2 antibody controls mouse 1° antibodies

The in vivo manipulation of specific type 2 cytokines using anticytokine monoclonal antibodies, or mouse strains with targeted deletions in cytokine and/or cytokine receptor genes, has proved a fruitful approach in identifying the importance of individual cytokines and the responses that they control in contributing to host resistance. These studies have identified important roles for IL-4, IL-9 and IL-13 in host protection against nematode infection, though the relative importance of each cytokine appears to be nematode-species dependent. 

Figure 8.1 The results of IHC of two experiments using Dynabeads (Dynal, New York, NY) coated with biotinylated anti-mouse IgG (first experiment) and protein S-100 (second experiment), (a) Positive control showing red color (S-100) localized in the melanoma cells, (b) Strong positive red color circles all beads coated with biotinylated anti-mouse antibody after the heating AR treatment (first experiment), (c) Using the heating AR treatment, S-100-coated polymer beads show positive red color around the beads as circles (second experiment), (d) Negative control of the first experiment. No red color could be seen for polymer beads (arrows) that had been treated with exactly the same protocol as that of slide (b), but omitting the avidin-biotin-peroxidase (label). Bar = 50pm. Reproduced with permission from Shi et al., J. Histochem. Cytochem. 2005 53 1167-1170. See color insert.

LSBL MAP MCB NDA NIH PPC PTC QA QC RAP WCB WCP WHO large-scale biosafety level mouse antibody production master cell bank new drug application National Institutes of Health post-production cells points to consider quality assurance quality control rat antibody production working cell bank well-characterized product World Health Organization... 

Fig. 1. Biodistribution of radiolabeled catalase conjugated with anti-ACE MAb 9B9 (hatched bars) or with control mouse IgG (open bars) 1 h after iv injection in rats. The data are shown as M SD, n = 3. The tissues are indicated as 1, blood 2, lung 3, liver 4, kidney 5, spleen 6, heart. Note that both conjugates demonstrate a similar biodistribution pattern in all inspected tissues, except the lung, where antibody-catalase conjugate displays about ten times higher accumulation than nonimmune counterpart.

Fig. 6. Effect of protein L injections on CD5+ B cells in the spleen. Transgenic mice expressing human Igs [46] were injected intraperitoneally five times with 1 mg of protein L every other day. On day 21, splenic lymphoid cell subpopulations were identified by immunofluorescent staining in control, HEL- and protein-L-treated transgenic mice. Splenocytes were stained with Cy-chrome-labeled anti-mouse B220 monoclonal antibody and phycoerythrin-labeled anti-mouse CD5 monoclonal antibody. All monoclonal antibodies and their corresponding isotype controls were purchased from BD PharMingen. The treatment did not affect B1 cells in the spleen.

Control line antibody The goat anti-mouse IgG is isolated from sera of goats immunized with mouse IgG and stored at -20°C in 1 mL aliquots. 

Two antibody controls are needed for labeled Fab E antibody Zenon immunocytochemistry (Table 12.4). These controls will show that each 1° antibody is labeled correctly. To generate a control and additional E antibody labeling reaction is needed, where the blocking normal mouse serum is added to the labeled Fab reagent (Zenon reagent) before the E antibody. In this incubation, the labeled... 

Fig. 14.1 Case No. 1 problem. Tissue is from the mouse spinal cord incubated with rabbit anti-ribosomal protein antibody at 1 1000 and goat anti-rabbit 488 at 1 1000, examined in a wide field fluorescence microscope, (a) This micrograph shows extremely light specific label (arrows) and a very high background, (b) A no 1° antibody control with high background and no specific labeling are shown...

The problem in case No. 2 was that the 1° antibody incubation solution dried on the section. The micrograph (Fig. 14.3b, arrows) showed the dried antibody on the microscope slide next to the tissue. The 2° antibody, goat anti-mouse 488 fluo-rophore, then bound to the attached 1° antibodies. The dried antibody is not possible to rinse off slides of sections, and when it occurs, the slides should be discarded. When this experiment was repeated and when antibody did not dry out, the labeling was as expected (Fig. 14.3d) with low background (Fig. 14.3d, asterisk), that matched low labeling seen in the no 1° antibody control (Fig. 14.3e)... 

