a detection line, and then placed in a drying oven at 20°C for 12 hours;

[0011] ② Quality control line coating: Spray mouse anti-human IgG antibody solution on the nitrocellulose membrane to form a quality control line, parallel to the test line, with a spacing of 4mm, place in a drying oven at 20℃ for 12 hours, and store in a sealed and dry place;

[0012] S2, Gold Standard Pad Package:

[0013] ① Gold label pad treatment: The glass cellulose membrane / polyester membrane was immersed in a buffer solution (20 mmol / l PBS, 1% casein, 1% PVP, 1% sucrose solution, 0.2% Triton) for 3 hours, and then taken out and dried at 25°C for 8 hours.

[0014] ② Gold label pad coating: Spray the Kim-1 monoclonal antibody colloidal gold complex solution, which is different from that used in the test line, evenly on the treated gold label pad at 1 μl / cm with a gold sprayer, dry at 25°C for 8 hours, and seal and store in a dry place;

[0015] S3. Sample pad preparation:

[0016] The sample pad was immersed in 0.1M PBS buffer solution for 3 hours, and then taken out and dried at 25°C for 8 hours;

[0017] S4. Preparation of absorbent pad:

[0018] Cut the absorbent paper into 30cm*2.7cm;

[0019] S5. Colloidal gold test strip assembly:

[0020] Plastic backing, sample pad and absorbent paper are common components of this type of products. The sample pad, gold label pad, nitrocellulose membrane and absorbent pad are pasted on the plastic backing, and the pasted large plate is cut into 5.6 cm test strips with a chopper and assembled into kim-1 colloidal gold test strips.

[0021] Furthermore, the S1 kim-1 monoclonal antibody solution is diluted to a concentration of 1.8 mg / ml to 2.2 mg / ml using a PB buffer solution of 18 mmol / L to 22 mmol / L and pH 7.2 to 7.5.

[0022] Furthermore, the concentration of mouse anti-human IgG antibody coated on the quality control line of S1 is 3 mg / ml to 5 mg / ml.

[0023] Furthermore, the preparation of the colloidal gold-antibody complex solution of S2 comprises the following steps:

[0024] a. Preparation of colloidal gold: Heat 100 ml of 0.01% chloroauric acid aqueous solution to boiling, quickly add 1 ml of 1% trisodium citrate aqueous solution under stirring until the color of the solution changes from golden yellow to red, continue boiling for 5 minutes until the color remains stable, cool and restore to the original volume, add 0.02% sodium azide (NaN3), filter and sterilize again through a 0.22 μm filter membrane to obtain a colloidal gold solution, and store at 4 ° C for use;

[0025] b. Affinity purification: Use 0.2 mol / L potassium carbonate solution to adjust the pH of the colloidal gold solution to 7.8, then add avidin to form an avidin-colloidal gold solution, the mass volume ratio of the avidin and the colloidal gold solution is 10:1 μg / ml, and react in a 25°C water bath for 30 minutes; then add 5% BSA to block for 20 minutes, centrifuge to obtain the precipitate, use 5% BSA to restore the precipitate to a final concentration of 1% avidin-colloidal gold solution, and store at 4°C for use;

[0026] c. Preparation of colloidal gold-antibody complex solution: Add monoclonal antibody-biotin to the colloidal gold solution prepared in step b above, wherein the mass ratio of monoclonal antibody-biotin is 20:5, and the mass volume ratio of the antibody to the solution is 50:1 in μg / ml. Take 100 ml and stir for reaction at 37°C for 40 minutes, then add 5% BSA and stir for 20 minutes, centrifuge at 6000 rpm for 20 minutes, discard the supernatant, restore the precipitate to the original volume with gold-labeled antibody preservation solution, and store the final product at 4°C to obtain a colloidal gold-antibody complex solution.

[0027] Furthermore, the chloroauric acid aqueous solution in step a is prepared by dissolving 1 gram of chloroauric acid in 100 ml of ultrapure water to form a 1% chloroauric acid aqueous solution, which is then dissolved at room temperature, filtered through a 0.22 μm filter membrane for sterilization, and stored at 4° C. for standby use; and diluted to a concentration of 0.01% before use.

