Denatured IgG and rheumatoid factor immune antigen and preparation method thereof
A technology of rheumatoid factor and immune antigen, applied in the biological field, can solve the problems of incomplete epitope research, insufficient denaturation, and too long heating time, so as to facilitate large-scale production, reduce batch-to-batch variation, Improve the effect of agglutination problems
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[0041] The invention relates to a preparation method of denatured IgG, which comprises denaturing IgG in the presence of a protein protecting agent to obtain denatured IgG.
[0042] The preparation method of denatured IgG provided by the present invention avoids the aggregation of denatured IgG during the denaturation process by adding a protein protective agent during the denaturation process, improves the stability of denatured IgG, makes IgG denature more fully, and exposes more antigenic epitopes , to increase antigenic activity. The method makes the denaturation process stable, and the obtained denatured IgG has good monodispersity, sufficient denaturation, high activity, ensures batch-to-batch difference, and is simple to operate, low in cost and convenient for large-scale production.
[0043] In a preferred embodiment, the protein protecting agent includes disaccharides, such as sucrose, trehalose, maltose, lactose and the like. In the present invention, it is found th...
Embodiment 1
[0063] The influence of embodiment 1 sucrose concentration on denaturation effect
[0064] 1. Prepare sucrose solutions containing 0w / v%, 10w / v%, 20w / v%, 30w / v%, 40w / v%, and 50w / v% respectively with 10mmol / L PBS (PH7.4) buffer solution;
[0065] 2. Dilute the IgG (50mg / ml) to 10mg / ml with the buffer in step 1;
[0066] 3. Heat the solution in step 2 in a constant temperature water bath at 63°C for 60 minutes;
[0067] 4. Put the heated solution in step 3 in an ice-water bath and keep it away from light for 2 hours;
[0068] 5. Observe the solution in step 4 for turbidity, see the results figure 1 , the sucrose concentration from left to right is 0w / v%, 10w / v%, 20w / v%, 30w / v%, 40w / v%, 50w / v%.
[0069] from figure 1 The results show that when the sucrose concentration is less than 30w / v%, agglutination is likely to occur during the denaturation process.
Embodiment 2
[0070] Embodiment 2 Denatured IgG coupling latex
[0071] The turbid solution in Example 1 was used for latex coupling.
[0072] Coupling scheme:
[0073] 1. Take 160 μl of 10% polystyrene carboxyl latex with a particle size of 188nm, and dilute to 2ml with 10mmol / L HEPES (PH7.5) buffer solution;
[0074] 2. Slowly add 50 μl (10 mg / ml) EDC solution and 50 μl (10 mg / ml) NHS solution to step 1, stir and react at room temperature for 20 minutes, then dilute to 10 ml with 10 mmol / L HEPES (PH7.5) buffer solution;
[0075] 3. Take 70 μl of denatured IgG solution without turbidity in Example 1, add urea 200mmol / L (final concentration) and BSA 0.05% (final concentration) to dilute;
[0076] 4. Slowly add the denatured IgG solution diluted in step 3 to step 2, and stir at room temperature for 3 hours;
[0077] 5. Centrifuge at 15000rpm for 40min, resuspend by ultrasonication in 10mmol / L PBS (containing 0.2v / v% Tween 20, 0.1w / v%BSA, 10w / v% sucrose), which is the RF detection reagent ...
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