Preparation Of Monoclonal Antibody Against SEA And Its Application In Immunochromatographic Test Strip

Staphyloccocus aureus is a spherical and facultative anaerobic gram-positive bacterium that widely distributed in nature.This pathogen does not cause fatal in natural state,but can cause severe infection to the damaged skin.When its concentration exceeds 10~5 CFU/m L,the staphylococcus aureus can produces the staphylococcus aureus enterotoxins(SEs),including SEA,SEB,SEC,SED,SEE and other SEs under appropriate condition.SEs are a class of small molecular proteins with the molecular weight ranging from 26 to 30 k Da.Accidental consumption of SE contaminated foods can produce food poisoning symptoms,including dizziness,abdominal pain,and vomiting.In recent years,the food poisoning incidents caused by staphylococcus aureus and its enterotoxins have occurred frequently around the world,and thereby posing a serious threat to human health.In this study,an anti-SEA monoclonal antibody(anti-SEA m Abs)was prepared,and a cell line namely 4C6,which can secret the anti-SEA m Abs,was obtained by the following processes,such as injecting the SEA protein in BALB/c mice,determining the titer of anti-SEA serum,fusing the spleen cells with the mouse myeloma cells,screening and subcloning the positive hybrids.Then,the binding affinity,subtype and specificity of anti-SEA m Abs 4C6 were evaluated by the enzyme-linked immunosorbent assay.The results show that the affinity constant of m Abs is 4.9×10~7mol/L,and the subtype of this antibody is Ig G1.In addition,the obtained anti-SEA m Abs also shows an excellent specificity to SEA with a negligible cross-reaction to SEB,SEC,SED,and SEE.Subsequently,a fluorescent immunochromatographic assay with competitive format was developed for the SEA determination in pasteurized milk by using quantum dot beads(QB-ICA)as signal amplifier.Various parameters that could influence the detection performance of QB-ICA were optimized.The reuslts showed that the optimal p H condition for coupling m Abs on the surface of QBs was 5.0,and the labeling amount of anti-SEA ascites was 400μg per mg QBs.The SEA concentration sprayed on T-line was 0.25 mg/m L,and the interpretation time of the strip for SEA quantitative analysis was 15 min.Under the optimal conditions,the proposed method exhibited a good dynamic linearity for SEA quantitative detection from 2 ng/m L to 150 ng/m L,which was presented by a regression equation of y=-17.2 ln(x)+100.95(R~2=0.9942),and the limit of detection(LOD)was calculated at1.89 ng/m L.In addition,the average revoveries of intra-and inter-assays ranged from85.5%to 128.1%for SEA spiked milk samples,while the coefficient of variations(CV)of intra-and inter-assays ranged from 4.6%to 14.2%,indicating an accepted accuracy for SEA quantitative determination in real milk samples.Then,a phenylboronic acid(PBA)modified aggregation-induced emission fluorescent microspheres(AIE FM)was used as the fluorescence probe to enhance the sensitivity of ICA with sandwich format for SEA determination in milk samples.The anti-SEA m Abs was directly coupled on the surface of AIE FM via the boric acid affinity reaction of PBA to cis-diol structure on Fc fragment of antibody.The optimal p H condition for coupling the m Abs on AIE FM was 7.0,while the optimal dosage of anti-SEA m Abs labeled on per mg AIE-FM was 20μg;the anti-SEA m Abs sprayed on NC membrane as T line was 2 mg/m L,and the dosage of AIE probe for each test was 0.37μg.Under the optimal conditions,the standard curve of established AIE-ICA was y=0.0465x+0.142(R~2=0.9908),with the dynamic linearity for SEA quantitative determination ranging from 0.1 ng/m L to 80 ng/m L.The LOD value was calculated as low as 0.04 ng/m L.In addition,the proposed method also exhibited an accepted accuracy and precision for SEA quantitative detection in real milk sample with average recoveries ranging from 91.3%to 117.6%and CV values ranging from5.9%to 11.8%,respectively.In conclusion,an anti-SEA m Abs was prepared by injecting SEA protein in Balb/c mices,and then was coupled with QBs and AIE FM as the highly luminous probes to develop the ICA method with competitive and sandwich format,respectively,for the rapid and sensitive determination of SEA in milk samples.Both methods show the unique advantages,including widely dynamic linearity,high sensitivity,and short time-consuming for SEA quantitative detection,and thereby providing a potential analytical tool for the on-site detection of SEA contamination in resource-limited regions.