Need a simplified SOP for this procedure please keep any measurements in microli

Question

Need a simplified SOP for this procedure please keep any measurements in microliters.

Measurement of inhibition of GlpQ activity. (i) Chemicals and enzymes.

-NAD hydrate (NAD; Sigma-Aldrich), sn-glycero-3-phosphate dehydrogenase

(GpDH [EC 1.1.1.8]; Fluka), L--glycerophosphorylcholine (GPC) (Sigma), and

hydrazine monohydrate (Sigma-Aldrich) were all purchased from Sigma-Aldrich,

and glycine (Merck) and calcium chloride (CaCl2; Merck) were purchased from

VWR International. rPD was produced at GSK Bio as previously described (1).

(ii) GlpQ enzyme inhibition assay. To measure the inhibition of PD’s GlpQ

activity, twofold serial dilutions (1:2 to 1:32; 50 l per well) of serum in 0.8 M

hydrazine-0.2 M glycine-10 mM CaCl2 buffer, pH 9.0, were prepared in the wells

of a 96-well microtiter plate (Corning Costar). rPD diluted in 0.8 M hydrazine-

0.2 M glycine-10 mM CaCl2 buffer, pH 9.0, was added (0.5 g per ml; 50 l per

well), and the plate was incubated for 1 h at 22°C.

After incubation, GlpQ activity was determined by a coupled photometric

assay, essentially as described previously (14, 15), by adding 150 l of a reaction

mixture containing NAD, GpDH, and GPC in 0.8 M hydrazine-0.2 M glycine-10

mM CaCl2 buffer, pH 9.0, to each well. After 5 min of incubation at room

temperature, the rate of NAD reduction was measured by recording the absorbance

of each well at room temperature at 340 nm with a microplate reader

(Multiskan MS; Thermo Fisher Scientific [formerly called Thermo Electron

Corporation], Waltham, MA), using a kinetic processor (Ascent software;

Thermo Fisher Scientific) with a reading interval of 1 min for a duration of 25

min. The final reaction concentrations in the 0.25-ml assay mixture were 0.5 mM

NAD, 10 U/ml of GpDH, 0.8 mM GPC, and 0.1 g/ml of rPD, unless otherwise

stated. The assay and dilution buffer was 0.8 M hydrazine-0.2 M glycine-10 mM

CaCl2, pH 9.0. Reaction buffer was made fresh every week (14), and the final

reaction solutions were prepared fresh daily just before use from stock solutions

stored at 70°C (NAD and GPC) or 4°C (GpDH and rPD). For the calculation

of enzymatic activity (NADH production [pmol] per min), a molar absorbance

coefficient of 6,300 M1 cm1 was used for NADH absorbance at 340 nm.

In the absence of serum (wells containing rPD plus reaction buffer), the

enzymatic activity of rPD dropped during the 1-hour incubation period to about

one-half that without the incubation step. This was prevented by the addition of

normal human serum. Hence, as the reference wells to which the enzymatic

activities of the pre- and postvaccination serum samples were compared, a

twofold serially (1:2 to 1:32) diluted serum pool prepared from unimmunized

infant serum without detectable anti-rPD IgG antibody was used in duplicate.

The reagent control contained plain assay buffer without added rPD and serum,

and the serum control contained reference serum or test serum (both at a 1:2

dilution) without rPD.

For calculation of the inhibition index, the background readings for serum control

wells without PD were subtracted from those for the corresponding wells containing

rPD. The inhibition index was thereafter calculated for each well as follows: ([GlpQ

activityReference pool GlpQ activityTest serum]/GlpQ activityReference pool)    100.

On the basis of the distribution of inhibition indexes for serum samples taken

before vaccination (99th percentile, 16.1; n 69), the threshold for the inhibition

assay was set to 20. Thus, sera with inhibition indexes of 20 at a 1:2 dilution

were considered enzyme inhibition assay negative and sera with inhibition indexes

of 20 were considered enzyme inhibition assay positive.

Explanation / Answer

To measure the inhibition of PD's GlpQ activity. Firstly do Two fold serial dilutions (1:2 to 1:32; 50 l per well) of serum in (0.8 M hydrazine-0.2 M glycine-10 mM CaCl2 buffer, pH 9.0), Dilution prepare in the wells of a 96-well microtiter plate (Corning Costar). then add rPD diluted in (0.8 M hydrazine-0.2 M glycine-10 mM CaCl2 buffer, pH 9.0), and add (0.5 g per ml; 50 l per well), and then plate incubate for 1 h at 22°C. After incubation, GlpQ activity determine by a coupled photometric assay, by adding 150 l of a reaction mixture containing NAD, GpDH, and GPC in (0.8 M hydrazine-0.2 M glycine-10 mM CaCl2 buffer, pH 9.0), to each well. allow for 5 min incubation at room temp. After 5 min of incubation, the rate of NAD reduction measure by recording the absorbance of each well at room temperature at 340 nm with a microplate reader (Multiskan MS; Thermo Fisher Scientific [formerly called Thermo Electron Corporation], Waltham, MA), using a kinetic processor (Ascent software; Thermo Fisher Scientific) with a reading interval of 1 min for a duration of 25 min. The final reaction concentrations in the 0.25-ml assay mixture were 0.5 mM NAD, 10 U/ml of GpDH, 0.8 mM GPC, and 0.1 g/ml of rPD, unless otherwise stated. The assay and dilution buffer is 0.8 M hydrazine-0.2 M glycine-10 mM CaCl2, pH 9.0. Reaction buffer is make fresh every week, and the final reaction solutions were prepare fresh daily just before use from stock solutions stored at 70°C (NAD and GPC) or 4°C (GpDH and rPD). For the calculation of enzymatic activity (NADH production [pmol] per min), a molar absorbance coefficient of 6,300 M1 cm1 is use for NADH absorbance at 340 nm. In the absence of serum (wells containing rPD plus reaction buffer), the enzymatic activity of rPD dropped during the 1-hour incubation period to about one-half that without the incubation step. This was prevented by the addition of normal human serum. Hence, as the reference wells to which the enzymatic activities of the pre- and post vaccination serum samples were compared, a twofold serially (1:2 to 1:32) diluted serum pool prepared from unimmunized infant serum without detectable anti-rPD IgG antibody was used in duplicate. The reagent control contained plain assay buffer without added rPD and serum, and the serum control contained reference serum or test serum (both at a 1:2 dilution) without rPD. For calculation of the inhibition index, the background readings for serum control wells without PD are subtracte from those for the corresponding wells containing rPD. The inhibition index is there after calculated for each well as follows: ([GlpQ activityReference pool GlpQ activityTest serum]/GlpQ activityReference pool) × 100. On the basis of the distribution of inhibition indexes for serum samples taken before vaccination (99th percentile, 16.1; n = 69), the threshold for the inhibition assay was set to 20. Thus, sera with inhibition indexes of

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