[0028] Furthermore, the trisodium citrate aqueous solution in step a is prepared by weighing 1 g of trisodium citrate, dissolving it in 100 ml of ultrapure water to form a 1% trisodium citrate aqueous solution, filtering it through a 0.22 μm filter membrane for sterilization, and storing it at 4° C. for later use.

[0029] Furthermore, the potassium carbonate solution in step b is prepared by weighing 2.76 g of potassium carbonate, dissolving it in 100 ml of ultrapure water to form a 0.2 mol / L potassium carbonate solution, filtering it through a 0.22 micron filter membrane for sterilization, and storing it at room temperature.

[0030] Furthermore, the gold-labeled antibody preservation solution in step c is prepared by dissolving 15 g of sucrose and 20 μL of 0.02% sodium azide (NaN3) in 100 ml of an aqueous solution containing 1% bovine serum albumin (BSA) and a pH value of 7.6, filtering through a 0.22 μm filter membrane for sterilization, and storing at 4°C for future use.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] (1) Easy operation: The present invention uses the double antibody sandwich principle to qualitatively detect the kim-1 level. The specimen (urine) is dropped into the sample pad. After 5-10 minutes, the negative / positive result is judged according to the color (red) of the strip on the test line. If no red strip appears on the quality control line, the test strip is invalid.

[0033] (2) High accuracy and specificity: The monoclonal antibody solution that specifically recognizes human KIM-1 on the test line is paired with the KIM-1 monoclonal antibody solution labeled with colloidal gold on the gold label pad to form a stable and specific binding, thereby greatly improving the accuracy and specificity of the test and effectively avoiding false positive or false negative results caused by nonspecific binding.

[0034] (3) Rapid reporting of results: Since colloidal gold has a unique color development effect, when kim-1 is present in the sample to be tested, a clear color change will quickly form on the test line, thereby quickly giving the test results. This instant reporting feature enables doctors to make quick judgments and adjust treatment plans in a timely manner.

[0035] (4) Good stability: In the preparation process, the present invention strictly prepares and filters and sterilizes key raw materials such as chloroauric acid, trisodium citrate, potassium carbonate, etc., to ensure the stability of the colloidal gold antibody complex solution and the long-term storage effect of the test strip. In addition, by optimizing the antibody concentration and reaction conditions, the stability and reliability of the test strip are further improved. DETAILED DESCRIPTION

[0036] In order to better understand the technical content of the present invention, specific embodiments are provided below to further illustrate the present invention.

[0037] Unless otherwise specified, the experimental methods used in the embodiments of the present invention are all conventional methods.

[0038] Unless otherwise specified, the materials, reagents, etc. used in the embodiments of the present invention can be obtained from commercial sources.

[0039] Example 1 - Preparation of colloidal gold test strips for detecting kidney injury molecule 1 (kim-1)

[0040] 1. Preparation of colloidal gold-antibody complex solution:

[0041] (1) preparing a chloroauric acid solution: dissolving 1 g of chloroauric acid in 100 ml of ultrapure water to form a 1% chloroauric acid aqueous solution; after dissolving at room temperature, filtering through a 0.22 μm filter membrane for sterilization, and storing at 4° C. for later use; before use, diluting the 1% chloroauric acid aqueous solution to a concentration of 0.01%;

[0042] (2) Preparation of trisodium citrate solution: Weigh 1 g of trisodium citrate and dissolve it in 100 ml of ultrapure water to form a 1% trisodium citrate aqueous solution, filter through a 0.22 μm filter membrane for sterilization, and store at 4° C. until use;

[0043] (3) Preparation of potassium carbonate solution: Weigh 2.76 g of potassium carbonate and dissolve it in 100 ml of ultrapure water to form a 0.2 mol / L potassium carbonate solution. Filter through a 0.22 μm filter membrane to sterilize and store at room temperature.

