Production Of Anti-Bifidobacteria Lipoteichoic Acid Antibodies And Their Application In The Detection Of Bifidus Products

Objective : Preparing polyclonal antibodies against purified lipoteichoic acid(LTA) preparations of bifidobacterium ,using them to detect LTA and viable bifidobacterium in bifidus products,developing a new method which is more efficient and more easily being carried out.Methods: Enough Bifidobacterium bifidum (NO.86321) cells were harvested from BL broth, purified lipoteichoic acid(LTA) preparations of them by non-ionic detergent Triton x-114. Six to eight week-old male Balb/c mice were immunized by given subcutaneous injections of a emulsion of Freund complete adjuvant for first injection or Freund incomplete adjuvant for subsequent injections,containing TA precipita- -ted with mBSA, at three different concentration: -low ,middle and high dosage, ranged from the order of 50μg to the order of 100μg .We also use BCG vaccine for Multiclone stimulus before the first injection was done. Serum samples were obtained just before the first immunization to be used as negative control in specific antibody evaluation. Serum samples were also obtained one time a week after first injection and subsequently analyzed separately. All sera were analyzed by Indirect Enzyme-Linked Immunosorbent Assay (ELISA) procedures to determine specific antibody titer and test whether there were immune reaction with the following bacterium: Bifidobacterium bifidum, Bifidobacterium infantis,Streptococcus thermophilus,Lactobacillus bulgaricus,Staphylococcus epidermidis,Bacillus coli,Bacillus subtilis.LTA in dairy product was detected by double antibody sandwich Enzyme-Linked Immunosorbent Assay (ELISA). An original approach using a combination of the anti-LTA and a selective culture medium was developed for the specific concentration detection of viable cells of bifidobacteria in a certain dairy product.Results:Antibody to the LTA in mice was detectable by ELISA by day 6 or 7 ,there was no obvious difference of the antibody tite among each group,but the difference become larger along with the immunization time getting longer. Generally,the group with more dosage had higher antibody titer;but there was no obvious difference between the middle and high dose group.The maximal antibody titer was 1:1280 and it reacted strongly with Bifidobacterium bifidum,Bifidobacterium infantis, but got negative result with Streptococcus thermophilus,Lactobacillus bulgaricus,Staphylococcus epidermidis,Bacillus coli,Bacillus subtilis.In contrast,antibody to mBSA was either undetectable or negligible (ELISA titers, 0 to 16).High dosage TA-treated animals exhibited rapid onset of anaphylactoid symptoms,usually it happened within half an hour and can be observed the following phenomenon: hyperspasmia,dyspnea,mydriasis,pupil discoloration, heartbeat become weak or couldn't be touched.It was evident that animals had anaphylactoid responses resulting in a high rate of mortality,and the mice could recover their vital sign within 2～6 hours if it did not die.Other group never happended those cases.Moreover, detection of LTA and viable bifidobacterium are efficiently done, allowing the detection of at least 105 cfu/ml.Conclusions:Although most isolated,soluble LTA have been non-immunogenic, it confers immunogenicity ,when given as precipitates with methylated bovine serum albumin, it stimulated strong anti-LTA responses.The antiser has the bifidobacterial species specificity and can be used to detect other bifidobacteria species of human or animals origin bifidobacterium, it react very weakly with Bacterium lacticum, no cross reactivity with other bacterium . A immuno-culture method has been developed by combination of specific antibodies and plate culture to allow detection of viable bifidobacteria,it has good sensitivity and specificity. The whole procedure including purifying lipoteichoic acid(LTA) preparations of bifidobacterium,preparing polyclonal antibodies against it,using them to detect LTA and viable bifidobacterium in bifidus products ;it do not need specifc or expensive equipment and can be carried out in common laboratory ,simple and convenient ,it satisfy the market requirement well.