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Lyme Helicobacter IgG ELISA Kit
                           
(Germany IBL imported original: RE57201 )
Germany IBL China exclusive agent "Shenzhen Kerunda Biological Engineering Co., Ltd." dedicated to serve you
1. Use : This kit is used for qualitative/quantitative detection of anti-Lem IgG antibodies in serum or plasma, and can detect all infections of the subtype of Levospicious
3. Detection principle : The sandwich ELISA method is used to coat the antigen of the Helicobacter pylori on the bottom of the microplate. When testing, the diluted sample and the kit control are added to the microplate, and the anti-L. The IgG antibody binds to the antigen, is incubated, washed, and added with HRP-labeled anti-human IgG secondary antibody. After incubation and washing, HRP enzyme substrate TMB is added to terminate the color development, and the microplate reader reads the plate according to the standard. The curve or Cutoff value determines the positive of the sample.
4 , precautions and preventive measures
1) This kit is for in vitro diagnostics and professional use only.
2) Before starting the test, carefully and completely read the instructions, use the valid version of the package plug-in provided by the kit to ensure that all procedures are clear.
3) If the kit is damaged, please contact IBL or submit your application, but no more than one week after you get the kit. Do not use damaged reagents during testing to ensure your safety.
4) Do not mix reagents of different batch numbers according to the batch number and expiration date, and do not use expired reagents.
5) Follow good experimental guidelines and safety guidelines. Wear lab coat with rubber gloves and wear goggles if necessary.
6) This kit contains harmful reagents that can damage your eyes and skin. Please read the material source and label for details. The material safety data sheet for the product can be downloaded from the IBL homepage or directly from IBL.
7) According to the National Biohazardous Substances and Safety Regulations, all chemicals and prepared or used reagents should be treated as hazardous materials.
8) Avoid contact with the stop solution to avoid irritation or burning of the skin.
9) All reagents in the kit (including tested human serum/plasma) are negative for HIV/II, HbsAg and HCV, but such viruses (HIV/II, HbsAg and HCV) or others may appear in the reagents. The possibility of infectious pathogens cannot be completely ruled out. Therefore, all reagents should be treated as potentially biohazardous substances during use and handling.
5 , storage and stability
The kit and transport environment and storage environment temperature is 2-8 ° C, avoiding heat or direct sunlight. Sample storage and stability and reagent preparation will be described in the relevant sections. The unsealed coated slats are stable for three months, provided that the coated slats are sealed with a bag and stored at 2-8 °C.
6 , sample collection and storage
Serum/plasma (EDTA anticoagulation)
Venous blood collection is performed according to general principles. The integrity of the blood sample should be maintained from sample collection to testing. Do not use samples that are susceptible to blood, jaundice, and lipemia. If the sample is turbid, it should be centrifuged before the experiment to remove particulate matter.
Storage
2-8 ° C
≤ -20 ° C (packing frozen)
Avoid high temperatures and direct sunlight to avoid repeated freezing and thawing.
stability
5 days
12 months
7 , kit components
1×12×8
MTP
The coated plate is detachable and the micropores are coated with specific antigen.
1 x 12 mL
ENZCONJ
Enzyme-linked conjugate, green, ready-to-use, HRP-labeled anti-human IgG antibody
4 x 1.5mL
CONTROL AD
AD standard: The content is: 2; 10; 50; 200 U/mL, wherein standard B is used as the Cutoff standard, containing human anti-L. Helicobacter IgG antibody, stabilizer
1.5mL
CONTROL+
Positive control, ready to use, containing human anti-L. Helicobacter IgG antibody, stabilizer
1.5mL
CONTROL-
Negative control, ready to use, containing human serum and stabilizers.
1×100mL
DILBUF
Dilution buffer, blue, containing phosphate buffer, detergent, BSA and stabilizer
1×100mL
WASHBUF
Washing solution (10×) containing phosphate buffer, detergent and stabilizer.
1 x 12 mL
TMB SUBS
TMB substrate solution, ready to use, contains TMB.
1 x 12 mL
TMB STOP
TMB stop solution, ready to use, containing 1M H 2 SO 4
8 , the equipment required for the experiment (but the kit does not provide)
1) Pipette (Eppendorf pipette or similar instrument, CV < 3%), volume 5 μL; 100 μL; 1000 μL
2) Whirl mixer
3) 37 ° C incubator
4) Sample dilution test tube (1mL)
5) 8-channel pipette with reservoir.
6) Washing bottles, automatic or semi-automatic coated board washing equipment.
7) Microplate reader that can read at 450nm (reference wavelength: 600-650nm)
8) Double distilled or deionized water
9) Coordinate paper, sampling tip, chronograph.
9 , experimental operation and precautions
1) Unreasonable sample handling or any changes to the experimental operation will affect the results of the experiment. The sampling volume, incubation time, and processing steps must be performed exactly as described. Only standard pipettes and standard equipment can be used.
2) Once the experiment begins, all steps must be completed and no interruptions are allowed. Take all reagents and samples in a laboratory at 18-25 ° C. Gently shake the liquid reagent and sample before use. Avoid bubbles during mixing.
3) Do not touch reagents, pipettes, micropores/test tubes. When adding reagents and samples, use a new sampling tip to avoid cross-contamination. Unused reagents should be covered with a lid to avoid reuse.
