Immune-specific probes

To determine what cells make the immune-specific RNA we inoculated mid-third instar Drosophila larvae with bacteria and six hours later dissected them into fat bodies and fat body-free carcass. Total RNA was collected, separated by electrophoresis, transfered to nitrocellulose, and probed with labelled Pool 1 oligonucleotide. The results showed (Fig. 5) that intact inoculated larvae accumulate the immune-specific transcript while intact control larvae do not. The tissue dissection experiment showed that fat body cells of inoculated larvae contain transcripts homologous to the immune-specific probe, but the carcass does not. We conclude that fat body cells in Drosophila larvae accumulate a transcript that has homology to sarcotoxin when they have been inoculated with bacteria. We are currently cloning the responsible immune gene. 

Figure 4. The oligonucleotide recognizes an immune-specific transcript. melanogaster were inoculated with bacteria or left as controls and incubated for 11 or 30 hours as indicated. RNA was collected and separated on the gel shown at the right stained with ethidium bromide and then hybridized to the oligonucleotide probe in a northern blot on the left.

Figure 5. Drosophila fat body cells accumulate immune-specific RNA. Drosophila larvae were inoculated with bacteria and six hours later RNA was extracted from either whole larvae, from fat body, or from the carcasses minus fat body. These RNAs were separated on the gel shown on the right stained with ethidium bromide, then northern blotted and probed with the oligonucleotide on the left.

Some of the ABs may have structural homology to antibacterial proteins isolated from the flesh fly (20,21) since an oligonucleotide probe that can encode a portion of the sarcotoxin protein recognizes an immune-specific RNA in Drosophila fat body cells, the cellular origin of Drosophila s ABs (unpubl.). The induction mechanism must work relatively rapidly since we found immune-specific RNA in fat body cells within 6 hours after inoculation. Because of its homology to sarcotoxin, we assume that the immune-specific transcript detected by the oligonucleotide probe encodes an antibacterial protein. 

Different approaches have been used to probe the role of chemokines and their receptors in experimental models of arthritis for instance, peptide antagonists or immunization of the host to promote the generation of endogenous neutralizing Abs via the use of chemokine plasmid DNA vaccination (Table 4 and Ref. 50), injection of neutralizing antibodies (Table 5), or mice that lack specific chemokine or chemokine receptors (Table 6). 

The general method utilized to prepare E5-Ab solutions obviates the need for stocking large numbers of reagents which would be necessary if different activation methods were used for each antibody. A number of specific antibodies immobilized by this process have shown response similar to that of the same antibodies when adsorbed as immune complexes in the Stratus system. In addition, the dendrimer-coupled antibodies have shown dramatic improvements in sensitivity, flexibility and precision for the enzyme immunoassay system. Feasibility demonstration of an assay for DNA probes is a prelude to what can possibly be achieved with these dendrimer-based reagents. 

As discussed above, the LTS facilitates investigations of the mechanisms that enable the suppression of DTH by CD8-f Tregs because the assay is complete within 24 h. In other words, to probe the mechanisms and antigen specificity of suppression, manipulations can be made that only influence the suppression of the DTH response after the immunized mice are challenged. 

Fig. 4. Classification of reported noncompetitive immunoassays for haptens based on the assay principle. (A) Assays that include a chemical modification of hapten to allow sandwich-type detection. (B1) Improved single-antibody immunometric assays that separate immune complex and excess labeled antibody, either by using a hapten-immobilized affinity column or based on differences in their physical properties. (B2) A variation of single-antibody immunometric assays based on masking of unoccupied antibody by an immunoreactive macromolecule followed by selective capture and detection of the hapten-occupied antibody. (C) Assays employing a probe molecule specific to a hapten-antibody complex.

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