A Simple And Rapid Method For Preparation And Preliminary Application Of Monoclonal Antibody Against Salmonella Peg Fimbriae

Salmonella is an infectious zoonotic pathogen that faces challenges in the world.It is.infected through the digestive tract and mainly exists in the hosts' intestines.It can cause diarrhea,vomiting,and other diseases.It not only brings greater losses to the breeding industry but also poses a certain threat to human health.So far,it has been found that Salmonella contains more than 2,600 serotypes.Because of the provenance transmission and complex diversity of Salmonella infection in poultry production,the common D serotypes in poultry production in our country include Salmonella Pullorum,Salmonella Galinarum and Salmonella Enteritidis,etc.Peg fimbriae mediate the interaction between bacteria and host adhesion and colonization,as an important adhesion factor that affects the pathogenicity of group D Salmonella,it exists in pullorum,typhoid fever,and Salmonella enteritidis serotypes.Therefore,the preparation of anti-Peg fimbriae monoclonal antibody can be used to establish a Salmonella detection method,which lays a certain foundation for clinical rapid and accurate Salmonella detection.1.A simple and rapid preparation method of anti-Salmonella Peg fimbriae monoclonal antibodyTo prepare monoclonal antibodies about Salmonella Peg fimbriae,this study used a recombinant strain of S9H-Peg target antigen constructed in the laboratory for the first time(S9H is an inert carrier strain,which is preserved in the China Common Microbial Strain Collection Management Center.The deposit number is CGMCC No.2091)as the immunogen,adjust the concentration to 5×109 cfu/mL to immunize 6-week-old BALB/c mice without adding adjuvants.The dose is 100 ?L/mouse for a total of 4 immunizations.Three mice with the highest antibody titer against Peg fimbriae were selected for hybridoma cells production,and hybridoma cells were obtained by using mouse hybridoma cell fusion technology.Using S9H as a negative control,the hybridoma cells were selected by slide agglutination test,and 5 hybridoma cells were obtained,named as:72-?-F8,97-?-E4,97-?-B9,112-II-F5,152-I-D4.Three of them that can stably secrete monoclonal antibodies after 40 passages were selected,named as:97-?-E4,97-?-B9,and 152-?-D4.The titer of those cells' supernatant can reach at 1:2.Use them to prepare mouse ascites.The characteristics of the three monoclonal antibodies were determined:(1)The antibody subclass test showed that the three monoclonal antibodies were all IgG2b/?.(2)The results of titer determination showed:All three antibodies titer in ascites of mice are up to 1:25600 detected by indirect ELISA method(whole bacteria coated antigen concentration:1×108 cfu/mL)and 1:320,1:160,and 1:320 respectively detected by slide agglutination method(Antigen concentration:5×109 cfu/mL).(3)Specificity detection:? Using 11 other target antigens of Salmonella fimbriae Peg,Salmonella fimbriae Sef14,Salmonella fimbriae Saf,Salmonella fimbriae SipD,Salmonella type I fimbriae,E.coli fimbriae K88ac,E.coli fimbriae K99,E.coli fimbriae 987P,E.coli fimbriae F18ab,E.coli fimbriae F18ac,E.coli fimbriae Sfm,constructed in the laboratory to verify the specificity of monoclonal antibodies.The results of the slide agglutination test showed that the three monoclonal antibody had good specificity with Salmonella fimbriae Peg and the coincidence rate of specificity was 100%.But they can not be recognized by 11 other target antigens described above,and they can not be recognized by S9H inert carrier neither.? In the indirect immunofluorescence test,the prepared monoclonal antibody was used as the primary antibody and incubated with the S9H-Peg strain and S9H strain respectively.The goat anti-mouse IgG-HRP was used as the secondary antibody,and the results were observed under a fluorescence microscope.The prepared monoclonal antibody could specifically bind to the S9H-Peg strain,and specific green fluorescence fron the surface of the S9H-Peg strain can be observed;it could not bind to the S9H strain and the specifity fluorescence cannot be observed.?Western blot analysis showed that the prepared monoclonal antibody can react specifically with the Peg positive strain(S9H-Peg strain),and a specific band at 20kDa could be visible;but it does not react with the control strain S9H,showing no specific bands.In this study,a simple and rapid method was used to prepare monoclonal antibodies against Salmonella Peg fimbriae.The simplicity of this method is mainly reflected in the following aspects:(1)Simple immunogen preparation(no need for antigen extraction or prokaryotic expression and protein purification);(2)The immunization procedure is simple,less time-consuming and does not require adjuvants;(3)The antibody screening process is convenient and quick(only need to perform slide agglutination test,without the need for complex procedures such as ELISA detection).To a certain