The correct experiment was performed where the first 2° antibody was now a donkey anti-mouse 488 fluorophore. The results show the expected labeling for both antibodies together (Fig. 14.6a, b, c) the no 1° antibody controls also show labeling only for the correct protein (Fig. 14.6d, e, f, g, h, i). 

The double-label experiment was performed, but no label was seen for the neu-rofllament with the 543 fluorophore (Fig. 14.7a, b, c. Problem experiment). The expected labeling for p38 was seen in the red channel but no neurofilament labeling was seen in the green channel. No 1° antibody control for each antibody (results not shown) confirmed that there was no labeling with mouse anti-p38 alone but samples with rabbit anti-neurofilament did not have labeling. 

Figure 4 is the computer image of the 96-well microtiter plate. Each well is illustrated as a circle, and the thickness of the line used to draw the circle graphically displays the measured optical density in the wells. In the experiment illustrated, rows of weUs "A" and "H" were unused and are blmk. For each individual row, wells 3 through 10 illustrate a two-fold serial dilution of competitor. Wells in columns 1 and 2 for rows "B" through "G" are controls where the MelQx-albumin was omitted. The absence of color in these wells demonstrates the lack of non-specific binding of the anti-MelQx and peroxidase-anti-mouse antibodies, and defines experimentally "complete inhibition" for the competitive ELISA. Wells "Bll" through "G12" define the other extreme experimental condition, that of no inhibition. In these wells, no competitor was added and all of die anti-MelQx antibody was available to bind to the solid phase. 

Figure 4 Analysis of neutrophils in synovial fluid by flow cytometry. Synovial fluid was obtained from a control mouse and a mouse with established arthritis (days 7 after K/BxN serum injection). Cells were stained with Ly-6G antibody and analyzed by flow cytometry. Left panel green line indicates synovial fluid neutrophils from a mouse with established arthritis, red line shows synovial fluid neutrophils from control mouse, and black line reveals staining of established arthritis using an isotype control. The arthritic synovial fluid contains a large population of Ly-6G positive neutrophils while this population was absent in the synovial fluid of a control mouse. Right panel quantitation of the number of Ly-6G positive cells in control and arthritic joints (control mice n=2, arthritic mice n = 2). Data are the meaniSEM P<0.05 versus control mice.

Add diluted mouse anti-CCL21 and rabbit anti-CCR7 antibodies (in the Antibody Diluent provided with the PLA Probe kit) to the tissue. As negative controls, isotype-matched control IgG antibodies were added at same dilution. 

This vaccine is protective against invasive pneumococcal infections in immunocompetent adults [15]. Table 7 [14] lists the mouse IgG and IgM antibody response of four types of pneumococcal polysaccharide present in X-ray irradiated Polyvalent Pneumococcal Polysaccharide Vaccine Lot A and in a corresponding non-irradiated controls of the same polysaccharide type in Lot A. The type 9V, 14, 18C, and 19F polysaccharide samples were irradiated with X-rays at 24 kGy at room temperature and with X-rays at 13kGy at liquid nitrogen temperature. In each case for both the IgG and IgM mouse antibody responses there appeared to be no significant difference between the irradiated samples and the controls. 

Fig. 1. Effect of monoclonal antibodies and Fab fragments on enzyme activity. A, pifified poly(ADP-ribose) synthetase was preincubated with monoclonal antibodies at the molar ratios indicated. The enzyme activity was determined as described (4). Mab 137, a mouse monoclonal IgGl to prostaglandin E2, was used as a negative control. O, antibody G6 , antibody D4 A, antibody X3 , Mab 137. B, the enzyme was preincubated with Fab fragments. O, Fab from antibody G6 , Fab from antibody D4 A, Fab from antibody X3. A hundred-percent activity refers to the value obtained from control enzyme without antibody or Fab fragment.

B. Rfliovd, N. L. Krinick and J. Kopecek, Targetable photoacti-vatable drugs. 3. In vitro efficacy of polymer-bound chlorin-es toward human hepatocarcinoma cell-line (Plc/Prf/5) targeted with galactosamine and to mouse splenocytes targeted with anti-Thy 1.2 antibodies,/. Control. Release, 25, 71-87 (1993). 

---