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Forest encephalitis IgG ELISA kit instructions
Forest encephalitis IgG ELISA kit instructions
(Germany IBL imported original: RE57401)
Germany IBL China exclusive agent "Shenzhen Kerunda Biological Engineering Co., Ltd." dedicated to serve you
ã€expected usage】
The kit can be used to quantitatively detect anti-encephalitis virus (TEBV) IgG antibodies in human serum plasma and cerebrospinal fluid, thereby controlling humoral immune status and determining seroconversion after vaccination. Identification of significant and potential encephalitis virus infections, usually accompanied by detection of IgM against encephalitis virus.
In Europe, forest encephalitis and Lyme disease are the most common vector-borne diseases. Lyme disease is widely distributed, but forest encephalitis exists only in special places (Southern Germany, Thuringia, Austria, Switzerland, Hungary, Sweden, Czech Republic, Slovak Republic, Croatia, Slovenia, and some former Soviet Union regions, etc.).
The infections of both diseases are similar to their development and contain two or more stages. The viremia phase of forest encephalitis contains an incubation period of 3 to 14 days, and symptoms of influenza are similar at the beginning of this phase (1-8 days). After an interval of no fever for a week, the infection enters the second phase, characterized by changes in the intensity of the symptoms of the nervous system, which may last for several weeks.
In the early stages of the second phase of the disease, IgM antibodies against forest encephalitis are usually detected. Antibody levels peaked after 2 to 6 weeks. It may take up to 10 months for the antibody level to be reduced below detectable. IgG antibodies against forest encephalitis are detected at the same time as or after the emergence of IgM antibodies. Infection means the presence of immunity that usually lasts for a lifetime. Vaccination can also prevent disease.
Forest encephalitis-specific antibodies in cerebrospinal fluid may be caused by dysfunction of the blood-brain barrier or by a local immune response when the immune response to the forest encephalitis antigen is preceded. Therefore, fluctuations in antibody levels in cerebrospinal fluid may be different from those in serum or plasma.
RF factor and specific IgG do not interfere with the detection of IgM due to the addition of RF/IgG adsorbents. The combination of these two detection systems can be used for humoral immune follow-up or infection after vaccination with forest encephalitis, early forest encephalitis infection and monitoring of antibody levels in human serum, plasma and cerebrospinal fluid.
[Detection principle]
This kit uses a two-step ELISA. The micropores are coated with inactivated forest encephalitis virus. The diluted serum, plasma or cerebrospinal fluid sample is incubated in the microwell. During the incubation period, the forest encephalitis-specific antibodies are combined with the forest encephalitis virus, and the unbound fraction is washed away by washing. The enzyme-linked conjugate is added for incubation, and the HRP-labeled anti-human IgG antibody specifically binds to the human IgG antibody. After adding the substrate solution, the color changes to blue. After incubation for a while, the reaction solution is terminated by adding a stop solution, the color is changed from blue to yellow, and finally read on the microplate reader.
This kit is shipped at room temperature and stored at 2 – 8 °C. Avoid high temperatures or direct sunlight. The storage and stability of the sample and prepared reagents are set forth in the corresponding sections. The coated slats remain stable during the life of the slats and should be sealed in a sachet with the desiccant when stored at 2 – 8 °C.
[sample collection and storage]
Serum, plasma or cerebrospinal fluid
Obey the general rules of venipuncture, it is very important to preserve the integrity of the sample from collection to the experiment. Be careful not to use samples of hemolysis, lipemia or jaundice. The turbidity of the sample should be centrifuged before the experiment and all particles removed. substance.
store
2~8°C
≤ -20 ° C (small bottle dispensing)
Avoid high temperatures and direct sunlight, avoid repeated freezing and thawing
stability
5d
12 months
[reagent composition]
Quantity
Identification
Component
1×12×8
MTP
Coated board
Ready-to-use, detachable, coated with inactivated TBE virus
2 x 75mL
DILBUF
Dilution buffer
Ready to use, red
1 x 0.6 mL
ENZCONJ CONC
Enzyme complex
Blue, enzyme-labeled anti-human IgG antibody
5×0.35mL
CAL 1-5
Concentrated calibrator
Containing human serum, stabilizers, preservatives
2×0.35mL
Control LL
Control HL
Quality Control LL+HL Concentration
Positive control serum, LL "low level" HL "high level"
Contains: human serum, stabilizers, preservatives.
The multiple of the concentration varies with different batch numbers. For details, please refer to the label on the bottle.
1×100mL
WASHBUF
CONC
Washing solution, 10 times concentrated
Phosphate buffer
2×12mL
TMB SUBS
TMB substrate solution
Ready to use, including TMB
1 x 12 mL
TMB STOP
TMB stop solution
Ready to use, containing 0.5M sulfuric acid
2×
FOIL
Sealing plate
Note: Washing liquid, substrate liquid, and stop solution can be exchanged with the following products: Diphtheria (RE57431) Forest Encephalitis IgM (RE57411) Tetanus (RE57441)
1. Single-use 5;25;50;100;500μl loading gun head
2. Whirl mixer
3. Sample dilution tube
4. Orbital oscillator (200-900rpm)
5. 8-channel micropipette and sample tank.
