Evaluation of Specificity of Antibodies Using ELISA

The reactivity of the anti-heparan sulfate antibodies with other molecules can be analyzed by ELISA in two ways: (1) by application of antibodies to wells of microtiter plates coated with the test molecule, or (2) by an inhibition assay in which the antibodies are incubated with the test molecule.

Method 1

1. All incubation steps are carried out at room temperature.

2. Coat wells with test molecules (see step 9, Subheading 2.8.) by incubation with 10 [g of test molecules/mL solution for 16 h at 4°C.

3. Wash the wells 6 times with PBS containing 0.1% (v/v) Tween-20.

4. Block the free binding sites with 200 [L of blocking solution for 1 h.

5. Wash the wells 6 times with PBS containing 0.1% (v/v) Tween-20.

6. Incubate the wells with 100 [L of anti-heparan sulfate (primary) antibody solution for 60 min.

7. Wash the wells 6 times with PBS containing 0.1% (v/v) Tween-20.

8. Incubate the wells with 100 [L of mouse anti-cMyc antibody solution for 60 min.

Fig. 3. Specificity of anti-heparan sulfate antibody HS3G8. Rat kidney cryosections were treated with heparinase III (A), heparinase III incubation buffer (B), chondroitinase ABC (C), and chondroitinase ABC incubation buffer (D). Next, sections were incubated with periplasmic fraction containing the antibody. Bound antibodies were visualized using mouse anti-cMyc IgG followed by Alexa 488-conjugated goat anti-mouse IgG. Bar= 50 ^m. Source: from ref. 5.

Fig. 3. Specificity of anti-heparan sulfate antibody HS3G8. Rat kidney cryosections were treated with heparinase III (A), heparinase III incubation buffer (B), chondroitinase ABC (C), and chondroitinase ABC incubation buffer (D). Next, sections were incubated with periplasmic fraction containing the antibody. Bound antibodies were visualized using mouse anti-cMyc IgG followed by Alexa 488-conjugated goat anti-mouse IgG. Bar= 50 ^m. Source: from ref. 5.

9. Wash the wells 6 times with PBS containing 0.1% (v/v) Tween-20.

10. Incubate the wells with 100 ^L of alkaline phosphatase-conjugated rabbit anti-mouse IgG antibody solution for 60 min.

11. Wash the wells 4 times with PBS containing 0.1% (v/v) Tween-20.

13. Incubate the wells with 100 ^L of substrate solution (in the dark) until color development is optimal.

14. Read absorbance at 405 nm.

Method 2

1. Add 6 ^g of test molecule to 150 ^l antibody solution and incubate overnight at room temperature.

2. Transfer 100 ^l of this solution to a well coated with heparan sulfate and incubate for 60 min.

3. Proceed as under Method 1, starting with step 7.

Fig. 4. Immunostaining of rat kidney with three different anti-heparan sulfate scFv antibodies. Cryosections were incubated with periplasmatic fractions of bacteria expressing antibody HS4C3 (A), HS4D10 (B), HS3G8 (C), and anti-filaggrin (D)(control; filaggrin is not present in the kidney). Bound antibodies were visualized using mouse anti-cMyc IgG followed by Alexa 488-conjugated goat anti-mouse IgG. Bar=25 ^m. All three anti-heparan sulfate antibodies stain differently, indicating reactivity with different heparan sulfate species. G, glomerulus. Arrow in a: peritubular capillary. Source from ref. 5.

Fig. 4. Immunostaining of rat kidney with three different anti-heparan sulfate scFv antibodies. Cryosections were incubated with periplasmatic fractions of bacteria expressing antibody HS4C3 (A), HS4D10 (B), HS3G8 (C), and anti-filaggrin (D)(control; filaggrin is not present in the kidney). Bound antibodies were visualized using mouse anti-cMyc IgG followed by Alexa 488-conjugated goat anti-mouse IgG. Bar=25 ^m. All three anti-heparan sulfate antibodies stain differently, indicating reactivity with different heparan sulfate species. G, glomerulus. Arrow in a: peritubular capillary. Source from ref. 5.

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