In the past, I have coupled soluble antigens to SRBC for a variety of agglutination assays. I followed an old protocol described in Practical Immunology (Hudson & Hay, later editions Frank Hay & Westwood). Briefly, SRBC are washed 3 times with PBS by centrifugation (300 xg for 5 min) and a suspension of SRBC (4% v/v) in PBS is prepared. Then 50ml of tannic acid solution (2.5mg tannic acid in 50ml PBS) is added to 50ml of 4% SRBC suspension. Mix and incubate at 37°C for 15 minutes. Spin down cells very gently (100 xg for 15-20 min) to avoid cell aggregation. Wash tanned SRBC with PBS by gentle centrifugation and then resuspend cells in 100 ml PBS. A 50ml of this cell suspension can now be used for coating with soluble antigens. The rest of tanned SRBC can be used as control cells in agglutination assay. For antigen coating, add 50ml of antigen solution (for example, protein dissolved in PBS at 2mg/ml) to 50ml tanned SRBC suspension, incubate at 37°C for 30 minutes, and then wash in PBS. These cells can be treated further with formalin, by adding slowly and dropwise 10ml of 40% formalin to antigen-coated SRBC or tanned cells suspended in 100ml borate-succinate buffer (0.15M, pH 7.5), while mixing thoroughly. Leave the cells overnight at 4°C and then add additional 10ml formalin to cell suspensions. Allow the cells to settle for 24 hours (4°C), remove the supernatant, resuspend the cells in a large volume of borate-succinate buffer for further 24 hours and then wash the cells twice with borate-succinate buffer. For agglutination assays, antigen-coated SRBC (1% v/v) can be resuspended in PBS or Veronal-EDTA-gelatin buffer. I used SRBC in Veronal-EDTA-gelatin buffer for these assays because the background was usually very low in this buffer. The volumes of cell suspensions and solutions can be adjusted as required for the study.

thanks for answer Nasim Yousaf i used the same procedure with some modification as no formalin used so i will used the cells immediately or withen days the main concentration of tannic acid was 50 ug /ml or less as 1:20000 all these can give good result but how i can determined if antigen (LPS) was bounded or not ???

You should see clear differences between your controls and serum samples. SRBC and uncoated tanned SRBC should settle at the bottom of the plate (I used U well plates) like ‘buttons’ in the presence of buffer alone or rat serum. Agglutination pattern should only occur with coated SRBC in the presence of rat serum and you should be able to determine the titre. In this case, the antigen would be present on the cells. Reagents to detect LPS are available from a number of companies, such as ThermoFisher, InvivoGen, and others. However, I would also highlight another factor that you should keep in mind. Rats may express low titre heterophile antibodies to SRBC. In the past, I used PVG/c rats throughout my in vivo studies. At very early stage, we realized that normal rats exhibited antibody activities against both SRBC and Ox cells in vivo and activated the complement system. Bound immunoglobulins and C3 were detected on SRBC and Ox cells after incubation with normal rat plasma or serum by indirect immunofluorescence, but no immunoglobulin was detectable at 1:20 dilution of rat plasma. You may find this link useful:

https://www.researchgate.net/post/What_is_the_best_method_to_measure_endotoxin_LPS_in_serum_samples