ExerciseⅡ Precipitation Reaction
Precipitation reaction refers to the reaction of soluble antigen and corresponding antibody and yield precipitation line or circle.Common precipitation reactions include circle precipitation reaction，floccule precipitation reaction，agar diffusion and immune electrophoresis，etc.
Agar diffusion test refers to soluble antigen and antibody diffuse in agar and yield white precipitation line or circle，given that they correspond to each other and with appropriate proportion.Agar is an amylose of large molecule.Agar could be melt at the temperature above 100℃and concrete under 45℃to form reticulation formation，which allows antigen and antibody diffuse freely inside it.Agar diffusion test could be divided into single diffusion and double diffusion.The single diffusion refers to only diffusion of antigen or antibody，so it could be used as quantitative monitor.Double diffusion refers to diffusion of both antigen and antibody，so it could be used as qualitative analysis for antigen or antibody.
Single agar diffusion test
Principle
Single agar diffusion test is quantitative test.Usually it is used to measure unknown antigen by known antibody.Mix antibody of certain quantity with agar and layer on slide to make an agar plate contains antibody.Cut wells after agar concretion.Then add unknown antigen into wells.Antibody concentration is even for antibody and agar mixed before concretion.So antigen diffuses outward from wells.The farther from the well，the lower is the antigen concentration.So white precipitation circle is formed at the place where antigen and antibody are of appropriate proportion.This is called single diffusion for only antigen diffuses.
Observing the result，we can find the diameter of precipitation circle is proportional to the antigen concentration.If we define the diameter of precipitation circle，which is the reaction result of different concentration standard antigen and certain concentration anti-serum，as y-axis，and define the antigen concentration as x-axis，we can draw a standard curve.If we measure the diameter of precipitation circle of the unknown antigen，we can tell its concentration from the standard curve.This experiment could be used to monitor the concentration of immunoglobulin（Ig）or serum complement of the sample.
Materials
1.Diagnostic serum of human immunoglobulin IgG（freeze-dry sheep anti human IgG）.
2.Human Ig standard serum，examinee's serum.
3.Agar，slide，cutter，micropipette，moist chamber，37℃-temperature machine.
Methods and Results
1.Make plate：According to half of the serum titer，dilute anti-serum with 56℃pre-warm normal saline.Then add agar that cooled off to 56℃and mixed evenly.Draw 3 ml agar that contains anti-serum with pipette and layer the agar onto slide evenly.After agar concretion，put the slide in 4℃refrigerator for 5 mins.
2.Cut well：Cut well with cutter.The diameter of well is 3mm.Keep the distance among wells above 1cm.Cut 5 wells every row.
3.Dilute standard serum and sample serum following protocol.Dilute the sample to 1∶50，Standard serum dilute serially to 1∶12.5，1∶25，1∶50，1∶100，1∶200.
4.Add sample：Add standard serum of different dilution 10μL into wells of first row in turn，respectively.Add sample serum 10μL into wells of second row，respectively.
5.Keep agar slide in 37℃moist chamber for 24hours.Then measure the diameter of precipitation ring.
6.Draw standard curve on semilogarithmic paper.Define the diameter of precipitation ring as y-axis and IgG concentration of corresponding well as x-axis.Refer to the standard curve according to the precipitation circle of unknown serum.We could get the IgG concentration of unknown serum by counting the IgG concentration shown in curve with the dilution ratio.
Attentions
1.Layer the slide quickly and avoid air bubble.Anti-serum should be pre-hotted and agar should be cooled off to 56℃.
2.Do not destroy the wall of wells when add sample.
3.Distance among wells should be no less than 1cm.(https://www.daowen.com)
Double Agar Diffusion Test
Principle
Double agar diffusion test is a qualitative test.Add soluble antigen and corresponding antibody into wells on agar plate.Both antigen and antibody could diffuse.So visible precipitation line appears where antigen and antibody are of appropriate proportion.If antigen and antibody is not corresponding，precipitation line would not appear.This test is often used to analyze the purity and reciprocal relationship of antigen and antibody.
Materials
1.Normal human serum.
2.Sheep anti human IgG，unknown Ab1 and Ab2.
3.1%agar in normal saline.
3.Slide，cutter，moist chamber，37℃temperature machine.
Methods
1.Make plate：Layer the 1% melt agar 3ml onto a slide.
2.Cut wells：Cut on agar plate after the agar is concrete.
3.Add sample：Add sheep anti human IgG（Marked as Ab）into central well.Add Ag1 into wells up and down.Add Ag2 into wells left and right（Figure 1）.
4.Put agar plate into 37℃moist chamber.Observe result after 24hours.
Figure 1 Photo Indicating of adding sample
Results
Precipitation line appears among central well and surrounding wells，which means positive reaction.Positive result indicates that antibody reacted with corresponding antigen.If no precipitation line appears，the result is negative.Negative result indicates antigen have not met corresponding antibody.
Attentions
1.Agar plate should be layered once to be even and smooth.
2.Convert the bottle and hang in the air when add sample.Squeeze gently and avoid too much fluid overflow the wells，which would affect the precipitation line formation.