Enzyme-linked recovery

Watanabe et al. developed an enzyme-linked immunosorbent assay (ELISA) for the detection of inabenfide, a plant growth regulator, in rice. Specific monoclonal antibody (MAB) is used for this method. The effects of rice matrices on the sensitivity of ELISA can be reduced by adding 0.1% Tween 20. Good reproducibility and accuracy of the proposed ELISA were obtained for rice samples and the recovery was 92% at a fortification level of 5-500 xgkg . ... 

Enzyme-linked immunosorbent assay (ELISA). Li and Li developed an ELISA procedure for imidacloprid to determine its residues in coffee cherry and bean extracts. A 25-g amount of sample extracted with 300 mL of methanol and 1% sulfuric acid (3 1, v/v) for 3 min. An aliquot of the sample extract (0.5 mL) is mixed with 1 mL of water and a gentle stream of nitrogen is used to evaporate methanol. The solution is then extracted with 1 mL of ethyl acetate, the extract is reconstituted in 1 mL of PBST (phosphate-buffered saline containing 0.05% Tween 20) and competitive ELISA is performed to quantify imidacloprid in the extract. Eor methanol extracts of coffee cherries and beans fortified with imidacloprid at 0.5 mgL recoveries of imidacloprid by the ELISA method were 108 and 94, respectively. 

The successful of recovery of RNase A functional activity by a heat-induced AR method suggested the possibility of recovering RNase A immunoreactivity as well. The immunoreactivity of native RNase A and RNase A that was incubated at a concentration of 4 mg/mL in 10% neutral buffered formalin for 1 day and then freed of formaldehyde by dialysis against PBS was compared using capture enzyme-linked immunosorbent assay (ELISA). Selected fractions that... 

Note CHO = Chinese hamster ovary cells, ELISA = enzyme-linked immunosorbent assay, FACS = fluorescent-activated cell sorting or flow cytometry IHC = immunohistochemistry, LBI = ligand-binding inhibition, rec = recovery, RMN = rat micronucleus. 

An indirect enzyme-linked immunosorbent assay (ELISA) was developed for maduramicin in poultry feed. The assay utilized polyclonal anti-maduramicin antibody raised in rabbits, maduramicin monoamide with 1,6-hexane diamine-conjugated ovalbumin as the coating antigen, horseradish peroxidase conjugated goat anti-rabbit IgG and 2,2 azinobis(3-ethylbenzthiazoline) sulfonic acid (ABTS) for quantitation. Standard curves ranging from 0 to 80 ng/mL maduramicin were constructed. The assay did not cross-react with monensin, lasalocid, salinomycin, lincomycin, narasin, chlortetracycline or roxarsone. Broiler feed fortified at 4 to 7 ppm maduramicin were shown to be quantifiable by ELISA at an average recovery of 98.1%. This ELISA method for maduramicin in poultry feed is comparable to the established HPLC-F method. 

Linked with the previous basic description on antibodies, there is an other form of labelling, which employs an enzyme rather than a radioisotope. In this case the tag (the enzyme) is enormous with respect to the subject molecule. Since the concentrations are very low, recovery is undertaken using an antibody. The best known methodology of this type is the enzyme-linked immunosorbent assay... 

Antigen-capture enzyme-linked immunosorbent assay (ELISA) testing, IgM ELISA, polymerase chain reaction (PCR), and virus isolation can be used to diagnose a case of Ebola HF within a few days of the onset of symptoms. Persons tested later in the cour.se of the disease or after recovery can be tested for IgM and IgG antibodies the disease can al.so be diagnosed retrospectively in deceased patients by using iminunohistochemistry testing, virus isolation, or PCR. 

Jervis used porous silica coated with chemisorbed polyacrylhydrazide for immobilization of adenosine monophosphate (AMP) [117]. After periodate oxidation of its ribose residue the ligand was coupled to the carrier and used for isolation of lactate dehydrogenase from rabbit muscle. The specific capacity was 2 mg of protein/g adsorbent with a ligand content of 10 pmol/g, whereas recovery of enzymatic activity after elution was 85%. Hipwell et al. [118] found that for effective binding of lactate dehydrogenases on AMP-o-aminoalkyl-Sepharose the spacer arm length required at least 4 methylene links. Apparently, a macromolecule of polyacrylhydrazide acts itself like an extended spacer arm and thus allow AMP to bind the enzyme. 

All the enzymes used in the work described above are quite stable at room temperature and can be used in a free form. They can also be used in an immobilized form to improve the stability and to facilitate the recovery. Many immobilization techniques are available today (25). The recent procedure developed by Whitesides et al using water-insoluble, cross-linked poly(aerylamide-acryloxysuccinimide) appears to be very useful and applicable to many enzymes (37). We have found that the non-crosslinked polymer can be used directly for immobilization in the absence of the diamine cross-linking reagent. Reaction of an enzyme with the reactive polymer produces an immobilized enzyme which is soluble in aqueous solutions but insoluble in organic solvents. Many enzymes have been immobilized by this way and the stability of each enzyme is enhanced by a factor of greater than 100. Horse liver alcohol dehydrogenase and FDP aldolase, for example, have been successfully immobilized and showed a marked increase in stability. 

An early discovery by Frederick Richards that turned out to be useful was that the protein could be cleaved between residues 20 and 21 by the bacterial serine protease, subtilisin. The resulting two polypeptides were separated and purified. They were enzymatically inactive individually, but regained the activity of the native enzyme when they were recombined. This work shows that strong, nonco-valent interactions occur that can hold protein chains together even when one of the peptide links is cut. It also makes it possible to modify specific amino acid residues of the two polypeptide chains independently and to explore how each residue contributes to the reassembly of the protein and the recovery of enzymatic activity. 

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