6. Bottle washing, automated or semi-automatic washing machine
7. 450nm microplate reader
8. Double distilled or deionized water
9. Paper towels, pipettes, timers
[Preparation before experiment]
1. Preparation of concentrated reagents (example for 32 wells)
Dilution/dissolution
Component
The amount of diluent added
Diluent
proportion
Note
store
stability
80μL
ENZCONJ CONC
8mL
DILBUF
1:101
Mix carefully
18-25 ° C
1 hour
10mL
WASHBUF
CONC
90mL
Distilled water
1:10
Mix carefully
2-8 ° C
2 months
2. Dilution of standards, controls and samples
Dilution method
Diluent
Dilution ratio
Remarks
CAL 1-5
Control LL
Control HL
Conventional dilution
DILBUF
1:101
Example: 10μL calibrator / control + 1000μL
Serum, plasma
Conventional dilution
DILBUF
1:101
Example: 10 μL sample + 1000 μL
Cerebrospinal fluid
Conventional dilution
DILBUF
1:9
Example: 50μL sample +400μL
The standard curve method requires 1 to 5 calibrators and accused serum; the concentration of the sample should be further diluted if it is higher than the highest calibrator concentration. For the one-point calibration rule, only calibrator 4 and quality control serum are required.
[Experimental steps]
1. Add 200 μL of diluted calibrators, controls, and samples to the appropriate wells.
2. Seal the plate with a sealing plate and incubate for 1 hour at room temperature (18-25 ° C)
3. Remove the sealing plate, discard the reaction solution, wash the plate 3 times with the diluted washing solution (250 μL/well/time), and finally take the residual liquid through the clapper.
4. Add 200 μL of diluted enzyme conjugate to each well
5. Seal the plate with a sealing plate and incubate for 1 hour at room temperature (18-25 ° C)
6. Repeat step 3
7. Add 200 μL of TMB substrate to each well
8. Incubate for 30 minutes at room temperature (18-25 ° C)
9. Add 50 μL of Stop Solution to each well in the order of TMB substrate solution and mix gently.
10. Read at 450 nm ± 10 nm with a microplate reader within 10 minutes after the addition of the stop solution.
[calculation of results]
Standard curve method
On semi-logarithmic paper (or using automated instruments), the OD value of each standard is the Y-axis, and the corresponding concentration is the standard curve for the X-axis. It is recommended to use a 4-parameter logistic function (Y=d+(ab)/( 1+(x/c) b )).
The concentration of the sample should be read directly from the standard curve. If there is dilution, the calculation should be multiplied by the dilution factor. If the concentration of the sample is too high, the sample should be diluted according to the pre-experiment setting instructions.
2. One-point calibration
(1) Manual testing of sample concentration
The average OD value of each of the calibrator 4, the quality control serum, and the sample is required. The OD of calibrator 4 must be within the specified range (see the one-point calibration method evaluation form). A special correction factor must be calculated for each test. The theoretical OD values ​​of calibrator 4 are all measured in a specific batch (see the one-point calibration evaluation form). Multiply the OD of the quality control serum and sample by the correction factor:
The quality control serum and sample concentrations can be read from the batch reference curve.
example:
The theoretical OD value of calibrator 4 OD=1.377
Detected OD value of calibrator 4 OD=1.273
Correction factor F F=1.082
Sample detection OD value OD=0.937
Corrected OD value of the sample OD=1.014
Read concentration 0.5VIEU/mL
(2) Calculation of cerebrospinal fluid sample concentration (a little calibration method software)
Open the Excel file named "One Point Calibration TBE IgG Liquor" and fill in the following indicators of the QC certificate into Excel:
Theoretical OD value of calibrator 4
OD range of calibrator 4
Theoretical OD values ​​for critical 1 and critical 2
Critical 1 OD = lowest point of the gray zone
Critical 2 OD = highest point of the gray zone
The average OD of the detected calibrator 4 is filled in, and the identification and OD of the cerebrospinal fluid sample are inserted into the prepared calculation sheet. The evaluation and OD of cerebrospinal fluid samples are automatically completed.
[Determination of results]
Serum/plasma
<63VIEU/mL negative
63-126VIEU/mL gray area
>126VIEU/mL positive
Cerebrospinal fluid
OD<critical 1 theoretical OD negative
Critical 1 theory OD<OD<critical 2 theoretical OD gray zone
OD>critical 2 theoretical OD positive
[Description of results]
Vaccine
Seroconversion is demonstrated by detecting IgG antibodies against TBE in serum plasma
(1) Anti-TBE IgG negative
No seroconversion after vaccination
(2) Anti-TBE IgG gray area
There may be seroconversion, it is recommended to test again within 2~4 weeks
(3) Anti-TBE IgG positive
Seroconversion after vaccination
2. Infection
(1) Anti-TBE IgM and anti-TBE IgG negative
No infection
(2) Anti-TBE IgM negative and TBE IgG positive
This could be a potential immunization or an infection that is weeks or months ago.
(3) Anti-TBE IgM positive and TBE IgG negative
There may be a recent infection
(4) Both anti-TBE IgM and anti-TBE IgG are positive
Infected
This translation is for reference only, please refer to the original for details.
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