Product Informations

• Product No. BF06224
• Product Name Lentiviral Titer ELISA Test Kit (One-Step Method)
• Category 451
• Detection Range HIV-1 P24 抗原浓度：5-160pg/ml
• Precautions All components in the reagent kit taken out of the refrigerated environment and the specimens to be tested should be balanced at room temperature for 30 minutes before use. The microplate is vacuum-packed. Once opened, it is prone to failure due to moisture. Unused ones should be sealed and stored in self-sealing bags to remove air and be used within 3 days.
  If crystals appear in the washing solution, it can be dissolved at 37℃ before use.
  3. The result determination must be based on the reading of the microplate reader. It is recommended to use dual wavelengths of 450nm/630nm for detection and complete it within one hour after the reaction is terminated.
  4. The termination solution is a mixed strong acid. Safety must be observed when using it.
  5. When adding liquid, a liquid filler must be used and the accuracy of the liquid filler should be frequently checked.
  6. When washing, if done manually, add 300μl of washing solution to each well and shake for 30 to 60 seconds. If the plate washer is used for washing, each well must be filled with washing solution to prevent the presence of free enzymes at the well openings. When using a plate washer for washing, the soaking time should be set at 30 to 60 seconds.
  7. Operations should be carried out strictly in accordance with the instructions, and reagents of different batches must not be mixed
• Instruction Manual /files/uploads/instructions/2024/202409/BF06224_慢病毒滴度ELISA检测试剂盒(一步法)_说明书2400911.pdf
• Application 1、配液：将30 ml浓缩洗涤液（20倍）用蒸馏水稀释至600 ml；或取适量浓缩液按 需配制成1*洗涤液 本试剂盒采用ELISA 法可定性、定量检测HIV-1 P24蛋白，适用于血清、培养上清、样 品等中的慢病毒的滴度检测。检测范围为HIV-1 P24 抗原浓度：5-160pg/ml。仅限于实验 室研究。

Picture

Product Details

Kit composition
 

Microplate	8孔×12
Pyrolysis fluid	4ml
Positive control	0. 15ml
Enzyme conjugate	10ml
20× concentrated washing solution	30ml
Substrate solution	12ml
Termination solution	6ml

Storage: 2-8℃ (away from light), shelf life: 1 year


I. Principles
This kit uses the double antibody sandwich method to determine the HIV-1P24 protein. That is, the monoclonal antibody against HIV-1P24 is coated on the microplate. The sample to be tested is added to the coated reaction well and incubated. If the sample contains HIV-1P24 protein, this protein will form an antigen-antibody complex with the antibody in the well. Add the enzyme-labeled anti-HIV-1P24 monoclonal antibody, incubate, and then the enzyme-labeled antibody binds to the antigen on the antigen-antibody complex, and then reacts with the substrate to develop color. Finally, the reaction was terminated with the stop solution and the OD value was determined by an microplate reader.

Ii. Uses
This kit uses ELISA to qualitatively and quantitatively detect HIV-1 P24 protein and is suitable for the titer detection of lentivirus in cell culture supernatant samples. The detection range is HIV-1 P24 antigen concentration: 5-160pg/ml. Only for laboratory research.

Iii. Usage Method
1. Solution preparation: Dilute 30 ml of concentrated washing solution (20 times) with distilled water to 600 ml; Or take an appropriate amount of the concentrated liquid and prepare it into a 1* washing solution as needed.
2. Quantitative dilution of HIV-1 P24 series: The positive control (PC) contained 6.4 ng/ml of recombinant HIV-1 P24 antigen. Six concentrations of the positive control were diluted in a gradient series using complete DMEM or 1* washing solution (the dilution method is shown in the table below). Two replicate Wells were performed for each dilution concentration, and the sample to be tested was diluted using complete DMEM or 1* washing solution.
Note: If you use complete DMEM to dilute the positive control, please dilute the sample to be tested with complete DMEM. If you dilute the positive control with 1* washing solution, please dilute the sample to be tested with 1* washing solution.

Table 1 Quantitative dilution of HIV-1 P24 series

HIV-1 P24 antigen concentration (pg/ml)       Pipe number     Antigen （ ml）    Dilution solution（ ml）   
 

160 pg/ml	1#	0.01 ml PC	0.39ml
80 pg/ml	2#	0.2 ml 1#	0.2 ml
40 pg/ml	3#	0.2 ml 2#	0.2 ml
20 pg/ml	4#	0.2 ml 3#	0.2 ml
10 pg/ml	5#	0.2 ml 4#	0.2 ml
5 pg/ml	6#	0.2 ml 5#	0.2 ml

3. Numbering: Number the corresponding Wells of the samples in sequence. Each plate should have a total of four negative control Wells (it is recommended to have four Wells, with a minimum of two Wells) and two positive control Wells for each gradient. Among them, the negative control is the solution for diluting the sample to be tested and the positive control in step 2, and the positive control is the six tubes of HIV-1 p24 antigen that you gradient-diluted in Table 1.
4. Add 25μl of the lysis buffer to the reaction Wells.
5. Add 75μl of the sample or reference substance to be tested to the reaction well.
6. Add 75μl of the enzyme conjugate to the reaction well. Shake for 30 to 60 seconds to mix well, and incubate at 37 ° C for 50 minutes.
Wash the plate 5 times, pat dry and add 100μl of single-component TMB substrate to each well. Incubate at 37℃ for 10 minutes.
8. Add 50μl of stop solution per well, shake for 30 to 60 seconds to mix well. The OD value can stabilize within 1 hour.
9. Read with an microplate reader at a wavelength of 450nm(it is recommended to use a dual-wavelength microplate reader with a reference wavelength of 630nm).
Iv. Result Judgment

The normal range of the negative control: Under normal circumstances, the OD value of the negative control should be ≤0.05.
2. Normal range of positive control: Under normal circumstances, the OD value of a 160 pg/ml positive control is quantitatively determined to be ≥1.00
3. Critical value = average value of negative control +0.02;
4. Positive qualitative determination: If the OD value of the test specimen is less than the critical value, it is negative. If the OD value of the test specimen is greater than or equal to the cut-off value, it is positive.
5. Positive quantitative determination: Refer to the method shown in the following chart to draw the standard curve and conduct quantitative determination. It is recommended to use linear regression or four-parameter curve fitting.

Table 2. Positive Quantitative Determination

Pipe number      HIV-1 P24 antigen concentration (pg/ml)             ODvalue              Average OD value
 

1#	160 pg/ml	2.693	2.582	2.6375
2#	80 pg/ml	1.248	1.267	1.2575
3#	40 pg/ml	0.615	0.624	0.6195
4#	20 pg/ml	0.327	0.319	0.323
5#	10 pg/ml	0.149	0.15	0.1495
6#	5 pg/ml                                   0.076               0.078			0.077

NC1=0.023   NC2=0.022   NC3=0.022  NC4=0.021   平均值NCx=0.022 Cut  off=0.022+0.02=0.042