[0044] (4) Prepare the gold-labeled antibody storage solution: dissolve 15 g of sucrose and 20 μL of 0.02% sodium azide (NaN3) in 100 ml of an aqueous solution containing 1% bovine serum albumin (BSA) and a pH of 7.6, filter through a 0.22 μm filter membrane for sterilization, and store at 4°C until use;

[0045] (5) Preparation of colloidal gold: Heat 100 ml of the 0.01% chloroauric acid aqueous solution diluted in step (1) in a microwave oven until boiling, quickly and accurately add 1 ml of the 1% trisodium citrate aqueous solution prepared in step (2) while stirring, until the color of the solution changes from golden yellow to red, continue boiling at medium speed for 5 minutes until the color remains stable, return to the original volume after cooling, add 0.02% sodium azide (NaN3), filter through a 0.22 μm filter membrane again for sterilization, and store at 4°C for use;

[0046] (6) Affinity purification: first, the pH of the colloidal gold solution prepared in step (5) is adjusted to 7.8 using the 0.2 mol / L potassium carbonate solution prepared in step (3), and then avidin is added to form an avidin-colloidal gold solution, wherein the mass volume ratio of the avidin to the colloidal gold solution is 10:1 in μg / ml, and the solution is reacted in a 25°C water bath for 30 minutes; then, 5% BSA is added to block the solution for 20 minutes, and the precipitate is collected by centrifugation, and the precipitate is restored to a final concentration of 1% colloidal gold solution using 5% BSA, and the solution is stored at 4°C for later use;

[0047] (7) Preparation of colloidal gold-antibody complex solution: 50 μg monoclonal antibody-biotin is added per milliliter of colloidal gold solution. The monoclonal antibody-biotin is kim-1 monoclonal antibody and vitamin B7 in a mass ratio of 20:5. The antibody is added to the colloidal gold solution, 100 ml is stirred and reacted at 37°C for 40 minutes, and then 5% BSA is added to block and stir for 20 minutes. Centrifuge at 6000 rpm for 20 minutes, discard the supernatant, and use the gold-labeled antibody preservation solution prepared in step (4) to restore the precipitate to the original volume. The final product is stored at 4°C to obtain a colloidal gold-antibody complex solution.

[0048] 2.Colloidal gold test strip assembly

[0049] ⑴Nitrocellulose membrane coating

[0050] ① Detection line coating: Kim-1 monoclonal antibody was diluted with 1.8 mg / ml PB buffer solution at pH 7.5, and 1 μl / cm was used to draw a detection line on the nitrocellulose membrane, and then placed in a drying oven at 20°C for 12 hours.

[0051] ②Quality control line coating: Use mouse anti-human IgG antibody at a concentration of 3 mg / ml and 1 μl / cm to draw a quality control line on the nitrocellulose membrane, parallel to the test line with a spacing of 4 mm. Place it in a drying oven at 20°C for 12 hours and store it in a sealed and dry place.

[0052] ⑵Gold standard padding

[0053] ① Gold label pad treatment: The glass cellulose membrane / polyester membrane was immersed in a buffer solution (20 mmol / l PBS, 1% casein, 1% PVP, 1% sucrose solution, 0.2% Triton) for 3 hours, and then taken out and dried at 25°C for 8 hours.

[0054] ② Gold label pad coating: Spray the Kim-1 monoclonal antibody colloidal gold complex solution, which is different from that used in the test line, evenly on the treated gold label pad at 1 μl / cm using a gold sprayer, dry at 25°C for 8 hours, and seal and store in a dry place.

[0055] ⑶ Sample pad preparation:

[0056] The sample pad was immersed in 0.1 M PBS buffer solution for 3 hours, and then taken out and dried at 25° C. for 8 hours.

[0057] ⑷ Preparation of absorbent pad:

[0058] Cut the absorbent paper into 30cm*2.7cm.

[0059] ⑸Colloidal gold test strip assembly:

[0060] Plastic backing, sample pad and absorbent paper are common components of this type of products. The sample pad, gold label pad, nitrocellulose membrane and absorbent pad are pasted on the plastic backing, and the pasted large plate is cut into 5.6 cm test strips with a chopper.

