ELISA PS-PS (Identity /Ag content) clin.trial.

[ochernen]

Метод клинических исследований:

Название документа:
IDENTITY AND CONTENT OF NEISSERIA PS IN FINAL CONTAINER NIMENRIX
Я перевел как :
ОПРЕДЕЛЕНИЕ ПОДЛИННОСТИ И СОДЕРЖАНИЯ ПОЛИСАХАРИДОВ БАКТЕРИЙ РОДА НЕЙССЕРИЯ В КОНЕЧНОМ КОНТЕЙНЕРЕ С ВАКЦИНОЙ НИМЕНРИКС (NIMENRIX)

Сам метод:
Method : ELISA PS-PS (Identity /Ag content)

Что здесь ELISA PS-PS и т.д. (в условных обозначениях написано PS: Polysaccharide)??

Так и написать что-ли ИФА ПС-ПС (??), и если можно объясните чайнику чуть-чуть...в целях повышения образованности, что это за хрень??

Заранее благодарен.

[Shakhnin Dmitrii]

Тест, в котором и идентификация, и определение содержания антигена производится по специфическим полисахаридам. ELISA = ИФА, иммуно-ферментный анализ, при котором антитело, связывающееся со специфическим антигеном, несет на себе фермент, активность которого используют в качестве мерила количества связавшегося антитела, т.е. количества определяемого антигена.

[Dimpassy]

а нету там малость поширше описания про методику ELISA?

[ochernen]

Даю юольше описания:
5.6 Method : ELISA PS-PS (Identity /Ag content)

5.6.1 Putting in solution of standard, samples or internal control
- Reconstitute each freeze-dried sample in 625 µl of NaCl 0.15 M (physiological water).

5.6.2 Microplates coating
- Dilute(*) the monoclonal antibodies anti-PS (specific to the PS to quantify) in the carbonate buffer hco3/co32- 0.05M.
- Distribute 100µl of this solution in each well of the micmpiate.
- Cover the plates with an adhesive film or a lid.
- Incubate the microplates during 1 night at +4°C.
(*) the factors of dilution are defined in the annex.

5.6.3 Preparation of thesamples, the standard and the internal control
- Predilute the samples, the standard the internal control in the dilution buffer to be approximately at 40ng/mf in first well for PSA-C-W and approximately at 80 ng/ml for PSY.
- For a final container, the dilution is based on the theoretical PS content. For a final container ACWY-TT 1 dose (5/5/515): 10 µg/ml

5.6.4 Incubation of the samples, the standard and the internal control
Wash the microplates using the microplate washer (4 cycles of washing with 350µl of washing solution)
- Add 100µl of dilution buffer in each well, except in the first column
- Add 100µl of the different dilutions of samples, standard and internal control in columns 1 and 2 according to the scheme below:

Schemfeof theplate: 1 2 3 4 5 6 7 8 9 10 11 12
А Standard (dilution of 2 in 2)
В Blanks
C Internal control (dilution of 2 in 2)
D
E Sample 1 (dilution of 2 in 2)
F
G Sample 2 (dilution of 2 in 2)
н

- The standard, the internal control and the samples are then diluted serially (2-fold) from column 2 to column 11. Discard 100µ1 at the column 11. The column 12 corresponds to the blanks.
- Cover the plates with an adhesive film or a lid
- Incubate the microplates during 1 hour at 25°C under agitation

Note: the dilutions are carried out plate by plate as soon as possible after the deposit of the samples.

5.6.5 Incubation with the anti-PS coupledmith peroxidase
- Dilute(*) monoclonal antibody anti-PSA-PO in the dilution buffer containing 0.1 % of negative mouse serum.
- Dilute(*) monoclonal antibodies anti-PSC-PO, anti PSW-PO and anti PSY-PO in the dilution buffer.
- Wash the microplates 4 fold as previously described.
- Deposit 100µl of monoclonal antibody coupled with peroxidase in each well Cover the plates with an adhesive film or a lid.
- Incubate the microplates during 1 hour at 25°C under agitation. (*) the factors of dilution are defined in the" annex.

5.6.6 Revelation (oxidation of chromogen) "
- Wash the microplates PSA, PSC, PSW and PSY 4 fold as previously described.
- Add 100µl of coloring solution in each well.
- Incubate the microplates during 15 min at ambient temperature, in the dark.
- Stop the reaction with addition of 50µl H2SO4 in each well.

5.6.7 Riding
As soon as the region is stopped, read the optical densities of microplate with the spectrophotometr at 490 and 620 nm.

И еще:
anti-PSA-PO anti-PSC-PO, anti PSW-PO и anti PSY-PO это антитела к полисахаридам??

Заранее благодарен.

[Dimpassy]

это стандартная методика ИФА полисахаридов
anti-PSA-PO - антитела к полисахариду А, несущие пероксидазу (РО)

[ochernen]

Спасибо Огромное!!