Respiratory syncytial virus IgM ELISA kit instructions
(Germany IBL: RE56891)
Germany IBL China exclusive agent "Shenzhen Kerunda Biological Engineering Co., Ltd." dedicated to serve you
1 , the principle of experiment
This enzyme-free kit can be used for in vitro qualitative and quantitative detection of anti-RSV (respiratory syncytial virus) IgM antibodies in human serum.
2 , the preface
In a six-month-old infant with bronchiolitis or pneumonia, a clear association between respiratory RSV infection and specific clinical symptoms can be found. In older babies and children, the symptoms of infection are milder. More than 25% of RSV respiratory infections are detectable. Due to the possibility of re-infection of RSV, it is believed that these re-infected antibodies are associated with a slow development of these diseases in adults (similar to a cold). However, especially in the early years, serum antibodies are not effective protection against respiratory infections. Therefore, this pathogen may cause bronchiolitis or cause pneumonia in a four-year-old baby. Based on the relationship of respiratory syncytial virus antigens, we can classify respiratory syncytial viruses into two broad categories (A and B). Viral surface glycoproteins (G glycoproteins and fusion glycoproteins) produce viral suppressor antibodies. The G glycoproteins of RSV A and B viruses are significantly different from F glycoproteins. Complement binding experiments for the serological diagnosis of RSV have not been very satisfactory. The enzyme-free assay for serological diagnosis of RSV infection has high diagnostic value because the enzyme-free detection sensitivity is high and the immunoglobulins of different antigens can be distinguished. In RSV infection, the IgM antibody response may not occur or be so weak that reliable experimental results cannot be obtained. In an acute infection, IgG antibody detection in a single sample is not significant because IgA antibodies may persist for months or years. The serological test recommended for acute RSV infection is to detect IgG antibodies in serum (double, requiring a significant increase in concentration).
3 , the principle of experiment
The sandwich method principle utilized by this solid phase ELISA kit. The coating was coated with antigen. A sample-specific antibody that binds to the coated antigen is detected with an enzyme-labeled secondary antibody specific for human IgM. After substrate reaction, the intensity of the color displayed is directly proportional to the amount of IgM-specific antibody detected. The results of the samples can be obtained directly using the standard curve.
4 , the kit consists of:
4×2ml | Standard AD | 1;10;50;150U/mL, stand-by Standard A = negative control Standard B = critical quality control Standard C=weak positive control standard D=positive control Contains anti-RSV IgM antibody, PBS (phosphate buffer), stabilizer. |
1×14ml | Enzyme standard IgM | Red, stand by. Contains peroxidase-labeled anti-human IgM, protein buffer, stabilizer. |
1×12×8 | Coated board | Detachable, coated with specific antigen. |
1×14ml | TMB substrate solution | With TMB |
1×14ml | TMB stop solution | 0.5M H 2 SO 4 |
1×60ml | Diluent | PBS buffer, <0.1% NaN 3 |
1×60ml | Concentrated washing solution | Concentrated (10×), containing PBS buffer, Tween 20 |
2× | Sticky metal plate | Used to cover the coated board during incubation |
1× | Plastic bag | Used to store unused slats |
5 , the materials required for the experiment but the kit does not provide
1) RF adsorbent
2) Pipette, volume: 5; 50; 100; 500 μL
3) Calibrator
4) Sample dilution test tube (1ml)
5) 8-channel pipette with reagent reservoir
6) Washing bottles, automatic or semi-automatic washing machine
7) Microplate reader that can read at 450nm
8) Double distilled or deionized water
9) Absorbent paper, sampling tips and timer
6. Preparation instructions before the experiment
6.1 ingredient preparation
Dilution/dissolution | ingredient | Diluent | proportion | Remarks | Storage | stability | |
60ml | detergent | Add 600ml | Double distilled water | 1:10 | Heat to 37 ° C to dissolve Crystallize, fully mixed | 2-8 ° C | 8 weeks |
6.2 Sample dilution
sample | dilution | Diluent | proportion | Remarks |
Serum/plasma | Full dilution | Dilution buffer | 1:101 | Example: 5μL+500μL |
The IgM concentration in the sample above the highest standard should be further diluted.
6.3 Treatment with RF adsorbent:
note | To avoid interference with specific IgG and rheumatoid factors, the patient's serum should be treated with RF sorbent (RE59059). Do not use RF adsorbents for standards and controls. |
1. | 20 μL of RF adsorbent was added to 400 μL of the sample diluted 1:101. Mix well. |
2. | Incubation at room temperature (18-25 ° C) ≥ 1 min (<15 min) |
In order to avoid adsorption of specific antibodies, incubation times should be avoided > 15 min.
The pretreated sample may be cloudy.
7 , experimental steps:
1) Add 100 μL of standard and diluted sample to the corresponding wells and use standard B only for qualitative testing.
2) Incubate for 60 minutes at room temperature (18 ° C - 25 ° C) after sealing.
3) Remove the sticky metal plate and discard the liquid in the hole. The plate was washed 3 times with 300 μL of washing solution, and patted dry on absorbent paper.
4) Add 100 μL of enzyme conjugate to each well.
5) Incubate for 30 minutes at room temperature (18 ° C - 25 ° C) after sealing.
6) Remove the sticky metal plate and discard the liquid in the hole. The plate was washed 3 times with 300 μL of washing solution, and patted dry on absorbent paper. When adding the substrate and stop solution, an 8-well micropipette can be used if available. Make sure both the substrate and stop solution are added at the same time interval. Use the exact volume and avoid bubble formation.
7) Add 100 μL of TMB substrate solution to each well.
8) Incubate for 20 minutes at room temperature (18 ° C - 25 ° C) in the dark.
9) Add 100 μL of LTMB Stop Solution to each well to stop the substrate reaction. Gently shake the coated plate to mix the reagents evenly. At this point, the color blue turns yellow.
10) OD value was measured at 450 nm within 60 minutes after the addition of the stop solution.
8 , the result of calculation
A standard curve is drawn on semi-logarithmic paper or by an automatically generated method with the OD of the standard being the Y-axis and the concentration being the X-axis. Good experimental results can be obtained with a stereogram, a 4-parameter logogram or a log-log plot. For the standard curve, calculate it with each single value of the standard (the value of the sample result that is obviously abnormal must be deleted, and a more reasonable single value should be used). The concentration of the sample can be obtained directly from the standard curve. Once the sample has been diluted, it should be multiplied by the corresponding dilution factor. If the measured concentration of the sample is above the highest standard, the sample should be diluted and retested as described in the pre-experiment preparation instructions.
9. Result judgment: >12 U/ml is positive, 8 - 12 U/ml is suspicious, < 8 U/ml is negative
This test result is not the only factor determining the outcome of any treatment. It should be judged in combination with clinical observations and diagnostic results.
This translation is for reference only, please refer to the original for details.
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