Human Tetanus

FOR RESEARCH USE ONLY

96 determinations

Purpose

This kit allows for the determination of Tetanus expression in Human serum, ａnd other

biological fluids.

Principle of the assay

The kit assay Tetanus level in the sample，use Purified Tetanus antibody to coat microtiter

plate wells, make solid-phase antibody, then add Tetanus to wells, Combined With Tetanus,

after washing ａnd removing non-combinative antibody ａnd other components ,then Combined

Tetanus antibody which with HRP labeled become antibody – antigen - enzyme- antibody

complex, after washing Completely, Add TMB substrate solution,, TMB substrate becomes blue

color At HRP enzyme-catalyzed, reaction is terminated by the addition of a sulphuric acid

solution ａnd the color change is measured spectrophotometrically at a wavelength of 450 nm.

Compared with the CUTOFF value, according to this to judge Tetanus exist in the sample or

not.

Materials provided with the kit

1 wash solution 20ml×1bottle 7 Stop Solution 6ml×1 bottle

2 HRP-Conjugate reagent 6ml×1 bottle 8 Positive control 0.5ml×1 bottle

3 Microelisa stripplate 12well×8strips 9 Negative control 0.5ml×1bottle

4 Sample diluent 6ml×1 bottle 10 Instruction 1

5 Chromogen Solution A 6ml×1 bottle 11

Closure plate

membrane

2

6 Chromogen Solution B 6ml×1 bottle 12 Sealed bags 1

Specimen requirements

1. extract as soon as possible after Specimen collection,and according to the relevant

literature, ａnd should be experiment as soon as possible after the extraction. If it can’t,

specimen can be kept in -20 ℃ to preserve, Avoid repeated freeze-thaw cycles.

2. Can’t detect the sample which contain NaN3, because NaN3 inhibits HRP active.

Assay procedure

1.Number: to sample correspond microtitration well ａnd Number Sequence, each plate should

be set feminine comparison 2 wells, masculine comparison 2 wells, blank comparison 1

well（don’t add sample ａnd HRP-Conjugate reagent to blank comparison well, other each step

the operation are same）.

2.add sample：separately add Positive control ａnd Negative control 50μl to the Positive and

Negative well . add Sample dilution 40μl to testing sample well, then add testing sample 10μl.

add sample to the bottom of ELISA plates coated well , don’t touch the well wall as far as

possible, ａnd Gently mix.

3.Incubate: After closing plate with Closure plate membrane ,incubate for 30 min at 37℃.

4.Configurate liquid: 30-fold （or 20-fold）wash solution diluted 30-fold （or 20-fold） with distilled

water until 600ml,and reserve.

5.washing：Uncover Closure plate membrane, discard Liquid, dry by swing, add washing buffer

to every well, still for 30s then drain, repeat 5 times, dry by pat.

6.add enzyme：Add HRP-Conjugate reagent 50μlto each well, except the blank well.

7.incubate：Operation with 3.

8.washing：Operation with 5.

9.color：Add Chromogen Solution A 50ul ａnd Chromogen Solution B to each well, evade the

light preservation for 15 min at 37℃

10.Stop the reaction：Add Stop Solution50μl to each well, Stop the reaction（the blue color

change to yellow color）.

11. assay：take blank well as zero , Read absorbance at 450nm after Adding Stop Solution and

within 15min.

Determine the result

Test validity: the average of Positive control well≥1.00; the average of Negative control well

≤0.10.

Calculate Critical（CUT OFF） : Critical= the average of Negative control well + 0.15.

Negative control: sample OD< Calculate Critical（CUT OFF） is Tetanus Negative control.

Positive control: ample OD≥ Calculate Critical（CUT OFF） is Tetanus Positive control.

Important notes

1.Please according to use instruction strictly, Do not mix reagents with those from other lots.

2.The kit takes out from the refrigeration environment should be balanced 15-30 minutes in

the room temperature then use, ELISA plates coated if has not use up after opened, the

plate should be stored in Sealed bag.

3.washing buffer will Crystallization separation, it can be heated the water helps dissolve

when dilute . Washing does not affect the result.

4.Closure plate membrane only limits the disposable use, in order to avoid the overlapping

pollution

5.The substrate please evade the light preservation.

6.The test result determination must take the microtiter plate reader as a standard, when use

dual-wavelength to assay, Reference wavelength is 630nm.

7.All samples, washing buffer ａnd each kind of reject should according to infective material

process. Stopp Solution is 2M sulphuric acid. You must pay attention to safe when use .

Storage ａnd validity

1．Storage： 2-8℃.

2．validity： six months.Human Tetanus