one-way immunodiffusion assay
one-way immunodiffusion assay
A single immunodiffusion assay can be used to: measure IgG, IgA and IgM levels in the serum of normal people or patients.
Operation method
Example method for the determination of normal values of serum IgG in the population
Principle
In the agar plate containing specific antibody, and add quantitative antigen into the wells, when the antigen spreads to the surrounding area and combines with the antibody in the agar, a white precipitation ring is formed, whose diameter or area is positively correlated with the concentration of the antigen. The diameter or area of the ring is positively correlated with the antigen concentration. At the same time, a standard curve is made with a standard antigen or an international reference protein, which can be used to quantitatively detect the antigen concentration (mg/ml or U/ml) of an unknown specimen.
Materials and Instruments
Horse Anti-Human IgG Serum Move I. Experimental materials Schematic diagram of one-way immunodiffusion Caveat 1. When making a standard curve, at least two or more standard plates should be made to minimize errors. 2. When spiking, the plastic tip should be changed for each specimen drawn. Common Problems Bidirectional immunodiffusion refers to the diffusion of soluble antigens and their corresponding antibodies into each other in agar media, where they meet and form certain types of specific precipitation lines. The characteristics and positions of the precipitation lines depend not only on the specificity and ratio of the antigens and antibodies to each other, but also on their molecular size and speed of diffusion. When antigens and antibodies are present in multiple systems, multiple precipitation lines and even cross-reactions can occur. Several properties of the antigen or antibody, such as concentration, specificity, etc., can be understood based on the morphology, number, clarity, and location of the precipitation lines. Unidirectional immunodiffusion is performed by punching holes in an agar plate containing a specific antibody and adding a quantitative amount of antigen to the holes. When the antigen diffuses around and combines with the antibody in the agar, a white precipitation ring is formed, the diameter or area of which correlates positively with the concentration of the antigen. The diameter or area of the ring is positively correlated with the antigen concentration. At the same time, a standard curve is made with a standard antigen or international reference protein, which can be used to quantitatively detect the antigen concentration (mg/ml or U/ml) of an unknown specimen. This method can be used to detect the levels of IgG, IgA and IgM in the serum of normal people or patients. Source: Department of Immunology, School of Basic Medical Sciences, Peking University. For more product details, please visit Aladdin Scientific website.
Ion Agar Physiological Saline Agar PBS Standard Reference Protein
Micro-sampler Perforator Wet box Agar plate Gauze Foam
2% ionized agar or saline agar (containing 2% NaN3 ).
Standard horse anti-human IgG serum (antibody) (produced by Beijing Research Institute of Biological Products)
Working standard reference protein (produced by Beijing Research Institute of Biological Products)
pH7.2 PBS
Perforator (hole diameter 3 mm) and perforation template
Microsampler
Wet box (container with wet gauze or foam)
Prepared agar plate containing 1% horse anti-human IgG antibody
Single-sample serum specimen to be examined at a dilution of 1/50
II. Experimental Methods
1. Preparation of Standard Curve
(1) According to the size of the slide, prepare 1% ionized agar for the agar plate. First of all, dispense 1/2 amount of 2% saline agar, for example, with ordinary slides need 1% ionic agar 4 ml, in the dispensing of 2% saline agar that is 2 ml. Dissolve in 56 ℃ ~ 60 ℃ water bath to equilibrate the temperature of standby.
(2) Dilute the antibody, dilute the standard anti-human IgG antibody with PBS at pH 7.2, and the final concentration is double the antibody potency. For example, if the serum potency is 1/140, the original concentrated serum should be diluted by 1/70. And the test tubes should be filled with an amount equal to the amount of 2% saline agar.
(3) Preparation of agar plate: preheat the diluted anti-human IgG antibody in a water bath at 56℃ for about half a minute, then pour it into a 2% saline agar tube that has been dissolved and maintained at 56℃~60℃, and plug the mouth of the tube tightly with the thumb. Turn the tube 1-2 times, mix the antibody and agar evenly (Note: Do not produce bubbles when mixing the antibody and agar), pour it on the slide immediately, and wait until it solidifies.
(4) Punching: Place the agar plate on the template and punch 5 holes in the same straight line with a hole punch at a distance of 10 mm.
(5) Dilution of different concentrations of standard reference proteins (working standards): Dilution should be carried out according to the instructions of the product, for example: the immunoglobulin content of the working standard, IgG is 100 units/ml, 80.4 micrograms/unit, and its dilution range is 1/10, 1/20, 1/40, 1/80 and 1/40 of the working standard. The dilution range is 1/10, 1/20, 1/40, 1/80 and 1/160.
(6) Add samples: Add 10 ul of the diluted working standard proteins of different concentrations to each well in turn with a micro-sampler (Note: the plastic tip should be changed for each dilution).
(7) Put the spiked agar plate in a wet box and place it in a 37℃ incubator to observe the result after 24 hours.
(8) Measure and record the diameter of the precipitation ring with a goniometer, then use the diameter of the precipitation ring as the vertical coordinate and the amount of the standard reference protein (unit/ml) as the horizontal coordinate to plot the standard curve.
2. Determination of the normal value of IgG in human serum
(1) Place the prepared antibody agar plate on the punching template, and punch 4 holes in each agar plate (pore diameter of 3 mm, pore spacing of 10 mm).
(2) Dilute one unit of normal human serum 1/50 with pH 7.2 PBS.
(3) Use a micro-sampler to take 10 μl of the 1/50 diluted single human serum specimen and add it to the wells, each specimen should be added to two wells.
(4) Mark and put in the wet box, set in 37℃ temperature box, observe the result after 24 hours.
Experimental results
Measure the diameter of the precipitation ring of each specimen and record the results, then use the standard curve to measure the amount of IgG contained in each specimen (U/ml,) and converted to mg/ml. 