[0061] Example 2-Use of colloidal gold test paper for detecting kidney injury molecule 1 (kim-1)

[0062] Drop the sample to be tested onto the sample pad of the test strip. After 3-5 minutes, if a red strip appears at the test line and the quality control line of the test strip respectively, it means that the sample to be tested contains the corresponding antigen, which is a positive sample; if a red strip appears only at the quality control line and the test line does not change, it means that the sample to be tested does not contain the corresponding antigen, which is a negative sample; if no red strip appears at both the quality control line and the test line of the test strip, it means that the test is invalid.

[0063] Test Example 1

[0064] The clinical equivalence of the product "Colloidal gold test strip for detecting kidney injury molecule 1 (KIM-1)" of the present invention was evaluated by blind synchronous testing with the "gold standard" for diagnosing kidney disease. 1047 valid samples were used in the Affiliated Hospital of Jiangxi University of Traditional Chinese Medicine. 292 cases were diagnosed / detected positive by both methods, and 675 cases were diagnosed / detected negative by both methods; 31 cases were diagnosed positive by the product of the present invention and negative by the gold standard; 49 cases were diagnosed negative by the product of the present invention and positive by the gold standard. The test results are shown in the table below.

[0065] Table: Summary of test results of two methods (n = 1047 cases)

[0066]

[0067] The positive compliance rate of the test results of the product of the present invention is 90.4%, and the negative compliance rate is 93.2%.

[0068] The diagnostic consistency rate of the two methods = 967÷1047 = 92.4%.

[0069] Comparison of the consistency of the detection results of the two methods;

[0070] After statistical calculation, the Kappa value of the detection results of the two methods was 0.824, indicating that the detection results of the two methods were very consistent.

[0071] The Kappa consistency test was statistically significant (P<0.01), indicating that the diagnosis / detection results of the two methods were consistent. Conclusion: The product of the present invention is clinically equivalent to the gold standard.

[0072] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A colloidal gold test strip for detecting kidney injury molecule 1 (kim-1), characterized in that: The invention comprises a plastic backing, a nitrocellulose membrane, a gold label pad, a sample pad and a water-absorbing pad which are connected in sequence. The nitrocellulose membrane is provided with a detection line and a quality control line which are parallel to each other. The detection line contains a kim-1 monoclonal antibody liquid, and the quality control line contains a mouse anti-human IgG antibody. The gold label pad is coated with a colloidal gold antibody complex solution, and the solution contains a kim-1 monoclonal antibody liquid. The kim-1 monoclonal antibody liquid on the detection line and the colloidal gold-labeled kim-1 monoclonal antibody liquid on the gold label pad can be paired with each other to form a double antibody sandwich structure.

2. A colloidal gold test strip for detecting kidney injury molecule 1 (kim-1) as claimed in claim 1, characterized in that: The acid cellulose membrane, the gold label pad, the sample pad and the water-absorbing pad are pasted on the plastic backing.

3. A method for preparing a colloidal gold test strip for detecting kidney injury molecule 1 (kim-1) as claimed in claim 1, characterized in that: The following steps are involved: S1, the nitrocellulose membrane is sprayed with kim-1 monoclonal antibody solution to form the test line, and the nitrocellulose membrane is sprayed with mouse anti-human IgG antibody solution to form the quality control line; S2, adding kim-1 monoclonal antibody solution to colloidal gold to obtain a colloidal gold-antibody complex solution, and spraying the complex solution onto a glass cellulose membrane or a polyester membrane to obtain a gold label pad; S3. Paste the plastic backing, nitrocellulose membrane, gold label pad, sample pad and absorbent pad to assemble a colloidal gold test strip for detecting kim-1.

4. A method for preparing a colloidal gold test strip for detecting kidney injury molecule 1 (kim-1) as claimed in claim 3, characterized in that: The S1 kim-1 monoclonal antibody solution is diluted to a concentration of 1.8 mg / ml to 2.2 mg / ml using a PB buffer solution of 18 mmol / L to 22 mmol / L and pH 7.2 to 7.

