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Instructions for use of the human rabies virus IgG antibody (RV IgG) enzyme-linked immunoassay kit

 Last Update: 2022-09-14
 Source: Internet
 Author: User

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Instructions for the use of the human rabies virus IgG antibody (RV IgG) enzyme-linked immunoassay
kit

This kit is for research use
only.

Detection range: 96T 1.
6 IU/L -70 IU/LPurpose of use:
This kit is used to determine the content of rabies virus IgG antibody (RV IgG) in human serum, plasma and related liquid samples
.
Experimental Principles
This kit uses the double antigen sandwich method to determine the level
of IgG antibody (RV IgG) of human rabies virus in the specimen.
The microplate is coated with purified antigens to make solid-phase antigens, and rabies virus IgG antibodies (RV IgG) are added to the coated microwells in turn, and then combined with HRP-labeled antigens to form an antigen-antibody-microplate antigen complex, which is added to the substrate TMB color
after thorough washing.
TMB is converted to blue under the catalysis of HRPase and converted to a final yellow
color under the action of acid.
The shade of color was positively correlated with the rabies virus IgG antibody (RV IgG) in the
sample.
Absorbance (OD value) is determined at a wavelength of 450 nm with a microplate reader and the concentration
of human rabies virus IgG antibody (RV IgG) in the sample is calculated by standard curve.
Kit composition

1	30x concentrated wash solution	20ml × 1 bottle	7	Termination solution	6 ml × 1 bottle
2	Microplate reagents	6 ml × 1 bottle	8	Standard (120IU/L)	0. 5 ml × 1 bottle
3	Microplate-coated plates	12 holes × 8	9	Standard dilution	1. 5 ml × 1 bottle
4	Sample dilution	6 ml × 1 bottle	10	Instructions	1 serving
5	Color developer A liquid	6 ml × 1 bottle	11	Sealing film	2 sheets
6	Color developer B solution	6ml × 1/bottle	12	Sealed bags	1 pcs

Specimen requirements
1.
Specimens should be extracted as soon as possible after collection, and the extraction should be carried out according to the relevant literature, and experiments
should be carried out as soon as possible after extraction.
If the test cannot be performed immediately, the specimen can be stored at -20 °C, but repeated freeze-thaw
should be avoided 2.
Samples containing NaN3 cannot be detected, because NaN3 inhibits the activity
of horseradish peroxidase (HRP).
Procedure

Dilution of standards: This kit provides a standard in the original times, and the user can dilute
it in a small test tube according to the following diagram.

60 IU/L	Standard No. 5	150 μl of the original standard is added to 150 μl of the standard dilution
30 IU/L	Standard No. 4	150 μl of Standard 5 is added to 150 μl of standard dilution
15 IU/L	Standard No. 3	150 μl of Standard 4 was added to 150 μl of standard dilution
7. 5 IU/L	Standard No. 2	150 μl of Standard 3 was added to 150 μl of standard dilution
3. 75 IU/L	Standard No. 1	150 μl of Standard 2 was added to 150 μl of standard dilution

Dosing: Blank wells are set up separately (blank control wells do not add samples and microplate reagents, and the other steps are the same), standard wells, and sample wells
to be measured.
Accurately add 50 μl of the standard on the microplate-coated plate, first add 40 μl of the sample dilution to the well of the sample to be measured, and then add 10 μl of the sample to be measured (the final dilution of the sample is 5 times).

Add the sample to the bottom of the microplate plate well, trying not to touch the well wall, and gently shake the mixture
.
Incubation: Incubate at 37 °C after sealing the plate with sealing membrane for 30 min
.
Dosing: Dilute 30x concentrated wash solution with distilled water 30x and set aside
Washing: Carefully remove the sealing film, discard the liquid, shake dry, fill each well with washing solution, let stand for 30 seconds and discard, repeat 5 times, pat dry
.
Add enzymes: Add 50 μl of microplate reagent per well, except for blank wells
.
Incubation: Operation same as 3
.
Washing: Operation with 5
.
Color rendering: First add color developer A50μl to each well, then add color developer B50μl, gently shake and mix well, 37 °C avoid light for 10 minutes.
Stop: Add 50 μl of stop solution per well to terminate the reaction (at this point the blue vertical to yellow)
is added.
Assay: Zero out the blank wells, and measure the absorbance (OD value)
of each well sequentially at a wavelength of 450 nm.
The assay should be performed
within 15 min after adding the stop solution.

Summary of operation procedure:Calculation
Takes the concentration of the standard as the abscissa and the OD value as the ordinate coordinate, draws the standard curve on the coordinate paper, and finds the corresponding concentration according to the OD value of the sample by the standard curve; Multiply by the dilution multiple; Or use the concentration of the standard and the OD value to calculate the straight line regression equation of the standard curve, substitute the OD value of the sample into the equation, calculate the sample concentration, and then multiply by the dilution multiple, that is, the actual concentration
of the sample.
Precautions
1.
The kit should be equilibrated at room temperature for 15-30 minutes before use, and if the microplate package is not used after the plate is opened, the plate should be stored in a sealed bag
.

2.
The concentrated washing liquid may have crystallization precipitation, and when diluted, it can be warmed and dissolved in the water bath, and the washing will not affect the result
.

3.
The dosing device should be used for each step of the sample, and the accuracy should be checked frequently to avoid test errors
.
A dosing time is best controlled within 5 minutes, such as the number of specimens, it is recommended to use a platoon gun to dosing
.

Please do a standard curve at the same time as each measurement, preferably a re-hole
.
If the content of the substance to be measured in the specimen is too high (the sample OD value is greater than the OD value of the first well of the standard well), please dilute a certain multiple (n times) with the sample dilution solution before measuring, and then multiply the total dilution multiple (×n×5)
when calculating.
The sealing membrane is limited to single use only to avoid cross-contamination
.

6.
Please store
the substrate away from light.

7.
Strictly in accordance with the operation of the instructions, the test results must be determined by the microplate reader reading.

8.
All samples, washing liquid and various wastes should be treated
as infectious substances.

9.
The components of different batch numbers of this reagent shall not be mixed
.
Storage conditions and validity period
1.
Kit preservation:; 2-8℃
。
2.
Validity: 6 months

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