（请教）Competitive ELISA

一篇文献中的ELISA方法如下：

Control peptides (14–17 amino acid sequence near the N-terminal or C-terminal of the studied protein) and the reactive rabbit anti-defensin antibodies (affinity pure) were obtained from Alpha Diagnostics .

CoStar flat-bottom high-binding ELISA plates were coated with control peptides at 5ug/mL in 0.05 M of carbonate/bicarbonate buffer (pH 9.5). After overnight incubation at 4°C, the plates were brought to room temperature and washed four times with PBS, 0.05%Tween 20.

Nonspecific binding was blocked with 100 ul/well of blocking solution PBS and 1% BSA and incubation for 2 hours at room temperature followed by four times of washing with PBS and 0.05% Tween 20.

A fixed amount (25 uL) of antibody from 4 ug/mL of solution was reacted with bound (coated) antigen in the presence of known varying concentrations (0–40ug/mL) of the same control peptide antigen (standards) or the unknown (samples) all in a final volume of 100 uL/well in dilution buffer (PBS, 1% BSA, and 0.05% Tween 20) in duplicates.Blank wells contained only 100uL of dilution buffer. After 2 hours of incubation at room temperature, the wells were washed four times with PBS and 0.05% Tween 20.

Subsequently, goat anti-rabbit immunoglobulin G whole-molecule peroxidase conjugate affinity isolated antibody adsorbed with human serum proteins (Sigma Aldrich, Inc., Saint Louis, MO) diluted 1:10,000 with dilution buffer (PBS-1% BSA 0.05% Tween 20) was added (100 uL) to each well and plates were incubated for 2 hours at room temperature.

After washing four times with PBS and 0.05% Tween 20, 100uL of stabilized tetramethylbenzidine substrate solution (Sigma Aldrich, Inc.) was added to each well. After 30 minute of incubation at room temperature, 50 uL of stop solution (2 N of H2SO4) was added to each well and absorbance A450–620 was read using a Lab Systems Plus Plate Reader.

The amounts of protein in the unknowns were calculated with reference to the standard curve obtained with the known concentrations of peptide antigens. By optimizing the concentration of coating antigen and the antibody as given previously the ELISA could detect defensins as low as 0.5–1 ug/mL.

我没接触过ELISA实验,请教一下这个竞争法是不是这样的?

用等浓度抗原包被,然后加等浓度抗体和含未知浓度蛋白的样本,因此,固相抗原和样本中的抗原竞争结合加入的一抗. 加入标记的二抗后,与固相抗原结合的抗体可以显色.如果样本中抗原含量高,一抗和样本抗原结合的复合物被洗脱,因此与固相抗原结合的一抗减少,显色程度降低, 显色程度和样本抗原含量成反比,由此进行定量.

在这个实验中,如果要人工合成多肽作为固相抗原，合成的纯度多少适宜?一抗是多抗还是单抗?(以上文献中没有说明)　另外，如果我想用抗体包被，然后人工合成生物素标记抗原肽和标本中的抗原竞争结合固相抗体，是不是更加简便可行？

请战友多多指教!谢谢!