extent,the preparation process of monoclonal antibodies is simplified,the workload is reduced,and the preparation efficiency of monoclonal antibodies is improved.This study provides a successful case of applying an inert carrier carrying a foreign antigen(Peg)to the immunized antigen preparation and easy screening of monoclonal antibodies and provides a new idea and realistic basis for the preparation strategy of monoclonal antibodies.At the same time,it provides a material and experimental basis for the Salmonella diagnostic technology targeting Peg fimbriae.2.Establishment and preliminary application of a slide agglutination detection method for Salmonella PullorumIn the previous chapter,three monoclonal antibodies against Salmonella Peg fimbriae were successfully constructed.It has been verified that all three monoclonal antibodies have slide agglutination characteristic,and the previous tests have shown that the combined use of the three monoclonal antibodies is better than the single-use.Based on the above experimental background,the three monoclonal antibodies prepared in Chapter 1(97-?-E4,97-?-B9,152-?-D4)were used to establish a slide agglutination method for Salmonella Pullorum.First,the sensitivity of the established method was tested.The results showed that the detection limit of the method for the pure culture of Salmonella Pullorum NCTC5776 was 104 cfu/reaction.Second,Salmonella expressing peg fimbriae(including Salmonella Pullorum NCTC5776,Salmonella Pullorum ATCC700623),Salmonella that does not express peg fimbriae(including Salmonella Typhimurium STM 43-3,Salmonella Typhimurium W32,Salmonella Saint Paul,Salmonella Aguna 36,Salmonella Choleraesuis 29,Salmonella Choleraesuis U80,Salmonella Tennessee)and non-Salmonella(including Escherichia coli APEC TW-XM,Escherichia coli ETEC C83901,Escherichia coli ETEC C83902,Escherichia coli ETEC C83903,Escherichia coli ETEC 987P-204,Escherichia coli ETEC 987P-205,Escherichia coli ETEC 987P-X)were used to verify the specificity of the established detection method,and the results showed that the detection method can specifically identify Peg-expressing Salmonella and does not have any cross-reactions with Salmonella that does not express Peg fimbriae and non-Salmonella,so the specificity is good.Then,the usage of this method was further explored:(1)For the detection of bottled drinking water,the samples were artificially contaminated with different concentrations of Salmonella,and then the monoclonal antibodies-based agglutination assay developed was used for detection.The results showed that when the samples were contaminated at 102 cfu/mL,Salmonella Pullorum can be detected by this method;(2)For the detection of simulated contaminated chicken feed samples,the single bacteria Salmonella Pullorum NCTC5776 was added to the sterile and antibiotic-free feed samples.After bacteria enrichment,the simulated contaminated feed was tested.The results showed that after 10 hours of bacteria pre-enrichment,this method can detect Salmonella Pullorum in the feed with a sensitivity of 4×104 cfu/reaction;(3)Clinically suspected 18 sick chickens infected with Salmonella Pullorum were tested for necropsy.The samples were prepared by grinding the focal organs(heart,liver,spleen,lung,kidney,and gallbladder)in a sterile environment.Salmonella in the sample was detected.The results showed that after the pre-enrichment process and the established slide agglutination method,a total of 5 Salmonella pullorum positive samples were detected from 18 samples,and the results were consistent with the traditional isolation and identification methods.In addition,the traditional isolation and identification method process is relatively cumbersome,but this new constructed method only needs to pre-enrich the sample before it can be used for detection,and the slide agglutination reaction takes only 2 minutes to obtain the result,which is convenient and rapid,showing the good practicability of this method.In summary,using the S9H-Peg target antigen recombinant strain constructed in the laboratory to finish the preparation of monoclonal antibody does not need antigen extraction,prokaryotic expression or protein purification.Also,this process does not require adjuvants,and is simple to screen.Monoclonal antibodies made according to this method are with good specificity and high sensitivity.Moreover,the combination of the three monoclonal antibodies was used as the antibody to establish a rapid detection method to detect Salmonella Pullorum by slide agglutination.After the specificity and sensitivity were tested,we simulated contamination sample artificially and collected clinical sample to test.Compared with the traditional national standard detection method,the newly established method is accurate,the steps are simpler and faster.In addition,it costs less and saves much time.It provides the way for further monitoring of Salmonella infection timely and has laid a profound research foundation.