1.7.
Antigen Preparation from
Bacteria
Santosh Kumar Yadav
Types of antigens prepared commercially
• Whole culture extract
• Native protein antigen
• Specific carbohydrate antigen
• Synthetic peptides
• Recombinant proteins
Methods and steps of antigen preparation
1. Whole culture extract:
• Organisms grown on suitable medium and extract
collected as antigens after some chemical treating.
2. Native protein antigens:
• Growing medium: usually grown on either in protein
containing liquid media e.g. meat infusion, trypticase
soy broth and solid media e.g. blood agar and serum
or egg agar specially useful for fastidious bacteria.
• Elimination of mixed culture medium protein or
other constituents. It can be achieved by
centrifugation, filtration.
• Cell disruption and production of initial crude
extract includes breaking of cells and brought
them into solution by using extraction buffer.
• It can be achieved by:
Using blenders
Grinding with abrasives: grinding in a paste and
mortar in presented of sand or aluminium and a
small amount of extraction buffer (usually Tris or
phosphate buffer).
Enzymatic methods:
• Gram positive bacteria: lysozymes form hen egg white – cleaves
peptidoglycan layers.
• Gram negative bacteria: lysozymes with EDTA.
Purification of extract:
• Filtration or low speed centrifugation to remove
insoluble material if presented.
– DNA and RNA removed by DNAase and RNAase or
precipitation by protamine sulphate
– Carbohydrate removed by precipitating using
ammonium sulphate, filtration out ppt. redissolved in
buffer.
• Thus clarified extract is further purified and the
targeted protein is obtained by different methods
e.g.
• Fraction
• Chromatography
• Gel electrophoresis
3. Specific carbohydrate antigen
• Group specific polysaccharides antigens
preparation is also important to classify micro-
organisms , vaccine preparation, etc.
Extraction methods
Lancefield’s acid extraction methods
Enzymatic extraction method
Fuller’s formamide extraction method
Acid extraction method:
• 50 ml overnight culture suspension in Todd-Hewitt
broth centrifuge.
• Deposit harvested and resuspended in 0.4 ml of 0.2N
HCL.
• Boiled in water bath for 10 min.
• Allow to cool and add 1 drop of 0.02% phenol red.
• Neutralize with 0.5N and 0.2N NaOH.
• Centrifuge that obtain the clear supernatant extract
with polysaccharides antigen.
Enzyme extraction method:
• A loopful of 18 hour blood agar culture growth
suspended in 0.25 ml enzyme solution
obtained from streptomyces albus.
• Heat at 50 degree C till clearing the contents
• Clear solution is used as extract.
• Suitable for streptococcal group A, C, G.
Fuller’s formide extraction method:
• Centrifuged deposit from 5ml of a culture in Todd-
Hewitt broth mix with 0.1ml formide and heat in oil
bath at 16c for 15min or till completely dissolved.
• Add 0.25ml ethanol containing 0.5% Hcl shake and
centrifuge.
• Discard precipitate and add 0.5ml acetone to the
supernatant shake centrifuge and discard supernatant.
• Dissolve ppt in 0.4ml saline and neutralize with 0.2N
NaOH using phenol red as the indicator.
4. Synthetic peptides
• Formed by linkage of two amino acids where the amine
group of one amino acid bind with carboxyl group of
another amino acids with the removal of water.
• Use of synthetic peptides containing specific amino
acids of interest as antigen has revolutionized the
laboratory diagnosis of several infectious diseases.
• Peptides that are bound to the carrier proteins or
bound to an inert solid surfaces are now synthetically
produced and used as antigens different serological
test e.g. ELISA and Immunization lab.
Synthesis of peptides:
• Purification from whole microbial proteins
• Genetically engineered or recombinant
• Artificial chemical synthesis
5. Recombinant proteins
• Proteins that have been produced by recombinant
DNA technology.
• Mostly useful for mass production of protein antigens
, vaccine preparation etc.
• Method:
Target genes cut by specific restriction endonuclease .
Both vector and target DNA fragment joined by DNA
ligase.
Hybrid DNA introduced to host cells (bacteria, yeast)
Cloned DNA formed.
Gene expressed and specific protein forms, harvested,
purified and used as antigen.
Antigen preparation of Salmonella
H- antigen:
• Inoculate single colony into Hazan broth and
incubate for 6 hrs.
• Prepare wet film to confirm all bacteria are
motile ensures sufficient flagellation by the
bacteria.
• Add 0.2% formaldehyde incubate for several hrs
at 37 degree C.
Somatic O-Antigen:
• Grow bacteria in Hard phenol agar.
• Suspend colony in saline and heat at 100
degree C (that will detach flagella and
fimbriae).
• Centrifuge remove supernant and resuspand in
sterile saline.
• Alternatively remove flagella by mixing a dense
saline suspension of the bacteria with equal
volume of 96% ethanol and incubate for 20 hrs.
• Before use, dilute in saline to a suitable density.
Streptolysin O antigen Preparation
• Family of lysine a protein of 67 k Da mol. Wt
formed by pyogenic streptococci.
• Activated by sulfhydro compound and inhibited
by cholesterol and related sterols.
• For better production, thus organisms should be
grown in the fat free and reducing agent
containing medium (kalbak broth).
Procedure:
• Grow organisms in Todd-Hewitt broth.
• Transfer 50 ml culture broth to 1000 ml of kalbak’s
broth.
• Incubate for 15 hrs at 37 degree C.
• Check the culture for haemolysin.
• Separate microbial cell by filtration.
• Preserve the filtrate by adding merthiolate.
• Store at 4 degree C.
• Streptolysin O present in filtrate got inactivated
after 1 month of preparation.
• Thus prepared SLO retains constant potency for
many years if properly stored.
Thank you
