| Ingredients | Test | «Positive control» | «Negative control» | |||||
| A1 A2 | B1 B2 | C1 C2 | D1 D2 | E1 E2 | F1 F2 | G1 G2 | H1 H2 | |
| 1. Buffer A (ml) | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 |
| Incubation at room temperature for 10 min. | ||||||||
| 2. The patient’s serum (ml) | 0.1 | -- | -- | -- | -- | -- | -- | -- |
| 3. Standard antigen 100 μg/ml (ml) | -- | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 pour out | -- |
| 4. Reagent «negative control» (ml) | -- | -- | -- | -- | -- | -- | -- | 0.1 |
| Incubation at 37oC for 20 min. | ||||||||
| 5. Conjugate (ml) | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 |
| Incubation at 37oC for 20 min. | ||||||||
| 6. Substrate (ml) | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 |
| Incubation at room temperature for 20 min. | ||||||||
| 7. 50% sulfuric acid (ml) | 0.05 | 0.05 | 0.05 | 0.05 | 0.05 | 0.05 | 0.05 | 0.05 |
| Results: (color/no color) | ||||||||
| Conclusion: the ELISA is ________________(positive/negative). The result means, that Hbs Ag is (not) detected in the patient’s serum. The concentration of HBs Ag in the patient’s serum is _______ μg/ml. |
Step 1.
A. Take the special plastic microtiter plate with numerous shallow wells. The anti-Hbs antibodies are adsorbed to its wells. Choose two vertical rows for performing the test.
B. Inoculate 0.1 ml of buffer A into each well in each vertical row (from A1 to H1 and from A2 to H2). Allow the plate to stand for 10 min. at room temperature.
C. Add equal amount (0.1 ml) of patient’s serum to the wells # A1 and # A2.
D. Add at 0.1 ml of standard antigen (initial concentration 100 μg/ml) to the wells B1 and B2, mix thoroughly and transfer 0.1 ml of the mixture into the next wells (C1 and C2 accordingly). Prepare the serial two-fold dilution in the next wells (from D1 to G1 and from D2 to G2). Pour out 0.1 ml of the mixture into the conteiner with disinfectant from the G1 and G2 wells.
E. Add at 0.1 ml of «negative control» to the wells H1 and H2.
F. Place the microtiter plate in the 37oC incubator. Allow plateto stand for 20 min.
Step 2.
A. Rinse off the plate with tap water.
B. Pipette 0.1 ml of conjugate (enzyme-labeled antiserum) into each well in each vertical row (A 1- H1 and A2 - H2).
C. Incubate the plate at 37oC for 20 min.
Step 3.
A. Wash off the plate with tap water three times before the next step.
B. Add 0.1 ml of substrate solution into each well (see Table 14-1).
C. Allow the plate to stand at room temperature for 20 min.
D. Add 0.05 ml of 50 per cent sulfuric acid into each well to stop the reaction.
BE CAREFUL: The step D is performed by lab technician!
E. Read the results visually. To do this, examine the color in the tested wells (A1 and A2), compare the color in these wells with the color of H1 and H2 (negative control ). Compare the intense of color in the A 1 and A2 (if the reaction in the tested well produces a visible color change) with the color in the wells with diluted standard antigen (B1 - G1 and B2 - G2).
PRACTICAL TASKS
1. Perform the ELISA to detect the Hbs Ag in the patient’s blood serum. Estimate the results. Make a conclusion. Draw the scheme of the test. Write down and fill in Table 14-1 in your protocol notebook.
2. Familiarize yourself the schemes of direct and indirect IF-test. For these schemes see wall display in the classroom.
LESSON 15
MEDICAL IMMUNOBIOLOGICAL PREPARATIONS
Prelab conference. Topics for discussion:
1. Acquired active and passive immunity.
2. Preparations to induce active artificial immunity.
3. Vaccines. Classification, application.
4. Vaccine strains. Requirements and preparation.
5. Toxoids. Preparation, titration, application.
6. Associated vaccines. Adjuvants.
7. Preparations to form artificial passive immunity.
8. Antibody-containing antisera for immunotherapy and immunoprophylaxis.
9. Serotherapy and seroprophylaxis complications. Mechanism, protection from complications.
10. Allergens. Their application.
Immunobiological preparations used for diagnosis, treatment and immunoprophylaxis of infectious diseases are listed in Table 15-1.
Table 15-1
Medical Immunobiological Preparations
| MIBP for (immuno)therapy and (immuno)prophylaxis | MIBP for infectious disease diagnosing |
| 1. Antigen-containing preparations (vaccines, toxoids) 2. Antibody-containing preparations (antisera, immunoglobulins, etc.) 3. Immunomodifiers and adaptogens 4. Eubiotics 5. Bacteriophages | 1. Components of serological reactions (diagnosticums, diagnostic immune antisera, complement, monoclonal antibodies, labeled antibodies, etc.) 2. Diagnostic reagents for microbiological examination v In vivo (allergens) v In vitro (bacteriophages - for phagotyping) |
¨ ACTIVE and PASSIVE IMMUNITY
Besides innate immunity, immunity may be acquired either actively or passively, depending on whether the immune person produces his own protective antibodies or T cells, or receives presynthesized antibodies or sensitized lymphocytes from another individual. Both active and passive immunity can be acquired either naturally or artificially (see Table 15-2).
Table 15-2