5.

5. A method for preparing a colloidal gold test strip for detecting kidney injury molecule 1 (kim-1) as claimed in claim 3, characterized in that: The concentration of mouse anti-human IgG antibody coated on the quality control line of S1 is 3 mg / ml to 5 mg / ml.

6. A method for preparing a colloidal gold test strip for detecting kidney injury molecule 1 (kim-1) as claimed in claim 3, characterized in that: The preparation of the colloidal gold-antibody complex solution of S2 comprises the following steps: a. Preparation of colloidal gold: Heat 100 ml of 0.01% chloroauric acid aqueous solution to boiling, quickly add 1 ml of 1% trisodium citrate aqueous solution under stirring until the color of the solution changes from golden yellow to red, continue boiling for 5 minutes until the color remains stable, cool and restore to the original volume, add 0.02% sodium azide (NaN3), filter and sterilize again through a 0.22 μm filter membrane to obtain a colloidal gold solution, and store at 4 ° C for use; b. Affinity purification: Use 0.2 mol / L potassium carbonate solution to adjust the pH of the colloidal gold solution to 7.8, then add avidin to form an avidin-colloidal gold solution, the mass volume ratio of the avidin and the colloidal gold solution is 10:1 μg / ml, and react in a 25°C water bath for 30 minutes; then add 5% BSA to block for 20 minutes, centrifuge to obtain the precipitate, use 5% BSA to restore the precipitate to a final concentration of 1% avidin-colloidal gold solution, and store at 4°C for use; c. Preparation of colloidal gold-antibody complex solution: Add monoclonal antibody-biotin to the colloidal gold solution prepared in step b above, wherein the mass ratio of monoclonal antibody-biotin is 20:5, and the mass volume ratio of the antibody to the solution is 50:1 in μg / ml. Take 100 ml and stir for reaction at 37°C for 40 minutes, then add 5% BSA and stir for 20 minutes, centrifuge at 6000 rpm for 20 minutes, discard the supernatant, restore the precipitate to the original volume with gold-labeled antibody preservation solution, and store the final product at 4°C to obtain a colloidal gold-antibody complex solution.

7. A method for preparing a colloidal gold test strip for detecting kidney injury molecule 1 (kim-1) as claimed in claim 6, characterized in that: The chloroauric acid aqueous solution in step a is prepared by dissolving 1 gram of chloroauric acid in 100 ml of ultrapure water to form a 1% chloroauric acid aqueous solution, which is then dissolved at room temperature, filtered through a 0.22 μm filter membrane for sterilization, and stored at 4° C. for standby use; and diluted to a concentration of 0.01% before use.

8. A method for preparing a colloidal gold test strip for detecting kidney injury molecule 1 (kim-1) as claimed in claim 6, characterized in that: In step a, the trisodium citrate aqueous solution is prepared by weighing 1 g of trisodium citrate, dissolving it in 100 ml of ultrapure water to form a 1% trisodium citrate aqueous solution, filtering it through a 0.22 μm filter membrane for sterilization, and storing it at 4° C. for later use.

9. A method for preparing a colloidal gold test strip for detecting kidney injury molecule 1 (kim-1) as claimed in claim 6, characterized in that: The potassium carbonate solution in step b is prepared by weighing 2.76 g of potassium carbonate, dissolving it in 100 ml of ultrapure water to form a 0.2 mol / L potassium carbonate solution, filtering it through a 0.22 μm filter membrane for sterilization, and storing it at room temperature.

10. A method for preparing a colloidal gold test strip for detecting kidney injury molecule 1 (kim-1) as claimed in claim 6, characterized in that: The gold-labeled antibody storage solution in step c is prepared by dissolving 15 g of sucrose and 20 μL of 0.02% sodium azide (NaN3) in 100 ml of an aqueous solution containing 1% bovine serum albumin (BSA) and a pH value of 7.6, filtering through a 0.22 μm filter membrane for sterilization, and storing at 4°C for later use.

Images