4) Calibrate the plate with a scaler.
5) Incubation time will affect the experimental results. The order and time of microwell loading must be the same. It is recommended to use an 8-channel pipette to load the sample.
6) Cleaning of the coated board is very important, and poor micropore cleaning will result in erroneous experimental results. It is recommended to use a multi-channel pipette or automatic micro-hole cleaning equipment for cleaning. Avoid microporous drying during each incubation interval. Avoid touching the coated holes when adding washing liquid and aspirating. Care should be taken when cleaning and adding reagents. When cleaning, the washing solution buffer is accurately added to the micropores while ensuring no residue in the micropores.
7) Humidity affects the coated board, so do not open the package when it has not reached room temperature. Unused coated sheets should be resealed in a desiccant bag.
8) This experiment can also be performed on an automated operating system. See “Performance” for specific information.
10 , preparation instructions before the experiment
10.1 Preparation of ingredients
Dilution/dissolution
ingredient
Diluent
proportion
Remarks
Storage
Validity period
100mL
detergent
1000mL
Double distilled water
1:10
If necessary, heat to 18-25 ° C to dissolve all crystals
2-8 ° C
2-3 months
10.2 sample dilution
10.2.1 Serum/plasma samples
sample
dilution
Reagent
proportion
Remarks
Serum/plasma
Dilute according to general rules
Dilution buffer
1:101
10 μL sample + 1 mL dilution buffer
Samples with IgG concentrations above the highest standard in the sample must be further diluted and tested repeatedly.
10.2.2 Cerebrospinal fluid sample: When testing the cerebrospinal fluid according to the Reiber method, it is necessary to make the serum and cerebrospinal fluid have the same concentration or the same Cutoff discrimination range (OD value is 1-0.1), which can be achieved by the following dilution:
sample
dilution
Reagent
proportion
Remarks
Serum/plasma
Dilute according to general rules
Dilution buffer
1:401
5 μL sample + 2 mL dilution buffer
Cerebrospinal fluid
Dilute according to general rules
Dilution buffer
1:4
50μL sample + 150μL dilution buffer
The Cutoff value can be corrected by a 1:101 dilution parameter, a 1:401 dilution of the serum Cutoff value, the dilution multiplied by 4. The 1:4 dilution of the cerebrospinal fluid Cutoff value, the dilution divided by 25.
If the OD value of the test sample is outside of 1.0 and 0.1, it should be tested by the following dilution.
Serum/plasma
1:100
1:200
1:400
1:800
1:1600
Cerebrospinal fluid
1:2
1:4
1:8
1:16
1:32
11 , experimental steps
2) Cover plate, incubated at 37 ° C for 60 min.
3) Discard the reaction solution in the micropore, wash each well with 300 ul of the diluted washing solution, and pat dry the plate on absorbent paper.
4) Add 100 μL of secondary antibody conjugate to each well.
5) Cover (new adhesive metal plate), incubate for 30 minutes at 37 °C.
6) Discard the reaction solution in the micropore, wash each well with 300 μL of the diluted washing solution, and pat dry the plate on absorbent paper.
7) If possible, use an 8-channel pipette to add substrate and stop solution. The time interval between the addition of the substrate and the stop solution should be the same, using an automatic displacement pipette and avoiding bubble generation.
8) Add 100 μL of TMB substrate solution to each well
9) Incubate in the dark at 18-25 °C for 30 min,
10) Add 100 ul of TMB Stop Solution to each well to stop the substrate reaction. After adding the stop solution, measure the OD value at 450 nm in 60 minutes (reference wavelength: 600-650 nm)
12 , quality control
The experimental results obtained in strict accordance with the instructions are valid. Users must strictly abide by laboratory optimization management regulations or other experimental standards. According to the requirements of the quality control sheet, all standard concentration must be within the allowable range. If the standard does not meet the requirements, the experiment is invalid and must be redone. Each laboratory should use samples of known concentration for further effective quality control. If the experiment deviates from the actual results, the following questions should be checked: reagent expiration date, storage conditions, sampler, experimental equipment, incubation time, cleaning method.
13 , calculation of results : calculation results can be qualitative or quantitative judgment
13.1 Qualitative judgment: The OD value of Standard B can be used as the Cutoff value to judge the positive result of Yin, and the OD of the sample is within 10% of the Cutoff value. The result is located in the gray area and is judged as suspicious. If it is higher than the gray area, it is judged as positive, and if it is lower than the gray area, it is judged as negative.
typical example:
Cut-off=OD (Standard B, Critical Standard) = 0.45
Sample OD=0.60
The critical indicator (COI) = 0.60 / 0.45 = 1.33. The sample is judged as positive
13.2 Quantitative judgment: On a semi-logarithmic coordinate paper or by an automatically generated method, a standard curve is drawn by taking the OD value of the standard as the Y-axis and the concentration as 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.
Typical standard curve
Example, cannot be used for calculation of experimental results
Standard
U/ml
Average OD
A
2
0.008
B
10
0.267
C
50
1.097
D
200
2.114
This translation is for reference only, please refer to the original for details.
Exclusive distributor in China: Shenzhen Kerunda Bioengineering Co., Ltd.
Company address : 6th Floor, No. 10, Yanshan Road, Shekou, Nanshan District, Shenzhen. Zip code : 518067
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