Hello everyone. I am sorry for the late post.
In this final post, I am going to share about one technique that I used in my project. I am designing antibody screening panels. This would require a lot of phenotyping of donor cells to create a good quality panel. One of the antigens that must be expressed in an antibody screening panel is the S antigen. This antigen is rare in the local population, with a frequency of 6%. Hence, the objective of this microplate technique is to screen for cells that are negative for the antithetical little s antigen. This means I would select cells that are either homozygous or heterozygous for S antigen to do full phenotyping.
Each week I would receive a batch of samples of about 60 and above. I need to screen all for little s negative antigen. Since the manufacturer insert of my antisera reagent does not recommend to be used for indirect coombs test, I cannot use the Techno TwinStation machine for this phenotyping. Instead, I used the microplate technique at room temperature, which allows screening of huge amount of samples at one go.
Procedures
1. Label all donor samples and position them according to the wells numbering in a rack.
2. Prepare 0.8% red cells suspension of each donor samples. The lower concentration would make it easier to read for agglutination.
3. Add 50ųl of red cells suspension into each well using a disposable pipette.
4. Add 50ųl of diluted antisera (1:1000) using the multi-dispenser pipette. The dilution factor of the antisera is derived from titration prior to the test.
5. Leave the microplate to stand without disturbance at room temperature for 30 to 40 minutes.
6. Record results.
Result Interpretation
If a well were positive for little s antigen, the red cells would form a monolayer suspension with no button formation observed. If a well were negative for little s antigen, the red cells would form a clear and compact button at the center of the well. To ensure valid results, positive and negative controls are used.
Picture retrieved from
http://www.hpa-midas.org.uk/images/page_images/agglutination_assay.gif
In this final post, I am going to share about one technique that I used in my project. I am designing antibody screening panels. This would require a lot of phenotyping of donor cells to create a good quality panel. One of the antigens that must be expressed in an antibody screening panel is the S antigen. This antigen is rare in the local population, with a frequency of 6%. Hence, the objective of this microplate technique is to screen for cells that are negative for the antithetical little s antigen. This means I would select cells that are either homozygous or heterozygous for S antigen to do full phenotyping.
Each week I would receive a batch of samples of about 60 and above. I need to screen all for little s negative antigen. Since the manufacturer insert of my antisera reagent does not recommend to be used for indirect coombs test, I cannot use the Techno TwinStation machine for this phenotyping. Instead, I used the microplate technique at room temperature, which allows screening of huge amount of samples at one go.
Procedures
1. Label all donor samples and position them according to the wells numbering in a rack.
2. Prepare 0.8% red cells suspension of each donor samples. The lower concentration would make it easier to read for agglutination.
3. Add 50ųl of red cells suspension into each well using a disposable pipette.
4. Add 50ųl of diluted antisera (1:1000) using the multi-dispenser pipette. The dilution factor of the antisera is derived from titration prior to the test.
5. Leave the microplate to stand without disturbance at room temperature for 30 to 40 minutes.
6. Record results.
Result Interpretation
If a well were positive for little s antigen, the red cells would form a monolayer suspension with no button formation observed. If a well were negative for little s antigen, the red cells would form a clear and compact button at the center of the well. To ensure valid results, positive and negative controls are used.
Picture retrieved from
http://www.hpa-midas.org.uk/images/page_images/agglutination_assay.gif
Advantages of microplate technique
1. It serves as a good preliminary screening method for phenotyping rare antigens.
2. It can handle large volume of donor samples at one time.
3. Produce rapid and reliable results.
4. Reduce wastage of disposable equipments in comparison to the tube technique.
5. Reduce cost, as the method is reliable for diluted antisera reagent.
Thank you.
Indah.
1. It serves as a good preliminary screening method for phenotyping rare antigens.
2. It can handle large volume of donor samples at one time.
3. Produce rapid and reliable results.
4. Reduce wastage of disposable equipments in comparison to the tube technique.
5. Reduce cost, as the method is reliable for diluted antisera reagent.
Thank you.
Indah.
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