In our lab, we use to incubate overnight at 4°C with agitation for the primary antibody. You could even try to incubate more than that at 4°C.

Do you know the kinetic constants for your antibody. Is the low affinity due to a reduced association or to a very high dissociation rate? That could change your possibilities. And what is the affinity vale that you call ''low''. Some ELISAs done with antibodies not having a very high affinity work perfectly. Is the low affinity a coating or a detection antibody?

Washing with PBS could result in non-specific background. I would try to play with the dilution factors instead. 

I do not know what you are working with, but if you have the luxury, I would put the antibody on a protein A capture chip and measure by SPR(Biacore).  The problm with ELISA is that if the Ab is low affinity (fast dissociating) it could easily completely dissocaite during the wash step.

I would rather try to reduce time and/or the blocking! However your working condition might not be optimum! try also to optimize and check all parameters; concentrations of antigen, primary, secondary antibodies, and times fro each step!

regards

Amended on 15 September 2017

I must sadly repeat about the differences in determination value between HPLC-photometric method and ELISA method (please see files in order to compare; SEC fucoidan determination and ELISA fucoidan).

The determined value of fucoidan (Okinawa-fucan) is c.a. 2,000-fold higher in the HPLC-photometric analysis as compared to notorious ELISA method (our experience together with Dr. Takeaki Nagamine, M.D., Graduate School of Health Sciences, Gunma University, 3-39-15 Showa-machi, Maebashi, Gunma 371-8514, Japan).   Dr. Nagamine has sadly thrown away the expensive fucoidan-assay kit of ELISA when he is retiring from the University by employing me (my unpublished observation or experience during my Arbeit at the University).

(1) Direct photometric assay without purification gives only 50% value as compared to HPLC-photometric assay due to violence against the Lambert-Beer's law (please see Fig. 10 of file "SEC fucoidan determination" again). 

(2) Further, binding protein is not so specific; i.e., avidin binds biotin (vitamin H), lipoic acid (thioctic acid), and amino acids, and binding is stronger to lipoic acid than biotin (please see file; Thioctic acid determination) .   Then, biotin assay by using avidin is very difficult since serum has 10-fold higher concentration of lipoic acid than biotin; i.e., lipoic acid may interfere the biotin binding reaction of avidin. 

(3) Furthermore, specificity of binding protein increases 10-fold by homo-dimerization(please see file; The Fascio Effect).   The specificity of IgG seems to be 100-fold lower than pentamer IgM due to the Fascio effect.

Then, 2 (1) x 10 (2) x 100 (3) = 2000-fold difference seems to be occurred between HPLC-photometric method and ELISA method. 

Therefore, notorious ELISA is not quantitative and not sensitive only giving false values at ng/mL.

Further, I must sadly repeat about the differences in determination value of biotin between HPLC-photometric method and binding assay method; i.e., such a biotin assay as

"Anal Biochem. 1986 Apr;154(1):367-70.

A sensitive enzyme assay for biotin, avidin, and streptavidin

Edward A. Bayer, Haya Ben-Hur, Meir Wilchek

Department of Biophysics, The Weizmann Institute of Science, Rehovot 76100, Israel

Reciprocal enzyme assays are described for the vitamin biotin and for the biotin-binding proteins avidin and streptavidin. The assays are based on the following steps: (a) biotinylated bovine serum albumin is adsorbed onto microtiter plates; (b) streptavidin (or avidin) is bound to the biotincoated plates: (c) biotinylated enzyme (in this case alkaline phosphatase) is then interacted with the free biotin-binding sites on the immobilized protein. For biotin assay, competition between the free vitamin and the biotinylated enzyme is carried out between steps (b) and (c). The method takes advantage of the four biotin-binding sites which characterize both avidin and streptavidin. The method is extremely versatile and accurate over a concentration range exceeding three orders of magnitude. The lower limits of detection are approximately 2 pg/ml (0.2 pg/sample) for biotin and less than 100 ng/ml (10 ng/sample) for either avidin or streptavidin."

gives only a false value with detection limit as 0.2 pg of biotin.

True HPLC-biotin determination method is presented in the file (Netherlands biotin).   HPLC-method gives a true value with detection limit 17 pg of biotin.  

By the way, I have recently found that homo-dimerization of human seum biotinidase has decreased Km for 10-fold and/or has increased 10-fold in the affinity to substrate biocytin (please again see file; The Fascio effect) with the help of Dr. Alaa Otaibi (Department of Prosthetic Dental Science, King Saud University, Riyadh, Riyadh, Saudi Arabia).  

Then, making multimer of IgG (from dimer to tetramer or pentamer oe hexamer) may be effective.   One example of mulimerization or aggregation has been previously presented with the collaboration of Dr. Claudio De Felice (Siena University, Siena, Italy) (please see file; CA agg Dr. De Felice).